CN102985561B - Normalizing chromosomes for the determination and verification of common and rare chromosomal aneuploidies - Google Patents

Normalizing chromosomes for the determination and verification of common and rare chromosomal aneuploidies Download PDF

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CN102985561B
CN102985561B CN201180022971.0A CN201180022971A CN102985561B CN 102985561 B CN102985561 B CN 102985561B CN 201180022971 A CN201180022971 A CN 201180022971A CN 102985561 B CN102985561 B CN 102985561B
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里查德·P·拉瓦
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Abstract

The present invention provides a method capable of detecting single or multiple fetal chromosomal aneuploidies in a maternal sample comprising fetal and maternal nucleic acids, and verifying that the correct determination has been made. The method is applicable to determining copy number variations (CNV) of any sequence of interest in samples comprising mixtures of genomic nucleic acids derived from two different genomes, and which are known or are suspected to differ in the amount of one or more sequence of interest. The method is applicable at least to the practice of noninvasive prenatal diagnostics, and to the diagnosis and monitoring of conditions associated with a difference in sequence representation in healthy versus diseased individuals.

Description

For determining and verifying normalization method karyomit(e) that is common and rare chromosomal aneuploidy
Invention field
The invention provides and a kind ofly can determine single or multiple fetal chromosomal aneuploidy in the maternal sample comprising fetus and maternal nucleic acids and the method for correct determination has been made in checking.The method is at least applicable to the enforcement of Non-invasive Prenatal Diagnosis, and is applicable to the diagnosis and the monitoring that contrast the symptom that sequence differential expression is associated in ill individuality with healthy individuals.
Background of invention
Obstetric and Gynecologic Department association of the U.S. (the American College of Obstetrics andGynecology announced for 2007; ACOG) implement notification number 77 to support to carry out dysploidy risk assessment to the first three months of all pregnant women's, this assessment is measured based on nuchal translucency and substitutes biochemical marker, in order to examination mongolism (Obstetric and Gynecologic Department association of the U.S. implements notification number 77 (ACOG Practice Bulletin No.77), Obstetric and Gynecologic Department (Obstet Gynecol) 109:217-227 [2007]).These examinations test only can provide risk to determine, this risk is determined indecisive and had the determination of non-optimal and high false positive rate.Nowadays, only have comprise chorionic villus sampling (CVS), the invasive methods of amniocentesis or umbilical cord puncture just provides clear and definite genetic information about fetus, but these programs are to mother and fetus all risky (people such as Odie ripple (Odibo), Obstetric and Gynecologic Department (Obstet Gynecol) 112:813-819 [2008]; The people such as Odie ripple (Odibo), Obstetric and Gynecologic Department (Obstet Gynecol) 111:589-595 [2008]; Ai Wensi (Evans) and Wa Puna (Wapner), perinatology collection of thesis (Semin Perinatol) 29:215-218 [2005]).Therefore, desirably a kind of being used for obtains the non-invasive means about the clear and definite information of fetal chromosomal state.
Extensive parallel DNA sequencing is carried out to the cfDNA obtained from Maternal plasma and produces millions of short data records labels, these short data records labels can be compared and be mapped to from the site with reference to human genome uniquely, and the counting of the label mapped may be used for determining chromosomal overexpression or expresses not enough (people such as model (Fan), institute of NAS periodical (Proc Natl Acad Sci USA) 105:16266-16271 [2008]; Wei Erketing (Voelkerding) and Lyons (Lyon), clinical chemistry (Clin Chem) 56:336-338 [2010]).But the order-checking degree of depth and subsequent counter statistics determine the sensitivity that fetus dysploidy is determined.Obviously the trisomy of more than one types can not be determined in test sample population, this situation highlights for being used for the demand (people such as Zhao (Chiu) of algorithm of the optimization determining chromosomal aneuploidy in Maternal plasma sample, BMJ (BMJ) 342, c7401 [2011]; The people such as Eric (Ehrich), U.S.'s journal of obstetrics and gynecology (Am J Obstet Gynecol) 2014:205 e1 [2011]).
Existing methodical limitation becomes the basis of the demand of the non invasive method for the best, the non invasive method of these the bests by for antenatal diagnosis and with copy number change the diagnosis of relevant Medical Condition and monitoring provide in specificity, susceptibility and suitability any one or all reliably to diagnose chromosomal aneuploidy.
Present invention achieves some in the demand, and particularly provide an advantage, namely provide a kind of method reliably, the method has enough susceptibilitys to determine single or multiple chromosomal aneuploidy, and correct determination has been made in the method checking.
Summary of the invention
The invention provides and a kind ofly can determine single or multiple fetal chromosomal aneuploidy in the maternal sample comprising fetus and maternal nucleic acids and the method for correct determination has been made in checking.The method is applicable to copy number variation (CNV) determining any interested sequence in multiple sample, these samples comprise the mixture of the genomic nucleic acids deriving from two different genes groups, and known or suspect that these two different genes groups are different in the amount of one or more interested sequence.The method is at least applicable to the enforcement of Non-invasive Prenatal Diagnosis, and is applicable to the diagnosis and the monitoring that contrast the symptom that sequence differential expression is relevant in ill individuality with healthy individuals.
In one embodiment, the method determines a kind of fetal chromosomal aneuploidy of presence or absence by following steps in the parent test sample comprising fetus and maternal nucleic acids molecule: (a) obtains the sequence information for fetus in maternal sample and maternal nucleic acids, to identify for a number of an interested chromosomal multiple sequence label and a number at least two chromosomal multiple sequence labels of normalization method; B () uses the number of sequence label to calculate for interested chromosomal first normalized value and second normalized value; And (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for interested chromosomal second normalized value and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in the sample to which.First and second threshold values can be identical, or they can be different.In the step (c) of this method, indicate presence or absence for described interested chromosomal a kind of dysploidy for described interested chromosomal first normalized value with comparing of threshold value, and for the comparatively validate presence or absence of described interested chromosomal second normalized value and threshold value for the determination of interested chromosomal a kind of dysploidy.In some embodiments, first normalized value is a first chromosome dosage, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is a second karyomit(e) dosage, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as described normalized karyomit(e) value (NCV).
In above-mentioned and all subsequent implementation schemes, the step obtaining order-checking information comprises order-checking (NGS) of future generation.NGS uses multiple reversible dye-terminators to carry out synthesis method order-checking (sequencing-by-synthesis).Alternately, NGS can be that connection method order-checking (sequencing-by-ligation) checks order.NGS can also be single-molecule sequencing.
Similarly, in above-mentioned and all subsequent implementation schemes, the normalization method karyomit(e) for karyomit(e) 21 is selected from karyomit(e) 9,11,14 and 1.In some embodiments, the normalization method karyomit(e) for karyomit(e) 18 is selected from karyomit(e) 8,3,2 and 6.In some embodiments, be selected from karyomit(e) 4, the group of karyomit(e) 2-6, karyomit(e) 5 and karyomit(e) 6 for the normalization method karyomit(e) of karyomit(e) 13.In some embodiments, the normalization method karyomit(e) for chromosome x is selected from karyomit(e) 6,5,13 and 3.In some embodiments, the normalization method karyomit(e) for karyomit(e) 1 is selected from karyomit(e) 10,11,9 and 15.In some embodiments, the normalization method karyomit(e) for karyomit(e) 2 is selected from karyomit(e) 8,7,12 and 14.In some embodiments, the normalization method karyomit(e) for karyomit(e) 3 is selected from karyomit(e) 6,5,8 and 18.In some embodiments, the normalization method karyomit(e) for karyomit(e) 4 is selected from karyomit(e) 3,5,6 and 13.In some embodiments, the normalization method karyomit(e) for karyomit(e) 5 is selected from karyomit(e) 6,3,8 and 18.In some embodiments, the normalization method karyomit(e) for karyomit(e) 6 is selected from karyomit(e) 5,3,8 and 18.In some embodiments, the normalization method karyomit(e) for karyomit(e) 7 is selected from karyomit(e) 12,2,14 and 8.In some embodiments, the normalization method karyomit(e) for karyomit(e) 8 is selected from karyomit(e) 2,7,12 and 3.In some embodiments, the normalization method karyomit(e) for karyomit(e) 9 is selected from karyomit(e) 11,10,1 and 14.In some embodiments, the normalization method karyomit(e) for karyomit(e) 10 is selected from karyomit(e) 1,11,9 and 15.In some embodiments, the normalization method karyomit(e) for karyomit(e) 11 is selected from karyomit(e) 1,10,9 and 15.In some embodiments, the normalization method karyomit(e) for karyomit(e) 12 is selected from karyomit(e) 7,14,2 and 8.In some embodiments, the normalization method karyomit(e) for karyomit(e) 14 is selected from karyomit(e) 12,7,2 and 9.In some embodiments, the normalization method karyomit(e) for karyomit(e) 15 is selected from karyomit(e) 1,10,11 and 9.In some embodiments, the normalization method karyomit(e) for karyomit(e) 16 is selected from karyomit(e) 20,17,15 and 1.In some embodiments, the normalization method karyomit(e) for karyomit(e) 17 is selected from karyomit(e) 16,20,19 and 22.In some embodiments, the normalization method karyomit(e) for karyomit(e) 19 is selected from 22,17,16 and 20.In some embodiments, the normalization method karyomit(e) for karyomit(e) 20 is selected from karyomit(e) 16,17,15 and 1.In some embodiments, the normalization method karyomit(e) for chromosome 22 is selected from karyomit(e) 19,17,16 and 20.
In another embodiment, the method determines a kind of fetal chromosomal aneuploidy of presence or absence by following steps in the parent test sample comprising fetus and maternal nucleic acids molecule: (a) obtains the sequence information for fetus in maternal sample and maternal nucleic acids, to identify for a number of an interested chromosomal multiple sequence label and a number at least two chromosomal multiple sequence labels of normalization method; B () uses the number of sequence label to calculate for interested chromosomal first normalized value and second normalized value; And (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for interested chromosomal second normalized value and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in the sample to which.First and second threshold values can be identical, or they can be different.In the step (c) of this method, show that presence or absence is for described interested chromosomal a kind of dysploidy for described interested chromosomal first normalized value with comparing of threshold value, and for the comparatively validate presence or absence of described interested chromosomal second normalized value and threshold value for the determination of interested chromosomal a kind of dysploidy.In some embodiments, first normalized value is a first chromosome dosage, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is a second karyomit(e) dosage, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as described normalized karyomit(e) value (NCV).Fetal chromosomal aneuploidy can be a kind of part or complete chromosomal aneuploidy.In these embodiments, fetal chromosomal aneuploidy can be selected from trisomy 21 (T21), 18 trisomys (T18), 13 trisomys (T13), X monosomy.In some embodiments, maternal sample obtains from a pregnant woman.In some embodiments, maternal sample is a kind of biological fluid sample, such as a blood sample or the blood plasma fractions that obtains from blood sample.In some embodiments, maternal sample is a plasma sample.In some embodiments, the nucleic acid in maternal sample is cfDNA molecule.In some other embodiments, parent test sample is the plasma sample obtained from a pregnant woman, and nucleic acid molecule is cfDNA molecule.
In another embodiment, the chromosomal aneuploidy that the method determination presence or absence at least two kinds is different.In one embodiment, the method by determining the fetal chromosomal aneuploidy that presence or absence at least two kinds is different at least two interested karyomit(e) repeating step (a)-(c) in the parent test sample comprising fetus and maternal nucleic acids molecule, wherein these steps comprise (a) and obtain sequence information for fetus in maternal sample and maternal nucleic acids, to identify for a number of an interested chromosomal multiple sequence label and a number at least two chromosomal multiple sequence labels of normalization method; B () uses the number of sequence label to calculate for interested chromosomal first normalized value and second normalized value; And (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for interested chromosomal second normalized value and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in the sample to which.First and second threshold values can be identical, or they can be different.In the step (c) of this method, show that presence or absence is for described interested chromosomal a kind of dysploidy for described interested chromosomal first normalized value with comparing of threshold value, and for the comparatively validate presence or absence of described interested chromosomal second normalized value and threshold value for the determination of interested chromosomal a kind of dysploidy.In some embodiments, first normalized value is a first chromosome dosage, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is a second karyomit(e) dosage, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as normalized karyomit(e) value (NCV) as described herein.In some embodiments, the method comprises and repeats the method to determine the fetal chromosomal aneuploidy that presence or absence at least two kinds is different for all karyomit(e).
In another embodiment, the chromosomal aneuploidy that the method determination presence or absence at least two kinds is different.In one embodiment, the fetal chromosomal aneuploidy of the method by determining that at least two interested karyomit(e) repeating step (a)-(c) presence or absence at least two kinds is different in the parent test sample comprising fetus and maternal nucleic acids molecule, wherein these steps comprise (a) and obtain sequence information for fetus in maternal sample and maternal nucleic acids, to identify for a number of an interested chromosomal multiple sequence label and a number at least two chromosomal multiple sequence labels of normalization method; B () uses the number of sequence label to calculate for interested chromosomal first normalized value and second normalized value; And (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for interested chromosomal second normalized value and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in the sample to which.First and second threshold values can be identical, or they can be different.In the step (c) of this method, show that presence or absence is for described interested chromosomal a kind of dysploidy for described interested chromosomal first normalized value with comparing of threshold value, and for the comparatively validate presence or absence of described interested chromosomal second normalized value and threshold value for the determination of interested chromosomal a kind of dysploidy.In some embodiments, first normalized value is a first chromosome dosage, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is a second karyomit(e) dosage, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as normalized karyomit(e) value (NCV) as described herein.In some embodiments, the method comprises and repeats the method to determine the fetal chromosomal aneuploidy that presence or absence at least two kinds is different for all karyomit(e).At least two kinds of different fetal chromosomal aneuploidies can be selected from T21, T18, T13 and X monosomy.In some embodiments, maternal sample obtains from a pregnant woman.In some embodiments, maternal sample is a kind of biological fluid sample, such as a blood sample or the blood plasma fractions that obtains from blood sample.In some embodiments, maternal sample is a plasma sample.In some embodiments, the nucleic acid in maternal sample is cfDNA molecule.In some other embodiments, parent test sample is the plasma sample obtained from a pregnant woman, and nucleic acid molecule is cfDNA molecule.
In another embodiment, the method by following steps checking in the parent test sample comprising fetus and maternal nucleic acids molecule presence or absence for the determination of interested chromosomal a kind of dysploidy: (a) obtains the sequence information for fetus and maternal nucleic acids in the sample to which, to identify for a number of the sequence label of an interested chromosomal multiple mapping and a number at least two chromosomal multiple sequence labels of normalization method; (b) use for interested chromosomal label number and determine for interested chromosomal first normalized value for the number of a chromosomal label of the first normalization method, and use for the chromosomal sequence label of the first normalization method number and determine for chromosomal second normalized value of the first normalization method for the number of a chromosomal sequence label of the second normalization method; And (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for chromosomal second normalized value of the first normalization method and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in the sample to which.First and second threshold values can be identical, or they can be different.In the step (c) of this method, show that presence or absence is for described interested chromosomal a kind of dysploidy for described interested chromosomal first normalized value with comparing of threshold value, and for the comparatively validate presence or absence of chromosomal second normalized value of described first normalization method and threshold value for the determination of interested chromosomal a kind of dysploidy.In some embodiments, first normalized value is a first chromosome dosage, it is the number for described interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is a second karyomit(e) dosage, it is the number for the chromosomal sequence label of the first normalization method and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as the normalized karyomit(e) value (NCV) of described calculating.
In another embodiment, the method verifies the determination of presence or absence for interested chromosomal a kind of dysploidy by following steps in the parent test sample comprising fetus and maternal nucleic acids molecule: (a) obtains the sequence information for fetus and maternal nucleic acids in the sample to which, to identify for a number of the sequence label of an interested chromosomal multiple mapping and a number at least two chromosomal multiple sequence labels of normalization method; (b) use for interested chromosomal label number and determine for interested chromosomal first normalized value for the number of a chromosomal label of the first normalization method, and use for the chromosomal sequence label of the first normalization method number and determine for chromosomal second normalized value of the first normalization method for the number of a chromosomal sequence label of the second normalization method; And (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for chromosomal second normalized value of the first normalization method and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in the sample to which.First and second threshold values can be identical, or they can be different.In the step (c) of this method, show that presence or absence is for described interested chromosomal a kind of dysploidy for described interested chromosomal first normalized value with comparing of threshold value, and for the comparatively validate presence or absence of chromosomal second normalized value of described first normalization method and threshold value for the determination of interested chromosomal a kind of dysploidy.In some embodiments, first normalized value is a first chromosome dosage, it is the number for described interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is a second karyomit(e) dosage, it is the number for the chromosomal sequence label of the first normalization method and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as the normalized karyomit(e) value (NCV) of described calculating.Fetal chromosomal aneuploidy can be a kind of part or complete chromosomal aneuploidy.In these embodiments, fetal chromosomal aneuploidy can be selected from T21, T18, T13 and X monosomy.In some embodiments, maternal sample obtains from a pregnant woman.In some embodiments, maternal sample is a kind of biological fluid sample, such as a blood sample or the blood plasma fractions that obtains from blood sample.In some embodiments, maternal sample is a plasma sample.In some embodiments, the nucleic acid in maternal sample is cfDNA molecule.In some other embodiments, parent test sample is the plasma sample obtained from a pregnant woman, and nucleic acid molecule is cfDNA molecule.
In another embodiment, the method by determining the fetal chromosomal aneuploidy that presence or absence at least two kinds is different at least two interested karyomit(e) repeating step (a)-(c) in the parent test sample comprising fetus and maternal nucleic acids molecule, wherein comprise (a) for step (a)-(c) of each in these at least two interested karyomit(e) and obtain sequence information for fetus and maternal nucleic acids in the sample to which, to identify for a number of the sequence label of an interested chromosomal multiple mapping and a number at least two chromosomal multiple sequence labels of normalization method, (b) use for interested chromosomal label number and determine for interested chromosomal first normalized value for the number of a chromosomal label of the first normalization method, and use for the chromosomal sequence label of the first normalization method number and determine for chromosomal second normalized value of the first normalization method for the number of a chromosomal sequence label of the second normalization method, and (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for chromosomal second normalized value of the first normalization method and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in the sample to which.First and second threshold values can be identical, or they can be different.In the step (c) of this method, for each in these at least two interested karyomit(e)s, show that presence or absence is for described interested chromosomal a kind of dysploidy for described interested chromosomal first normalized value with comparing of threshold value, and for the comparatively validate presence or absence of chromosomal second normalized value of described first normalization method and threshold value for the determination of interested chromosomal a kind of dysploidy.In some embodiments, first normalized value is a first chromosome dosage, it is the number for described interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is a second karyomit(e) dosage, it is the number for the chromosomal sequence label of the first normalization method and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as normalized karyomit(e) value (NCV) as described herein.In some embodiments, the method comprises and repeats the method to determine the fetal chromosomal aneuploidy that presence or absence at least two kinds is different for all karyomit(e).
In another embodiment, the method by determining the fetal chromosomal aneuploidy that presence or absence at least two kinds is different at least two interested karyomit(e) repeating step (a)-(c) in the parent test sample comprising fetus and maternal nucleic acids molecule, wherein comprise (a) for step (a)-(c) of each in these at least two interested karyomit(e) and obtain sequence information for fetus and maternal nucleic acids in the sample to which, to identify for a number of the sequence label of an interested chromosomal multiple mapping and a number at least two chromosomal multiple sequence labels of normalization method, (b) use for interested chromosomal label number and determine for interested chromosomal first normalized value for the number of a chromosomal label of the first normalization method, and use for the chromosomal sequence label of the first normalization method number and determine for chromosomal second normalized value of the first normalization method for the number of a chromosomal sequence label of the second normalization method, and (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for chromosomal second normalized value of the first normalization method and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in the sample to which.First and second threshold values can be identical, or they can be different.In the step (c) of this method, for each in these at least two interested karyomit(e)s, show that presence or absence is for described interested chromosomal a kind of dysploidy for described interested chromosomal first normalized value with comparing of threshold value, and for the comparatively validate presence or absence of chromosomal second normalized value of described first normalization method and threshold value for the determination of interested chromosomal a kind of dysploidy.In some embodiments, first normalized value is a first chromosome dosage, it is the number for described interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is a second karyomit(e) dosage, it is the number for the chromosomal sequence label of the first normalization method and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as normalized karyomit(e) value (NCV) as described herein.In some embodiments, the method comprises and repeats the method to determine the fetal chromosomal aneuploidy that presence or absence at least two kinds is different for all karyomit(e).At least two kinds of different fetal chromosomal aneuploidies can be selected from T21, T18, T13 and X monosomy.In some embodiments, maternal sample obtains from a pregnant woman.In some embodiments, maternal sample is a kind of biological fluid sample, such as a blood sample or the blood plasma fractions that obtains from blood sample.In some embodiments, maternal sample is a plasma sample.In some embodiments, the nucleic acid in maternal sample is cfDNA molecule.In some other embodiments, parent test sample is the plasma sample obtained from a pregnant woman, and nucleic acid molecule is cfDNA molecule.
In another embodiment, the method determines that presence or absence is selected from trisomy 21 by following steps in the Maternal plasma test sample comprising fetus and maternal nucleic acids molecule (such as cfDNA), 18 trisomys, 13 trisomys, and a kind of fetal chromosomal aneuploidy of X monosomy: (a) obtains the sequence information for fetus in maternal sample and maternal nucleic acids, to identify for a number of an interested chromosomal multiple sequence label and a number at least two chromosomal multiple sequence labels of normalization method, wherein obtain sequence information comprise use multiple reversible dye-terminators carry out extensive parallel synthesis order-checking, b () uses the number of sequence label to calculate for interested chromosomal first normalized value and second normalized value, and (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for interested chromosomal second normalized value and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in the sample to which.In some embodiments, first normalized value is a first chromosome dosage, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is a second karyomit(e) dosage, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as normalized karyomit(e) value (NCV) as described herein.In some embodiments, the method by determining that presence or absence is selected from the different chromosomal aneuploidy of trisomy 21,18 trisomys, 13 trisomys and X monosomy at least two kinds at least two interested karyomit(e) repeating step (a)-(c) in the Maternal plasma test sample comprising fetus and maternal nucleic acids molecule (such as cfDNA).The method may further include for all karyomit(e) repeating step (a)-(c) to determine presence or absence at least two kinds of fetal chromosomal aneuploidies.In some embodiments, maternal sample obtains from a pregnant woman.In some embodiments, maternal sample is a kind of biological fluid sample, such as a blood sample or the blood plasma fractions that obtains from blood sample.In some embodiments, maternal sample is a plasma sample.In some embodiments, the nucleic acid in maternal sample is cfDNA molecule.In some other embodiments, parent test sample is the plasma sample obtained from a pregnant woman, and nucleic acid molecule is cfDNA molecule.
In another embodiment, the method determines that presence or absence is selected from trisomy 21 by following steps in the Maternal plasma test sample comprising fetus and maternal nucleic acids molecule (such as cfDNA), 18 trisomys, 13 trisomys, and a kind of fetal chromosomal aneuploidy of X monosomy: (a) obtains the sequence information for fetus and maternal nucleic acids in the sample to which, to identify for a number of the sequence label of an interested chromosomal multiple mapping and a number at least two chromosomal multiple sequence labels of normalization method, wherein obtain sequence information comprise use multiple reversible dye-terminators carry out extensive parallel synthesis order-checking, (b) use for interested chromosomal label number and determine for interested chromosomal first normalized value for the number of a chromosomal label of the first normalization method, and use for the chromosomal sequence label of the first normalization method number and determine for chromosomal second normalized value of the first normalization method for the number of a chromosomal sequence label of the second normalization method, and (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for chromosomal second normalized value of the first normalization method and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in the sample to which.In some embodiments, first normalized value is a first chromosome dosage, it is the number for described interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is a second karyomit(e) dosage, it is the number for the chromosomal sequence label of the first normalization method and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as normalized karyomit(e) value (NCV) as described herein.In some embodiments, the method by determining that presence or absence is selected from the different chromosomal aneuploidy of trisomy 21,18 trisomys, 13 trisomys and X monosomy at least two kinds at least two interested karyomit(e) repeating step (a)-(c) in the Maternal plasma test sample comprising fetus and maternal nucleic acids molecule (such as cfDNA).The method may further include for all karyomit(e) repeating step (a)-(c) to determine presence or absence at least two kinds of fetal chromosomal aneuploidies.In some embodiments, maternal sample obtains from a pregnant woman.In some embodiments, maternal sample is a kind of biological fluid sample, such as a blood sample or the blood plasma fractions that obtains from blood sample.In some embodiments, maternal sample is a plasma sample.In some embodiments, the nucleic acid in maternal sample is cfDNA molecule.In some other embodiments, parent test sample is the plasma sample obtained from a pregnant woman, and nucleic acid molecule is cfDNA molecule.
In some above-mentioned embodiments and some subsequent implementation schemes, obtain to comprise for the sequence information of fetus and maternal nucleic acids in the sample to which and fetus in the sample to which and maternal nucleic acids molecule are checked order.
Combine by reference
All patents mentioned herein, patent application and other publications (comprising all sequences disclosed in these reference) all combine clearly by reference, and its combination degree just as each independent publication, patent or patent application definitely and be indicated as being individually and combine by reference.But, should not be understood to admit that it is about prior art of the present invention to the citation of any document.
Brief Description Of Drawings
Novel feature of the present invention in the dependent claims in addition singularity set forth.By reference to the better understanding that the detailed description of the invention and its accompanying drawing that set forth below the illustrative embodiments utilizing the principle of the invention will obtain feature and advantage of the present invention.
Fig. 1 provides a schema, shows and determines and verify two alternate embodiment of the method for presence or absence dysploidy.
Fig. 2 shows the normalized karyomit(e) value (example 1) for karyomit(e) 21 (zero), 18 (△) and 13 () determined in the sample from training set 1.
Fig. 3 shows the normalized karyomit(e) value (example 1) for karyomit(e) 21 (zero), 18 (△) and 13 () determined in the sample from test set 1.
Fig. 4 shows the normalized karyomit(e) value (example 1) for karyomit(e) 21 (zero) and 18 (△) using the method for normalizing of the people such as Zhao (Chiu) to determine in the sample from test set 1.
Fig. 5 shows the figure of the normalized karyomit(e) value for karyomit(e) 9 dosage using karyomit(e) 11 to determine in 48 samples of test set 1 (example 1) as normalization method karyomit(e).
Fig. 6 shows the figure of the normalized karyomit(e) value for karyomit(e) 8 dosage using karyomit(e) 2 to determine in 48 samples of test set 1 (example 1) as normalization method karyomit(e).
Fig. 7 shows the figure of the normalized karyomit(e) value for karyomit(e) 6 dosage using karyomit(e) 5 to determine in 48 samples of test set 1 (example 1) as normalization method karyomit(e).
Fig. 8 shows the figure of the normalized karyomit(e) value for karyomit(e) 21 dosage using karyomit(e) 9 (A), karyomit(e) 10 (B) and karyomit(e) 14 (C) to determine in 48 samples of test set 1 accordingly, this test set 1 comprises the sample of uninfluenced (zero) and influenced (△) (that is, trisomy 21).
Fig. 9 shows the figure of the normalized karyomit(e) value for karyomit(e) 8 dosage using karyomit(e) 2 to determine in test set 2 (example 4) as normalization method karyomit(e) (B) as normalization method karyomit(e) (A) and use karyomit(e) 7.
Detailed description of the invention
The invention provides and a kind ofly can determine single or multiple fetal chromosomal aneuploidy in the maternal sample comprising fetus and maternal nucleic acids and the method for correct determination has been made in checking.The method is applicable to copy number variation (CNV) determining any interested sequence in multiple sample, these samples comprise the mixture of the genomic nucleic acids deriving from two different genes groups, and known or suspect that these two different genes groups are different in the amount of one or more interested sequence.The method is at least applicable to the enforcement of Non-invasive Prenatal Diagnosis, and is applicable to the diagnosis and the monitoring that contrast the symptom that sequence differential expression is relevant in ill individuality with healthy individuals.
Except as otherwise noted, otherwise enforcement of the present invention relates to routine techniques conventional in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing and recombinant DNA field, and these technology are all in the technology category of this area.These technology are known to those of skill in the art, and be described in numerous textbook and reference (see people such as such as Pehanorm Brookers (Sambrook), " Molecular Cloning: A Laboratory guide (Molecular Cloning:A Laboratory Manual) ", the second edition (cold spring port (Cold Spring Harbor)), [1989]); With the people such as Ao Subaier (Ausubel), " up-to-date experimental methods of molecular biology compilation (Current Protocols in Molecular Biology) " [1987])
Numerical range comprises the numerical value limiting this scope.Run through each greatest measure limit that this specification sheets provides comprise each lower numerical limitation being intended that of this, clearly write out at this as this type of lower numerical limitation.Run through each minimum value limit that this specification sheets provides and will comprise each higher numerical limitation, clearly write out at this as this type of high value limit.Run through each numerical range that this specification sheets provides and will comprise each the narrower numerical range dropped in this type of wider numerical range, all write out clearly as this type of narrower numerical range herein.
Title provided herein is not the restriction to different aspect of the present invention or embodiment, and it can have by reference to as an overall specification sheets.Therefore, as above, the term directly defined hereinafter defines more fully by reference to as an overall specification sheets.
Unless defined separately at this, the identical meanings usually understood of the those of ordinary skill all had in field belonging to the present invention with the term of science of all technology as used herein.The different science dictionaries including the term comprised at this are know and be obtainable for those skilled in the art.Although similar or be equivalent to any method of those methods described herein and material and material have found purposes in enforcement or test the present invention, only illustrate some preferred method and materials.Therefore, namely the term directly defined hereinafter is illustrated more completely by being consulted as a whole by this specification sheets.Should be understood that the present invention is not limited to illustrated concrete grammar, code and reagent, because these can change, they to be got off use according to its situation by those skilled in the art.
Definition
As used in this, the term " " of odd number, " one " and " being somebody's turn to do " comprise plural reference, unless context clearly indicates in addition.Except as otherwise noted, nucleic acid from left to right to write by 5 ' to 3 ' direction and aminoacid sequence from left to right writes to carboxyl direction by amino.
Term " acquisition sequence information " in this article refers to and checks order to obtain the sequence information in sequence reads form to nucleic acid, and these sequence reads are identified as sequence label when being uniquely mapped to reference gene group.
Term " normalized value " in this article refer to determine for interested karyomit(e) and make the numerical value that the number for interested chromosomal sequence label is associated with the number for the chromosomal sequence label of normalization method.For example, " normalized value " can be a karyomit(e) dosage as other places description herein, or it can be the NCV (normalized karyomit(e) value) as other places description herein.
Term " interested karyomit(e) " in this article refers to a kind of karyomit(e) carrying out a kind of dysploidy of presence or absence and determine.Interested chromosomal example comprises karyomit(e) involved in common dysploidy (as trisomy 21), and karyomit(e) involved in rare dysploidy (as 2 trisomys).Any one in karyomit(e) 1-22, X and Y can be interested karyomit(e).
Term " multiple (multiple) and a plurality of (plurality) ", when using about chromosomal aneuploidy number and/or chromosome number, in this article refers to two or more dysploidy and/or karyomit(e).
Term " threshold value " in this article refers to and uses any numerical value that is that training dataset calculates and (such as dysploidy) diagnosis threshold that makes a variation as copy number in organism.If the result obtained from enforcement the present invention exceedes threshold value, so experimenter can be diagnosed as copy number variation (such as trisomy 21).Appropriate threshold value for method described herein can identify by analyzing the normalized value calculated for the training sample sets comprising qualified samples (that is, unaffected sample) (such as karyomit(e) dosage or NCV (normalized karyomit(e) value)).Threshold value can use qualified samples and be identified as having the sample of chromosomal aneuploidy (that is, affected sample) carries out setting (example see herein).In some embodiments, for identify the training set of appropriate threshold value comprise at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, or more qualified samples.The diagnostic tool using larger qualified samples collection to carry out improvement threshold may be favourable.
Term " order-checking (NGS) of future generation " in this article refers to the sequence measurement allowing the nucleic acid molecule increased with clonal fashion and single core acid molecule to be carried out to extensive parallel order-checking.The limiting examples of NGS comprises the synthesis method order-checking and connection method order-checking that use multiple reversible dye-terminators to carry out.
Term " reading " refers to a DNA sequence dna with sufficient length (such as at least about 30bp), and it may be used for identifying larger sequence or region, and such as it can be compared and specifically belong to karyomit(e) or genome area or gene.
Term " sequence label " in this article can with term " sequence label of mapping " exchange use with mention by than and belong to (that is, mapping) definitely and arrive sequence reads of larger sequence (for example, referring to genome).The sequence label mapped is uniquely mapped to a reference gene group, that is, they are attributed to the single position of reference gene group.The label (that is, the label do not mapped uniquely) of more than one position that can be mapped in reference gene group is not comprised in analysis.
Term " number of sequence label " is when using about the number for an interested karyomit(e) and/or the chromosomal label of one or more normalization method, in this article refer to and be mapped to this interested karyomit(e) and/or this or these chromosomal sequence label of normalization method, these sequence labels are the subsets of the multiple labels obtained for karyomit(e) all in sample.The number of tags obtained for a sample can be at least about 1 × 10 6individual sequence label, at least about 2 × 10 6individual sequence label, at least about 3 × 10 6individual sequence label, at least about 5 × 10 6individual sequence label, at least about 8 × 10 6individual sequence label, at least about 10 × 10 6individual sequence label, at least about 15 × 10 6individual sequence label, at least about 20 × 10 6individual sequence label, at least about 30 × 10 6individual sequence label, at least about 40 × 10 6individual sequence label, or at least about 50 × 10 6individual sequence label or at least about 60 × 10 6individual sequence label, or at least about 70 × 10 6individual sequence label or at least about 80 × 10 6individual sequence label, is included in the reading of (such as 36bp) between 20bp and 40bp, and each sample obtains by reading is mapped to reference gene group.The number being mapped to any one chromosomal label will depend on karyomit(e) size and chromosomal copy number.For example, the number being mapped to the label of the karyomit(e) 21 in trisomy 21 sample will be different from the number that (that is, being greater than) is mapped to the label of the karyomit(e) 21 in unaffected sample.Similarly, the number being mapped to the label of karyomit(e) 19 will be less than the number of the label being mapped to karyomit(e) 1 (it is about 4 times of karyomit(e) 19 size).The number being mapped to the label of interested sequence (such as karyomit(e)) is also referred to as " sequence label density ".
Term " sequence label density " in this article refers to the number of the sequence reads being mapped to reference gene group sequence, such as, are numbers of the sequence reads of the karyomit(e) 21 being mapped to reference gene group produced by sequence measurement for the sequence label density of karyomit(e) 21.Sequence label density can be determined for whole karyomit(e) or for chromosomal part.
As used herein, term " is compared ", " comparison " or " comparing " refer to and be identified as one or more sequences of mating with the known array from reference gene group with regard to their nucleic acid molecule order aspect.This kind of comparison can manually be carried out or be undertaken by computerized algorithm, and example comprises efficient few nucleotide that the part as hundred million sensible genomics analysis stream waterlines (Illumina Genomics Analysis pipeline) allots according to Local Alignment (Efficient Local Alignment of Nucleotide Data; ELAND) computer program.The coupling of the sequence reads in comparison can be 100% sequences match or be less than 100% (imperfections coupling).
As used herein, term " reference gene group " refers to any specific known group sequence (no matter be part or complete) of any organism or virus, and it may be used for doing reference to the sequence identified from experimenter.For example, the reference gene group for human experimenter and many other biological bodies sees American National Biotechnology Information center (National Center for BiotechnologyInformation) www.ncbi.nlm.nih.gov.
" genome " refers to the entire genetic information of organism or the virus represented with nucleotide sequence form.
Term " maternal sample " in this article refers to the biological sample obtained from the experimenter (such as women) of pregnancy.
Term " biological fluid " in this article refers to the liquid obtained from biogenetic derivation, and comprises such as blood, serum, blood plasma, phlegm, irrigating solution, cerebrospinal fluid, urine, seminal fluid, sweat, tears, saliva etc.As used herein, term " blood ", " blood plasma " and " serum " contain their part or the part through processing clearly.Similarly, when sample is when taking from examination of living tissue, swab, smear etc., " sample " contains the fragment through processing or part that derive from examination of living tissue, swab, smear etc. clearly.
Term " maternal nucleic acids " and " fetal nucleic acid " refer to the nucleic acid of the nucleic acid of pregnant female experimenter and the fetus entrained by pregnant female in this article accordingly.
Term " experimenter " in this article refers to human experimenter and nonhuman subjects, as Mammals, invertebrates, vertebrates, fungi, yeast, bacterium and virus.Although example herein relate to the mankind and words mainly for the relevant mankind, concept of the present invention is applicable to the genome from any plant or animal, and is applicable to the fields such as veterinary science, animal science and research laboratory.
The number that term " normalization method sequence " in this article refers to the sequence label that display is mapped on it between multiple sample and multiple order-checking batch has the sequence of variability, the variability of the number of this sequence label closest to it be used as normalized parameter for the variability of number of sequence label of interested sequence, and can best affected sample and one or more unaffected sample to be differentiated." normalization method karyomit(e) " is an example of " normalization method sequence ".
Term " sequence dosage " in this article refers to the parameter that the sequence label density of interested sequence is associated with the label densities of normalization method sequence." karyomit(e) dosage " is the number of the sequence label being mapped to karyomit(e) (such as interested karyomit(e)) and the ratio of number being mapped to the chromosomal sequence label of normalization method, and it is an example of sequence dosage." cycle tests dosage " is the parameter that the sequence label density of the interested sequence (such as karyomit(e) 21) making to determine in the test sample is associated with the sequence label density of normalization method sequence (such as karyomit(e) 9).Similarly, " qualified sequence dosage " is the parameter that the sequence label density of the interested sequence making to determine in qualified samples is associated with the sequence label density of normalization method sequence.
The number that term " karyomit(e) dosage " in this article refers to the sequence label being mapped to karyomit(e) (such as interested karyomit(e)) and the ratio of number being mapped to the chromosomal sequence label of normalization method.
Term " normalization method karyomit(e) " in this article refers to the karyomit(e) that number that display between multiple sample and multiple order-checking batch is mapped to its sequence label has variability, the variability of the number of this sequence label closest to it be used to obtain normalized value for the variability of number of interested chromosomal sequence label, and can best affected sample and one or more unaffected sample to be differentiated.
Term " interested sequence " in this article refers to the nucleotide sequence relevant with the sequence differential expression contrasted in healthy individuals in ill individuality.Interested sequence can be the sequence on the karyomit(e) of false demonstration in disease or hereditary conditions (that is, overexpression or expression deficiency).Interested sequence can also be a chromosomal part or karyomit(e) (that is, interested karyomit(e)).For example, interested sequence can be the karyomit(e) (such as karyomit(e) 13,18,21 and X) of overexpression in dysploidy symptom or the gene of the tumor-inhibiting factor expressing deficiency of encoding in cancer.Interested sequence to be included in total group of subject cell or subgroup overexpression or to express not enough sequence." interested qualified sequence " is the interested sequence in qualified samples." interested cycle tests " is the interested sequence in test sample.
Term " qualified samples " in this article refers to a sample of the mixture comprising multiple nucleic acids that compare with the nucleic acid in test sample, that exist with known copy number, and for interested sequence, normally (namely it be, aneuploid) sample, such as, the chromosomal qualified samples of normalization method for identifying for karyomit(e) 21 is the sample of a non-trisomy 21 sample.
Term " training set " and " training sample " are used in reference to the sample comprising nucleic acid that compare with the nucleic acid in test sample, that exist with known copy number in this article.Unless otherwise indicated, otherwise training set comprises qualified and affected sample.
Term " test sample " in this article refers to and comprises nucleic acid mixture and these nucleic acid comprise the sample of copy number at least one nucleotide sequence morphed under a cloud.The nucleic acid be present in test sample is called as " test nucleic acid ".
Term " dysploidy " refers to by losing or obtain whole karyomit(e) or a chromosomal part and the imbalance of the genetic material caused at this.
Term " karyomit(e) dysploidy " refers to the imbalance of the genetic material caused by losing or obtain whole karyomit(e) at this, and comprises germline dysploidy and mosaic dysploidy.
Term " part dysploidy " and " chromosome dyad dysploidy " refer to by losing or obtain a chromosomal part (such as at this, partial monoploidy and partial trisomy) and the imbalance of the genetic material caused, and contain the imbalance caused by transposition, deletion and insertion.
Term " nucleic acid molecule ", " polynucleotide " and " nucleic acid " are used interchangeably, and refer to a covalently bound nucleotide sequence (namely, the ribonucleotide of RNA and the deoxyribonucleotide of DNA), 3 ' position of the pentose of one of them Nucleotide is connected on 5 ' position of the pentose of next Nucleotide by a phosphodiester group, this comprises the sequence of any type of nucleic acid, including, but not limited to RNA, DNA and cfDNA molecule.Term " polynucleotide " comprise and be not limited to strand with the polynucleotide of double-strand.
Term " copy number variation (CNV) " in this article refers to and is present in nucleotide sequence copy number in test sample and the variation being present in the nucleotide sequence copy number in qualified samples (that is, normal sample) and comparing.Copy number variation comprises disappearance (comprising micro-deleted), inserts (comprising micro-insertion), copies, doubles, inversion, transposition and complicated multi-position make a variation.CNV covers the dysploidy of complete chromosomal aneuploidy and part.
Describe
The invention provides and a kind ofly can determine single or multiple fetal chromosomal aneuploidy in the maternal sample comprising fetus and maternal nucleic acids and the method for correct determination has been made in checking.The method is applicable to copy number variation (CNV) determining any interested sequence in multiple sample, these samples comprise the mixture of the genomic nucleic acids deriving from least two different genes groups, and known or suspect that these two different genes groups are different in the amount of one or more interested sequence.Interested sequence is included in hundreds of bases to dozens of megabase to the genome sequence within the scope of whole karyomit(e), and these genome sequences are known or be suspect to be relevant with heredity or disease condition.The chromosome segment (part 8 trisomy in such as acute myelocytic leukemia) that the example of interested sequence comprises the karyomit(e) relevant with the dysploidy known (such as trisomy 21) and doubles in disease (as cancer).
The inventive method is included in one or more parent test sample and obtains order-checking information, to calculate the karyomit(e) dosage for interested sequence (such as karyomit(e)), thus determine the single or multiple chromosomal aneuploidy of presence or absence, and comprise the determination that correct dysploidy is made in checking.Correctly determine that accuracy in the sample to which needed for presence or absence CNV (such as dysploidy) is based on the following: the variation (with batch order-checking variation) being mapped to the number of the sequence label of reference gene group between the multiple samples in order-checking batch, and in different order-checking batch, being mapped to the variation (between round order-checking variation) of the number of the sequence label of reference gene group, the impact of the distribution of the sequence label that these variations can make fetal chromosomal aneuploidy penetrate is not obvious.For example, for being mapped to the label of GC enrichment or the poor canonical sequence of GC, variation may be especially remarkable.In order to correct this kind of variation, the inventive method uses karyomit(e) dosage inherently to explain the order-checking variability of appearance based on the knowledge of normalization method karyomit(e) (or normalization method karyomit(e) group).
Normalization method karyomit(e) and karyomit(e) dosage
Use from the sequence information of one group of qualified samples obtained from experimenter to identify normalization method karyomit(e), these sample known packets are containing the cell had for the normal copy number of any one interested sequence (such as the diploid of karyomit(e) 21).The sequence information obtained from qualified samples is also for determining the identification (see example) having statistical significance of chromosomal aneuploidy in the test sample.In one embodiment, qualified samples obtains from the mother nourishing fetus, and this fetus has normal chromosomal copy number (such as the diploid of karyomit(e) 21) to have used cytogenetics means to confirm.Biology qualified samples can be a kind of biological fluid (such as blood plasma) or any applicable sample as mentioned below.In some embodiments, qualified samples comprises the mixture of nucleic acid molecule (such as cfDNA molecule).In some embodiments, qualified samples is the Maternal plasma sample of the mixture comprising fetus and parent cfDNA molecule.
By using any known sequence measurement, checking order at least partially of nucleic acid (such as fetus and maternal nucleic acids) is obtained for the chromosomal sequence information of normalization method.Preferably, the next generation of any one herein described in other places order-checking (NGS) method is used to check order in unit molecule or with the fetus of the molecular form of clonal fashion amplification and maternal nucleic acids.Millions of the sequence reads with predetermined length (such as 36bp) are produced by NGS technology, and are mapped to reference gene group to treat in counting as sequence label.The nucleic acid at least partially of each qualified samples is checked order, and the number being mapped to each chromosomal sequence label is counted.In some embodiments, the number being mapped to chromosomal sequence label can normalize to these interested qualified sequences, map them to length above.The sequence label density determined relative to the ratio of interested sequence length as label densities is referred to as label densities ratio in this article.Normalize to interested sequence length optional, but can be included as the step of the digital numbers be used in minimizing numerical value, understand for the mankind to simplify numerical value.When all qualified sequence label in each qualified samples is all mapped and when counting, being determined for the qualified sequence label density of interested sequence (such as clinically relevant sequence) in qualified samples, also being determined for being used for the sequence label density of the other sequence therefrom identifying normalization method sequence subsequently.
Based on calculated qualified label density, the qualified sequence dosage (such as karyomit(e) dosage) for interested sequence (such as karyomit(e) 21) is determined as the sequence label density for interested sequence and the ratio for the qualified sequence label density of the other sequence being used for subsequently therefrom identifying normalization method sequence separately.For example, karyomit(e) dosage for interested karyomit(e) (such as karyomit(e) 21) is determined with the ratio for the respective sequence label density of all the other karyomit(e)s all (that is, karyomit(e) 1-20, chromosome 22, chromosome x and karyomit(e) Y) as the sequence label density for karyomit(e) 21.Qualified sequence dosage can be determined for all karyomit(e).
Subsequently, in qualified samples, at least two normalization method sequences for interested sequence (such as karyomit(e) 21) are identified based on calculated sequence dosage.For example, the qualified normalization method sequence for karyomit(e) 21 identifies as the sequence of the sequence label Density Variation had in qualified samples closest to the sequence label Density Variation of karyomit(e) 21.For example, qualified normalization method sequence is the sequence with minimum variability.In some embodiments, plural normalization method sequence is identified.For example, the normalization method karyomit(e) with minimum variability for each in all karyomit(e) 1-22, chromosome x and karyomit(e) Y is determined.Table 9 in example 5 provides four normalization method karyomit(e)s, and these normalization method karyomit(e)s are confirmed as having four minimum variability for each in karyomit(e) 1-22, chromosome x and karyomit(e) Y.As shown in example, variability numerically can be expressed as the variation coefficient (CV%).Normalization method sequence can also be distinguish the sequence of one or more qualified samples and one or more affected sample best, that is, normalization method sequence is the sequence with maximum differentiability.Differentiability degree can be determined as the significant difference between the karyomit(e) dosage in the karyomit(e) dosage in qualified samples colony and one or more test sample.For example, differentiability numerically can be expressed as T test value, the karyomit(e) dosage in its expression qualified samples colony and the significant difference between the karyomit(e) dosage in one or more test sample.Alternately, differentiability numerically can be expressed as normalized karyomit(e) value (NCV), and it is the z score value for karyomit(e) dosage when NCV is normal distribution.Determining in z score value, average and the standard deviation of the karyomit(e) dosage in one group of qualified samples can used.Alternately, average and the standard deviation of the karyomit(e) dosage in the training set comprising qualified samples and affected sample can be used.In other embodiments, normalization method sequence is the sequence with minimum variability and maximum differentiability.
The method identifies congenitally to be had similar feature and tends to, between sample and order-checking batch, the sequence of similar variation occurs, and these sequences are applicable to determine to test the sequence dosage in sample.
Based on the identification to this in qualified samples or these normalization method sequences, the sequence information obtained for the nucleic acid in test sample is used to determine to test the one or more sequence dosage (such as karyomit(e) dosage) for interested sequence (such as karyomit(e) 21) in sample.In some embodiments, at least two the sequence dosage (such as karyomit(e) dosage) for interested sequence are determined.For example, use karyomit(e) 9 to determine a first chromosome dosage for karyomit(e) 21 as a first normalization method karyomit(e), and use karyomit(e) 11 to determine a second karyomit(e) dosage for karyomit(e) 21 as the second normalization method karyomit(e).Cycle tests dosage can be expressed as described NCV further.In some embodiments, can carry out by following steps the classification testing sample: directly will compare for interested chromosomal first cycle tests dosage and a first threshold and the second cycle tests dosage and a Second Threshold be compared to determine a kind of chromosomal aneuploidy of presence or absence in the test sample.For the comparatively validate determination of sample classification of interested chromosomal two karyomit(e) dosage.Select threshold value to classify sample as " normally ", " affected " or " " do not judge (no call) " sample according to user-defined reliability thresholds.In other embodiments, use a first normalization method karyomit(e) to determine for an interested chromosomal the first chromosome dosage, and use a second normalization method karyomit(e) to determine for the chromosomal second karyomit(e) dosage of the first normalization method.The classification testing sample can be carried out: the first chromosome dosage and a first threshold are compared and the second karyomit(e) dosage and a Second Threshold are compared to determine a kind of chromosomal aneuploidy of presence or absence in the test sample by following steps.For interested chromosomal karyomit(e) dosage and a first threshold compare determine in the test sample presence or absence for interested chromosomal dysploidy, and for the comparatively validate determination of sample classification of the chromosomal second karyomit(e) dosage of normalization method and a Second Threshold.Test chromosome dosage can be expressed as described NCV further, and wherein the first and second karyomit(e) dosage are expressed as the first and second NCV; And the classification testing sample is undertaken by a NCV and first threshold being compared and being compared by a 2nd NCV and Second Threshold.
Although example herein relates to complete chromosomal aneuploidy, concept of the present invention is applicable to the dysploidy of part.In one embodiment, interested sequence is the chromosome segment relevant with the dysploidy (such as chromosome deletion or insertion or unbalanced chromosome translocation) of part, and at least two normalization method sequences are chromosome segments irrelevant with the dysploidy of part, and the sequence label Density Variation of these two normalization method sequences is closest to the sequence label Density Variation of the chromosome segment relevant with the dysploidy of part.The dysploidy of part can use karyomit(e) dosage to determine (see the U.S. Patent application 12/958 that the International Application Serial No. PCT/US2010/058609 and 2010 submitted on December 1st, 2010 submits to 1, on December, 352, the title of these applications is all " for determining the method (Method for Determining Copy Number Variations) that copy number makes a variation " and is combined in this in full with it by reference).The dysploidy of at least two a kind of parts of normalization method sequence verification presence or absence can be used according to the inventive method.
Fig. 1 provides the schema of two exemplary of method 100, and the method is determined and verifies a kind of chromosomal aneuploidy of presence or absence comprising in the sample of two genomic mixtures (such as maternal sample).
In first embodiment, the method determines presence or absence fetal chromosomal aneuploidy by following steps in the parent test sample comprising fetus and maternal nucleic acids: (a) obtains the sequence information for fetus in maternal sample and maternal nucleic acids, to identify for the number of an interested chromosomal sequence label and the number at least two chromosomal sequence labels of normalization method; B () uses the number of sequence label to calculate for interested chromosomal first normalized value and second normalized value; And (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for interested chromosomal second normalized value and a Second Threshold, to determine presence or absence fetus dysploidy in the sample to which.First and second threshold values can be identical, or they can be different.In the step (c) of this method, show that presence or absence is for described interested chromosomal a kind of dysploidy for described interested chromosomal first normalized value with comparing of threshold value, and for the comparatively validate presence or absence of described interested chromosomal second normalized value and threshold value for the determination of interested chromosomal a kind of dysploidy.In some embodiments, the first normalized value is a first chromosome dosage, and it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method; And the second normalized value is a second karyomit(e) dosage, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as described normalized karyomit(e) value (NCV).
Step 110,120,130 and 140 according to method as shown in Figure 1 describes the first embodiment.The number (110) providing sequence label is checked order to the fetus obtained from maternal sample and maternal nucleic acids.The sequence label being mapped to an interested karyomit(e) (such as karyomit(e) 21) and the sequence label that is mapped to two normalization method karyomit(e)s (such as karyomit(e) 9 and karyomit(e) 11) to be counted and for calculating for interested chromosomal corresponding first and second normalized values (such as karyomit(e) dosage).In one embodiment, at least two karyomit(e) dosage are the normalized values determined for each interested karyomit(e).In one embodiment, be a first chromosome dosage for interested chromosomal first normalized value, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method; And be a second karyomit(e) dosage for interested chromosomal second normalized value, it is the number for interested chromosomal sequence label and the ratio (120) for the number of a chromosomal sequence label of the second normalization method.Will for interested chromosomal first normalized value (namely, the first chromosome dosage) compared with a first threshold, and for interested chromosomal second normalized value (i.e. the second karyomit(e) dosage) compared with a Second Threshold (130), and determination and the checking (140) of a kind of chromosomal aneuploidy of presence or absence will be carried out.Alternately, at least two karyomit(e) dosage are expressed as the first and second normalized karyomit(e) values (NCV), the average of the first chromosome dosage that the one NCV makes the first chromosome dosage corresponding in one group of qualified samples is associated, and the 2nd NCV makes the average of the second karyomit(e) dosage karyomit(e) dosage corresponding in same group of qualified samples be associated, as:
NCV ij = x ij - μ ^ j σ ^ j
wherein with the estimation average for the karyomit(e) dosage of jth in one group of qualified samples and standard deviation accordingly, and x ijfor the viewed jth of a test sample i karyomit(e) dosage.First and second normalized values (namely, NCV) compare (130) with a Second Threshold with a first threshold accordingly separately, and carry out determination and the checking (140) of a kind of chromosomal aneuploidy of presence or absence.The method can identify extremely rare (such as 9 trisomys) and more common chromosomal aneuploidy (such as trisomy 21), and can identify the multiple chromosomal aneuploidies from order-checking information, this order-checking information obtains from the single order-checking test sample nucleic (such as cfDNA) batch.As shown in example, although determine that the sequence information of presence or absence trisomy 21 discloses and there is not trisomy 21 for being used for of obtaining of sample, this sample comprises 9 trisomys.In some embodiments, chromosomal aneuploidy is identified in any one in karyomit(e) 1-22, chromosome x and karyomit(e) Y.Chromosomal aneuploidy can be identified in interested karyomit(e) and/or the first or second normalization method karyomit(e).In some embodiments, the method identifies the multiple chromosomal aneuploidies being selected from trisomy 21,13 trisomys, 18 trisomys and X monosomy.
In second embodiment, the method verifies the determination of presence or absence for interested chromosomal a kind of dysploidy by following steps in the parent test sample comprising fetus and maternal nucleic acids molecule: (a) obtains the sequence information for fetus and maternal nucleic acids in the sample to which, to identify for the number of the sequence label of an interested chromosomal mapping and the number at least two chromosomal sequence labels of normalization method; (b) use for interested chromosomal label number and determine for interested chromosomal first normalized value for the number of a chromosomal label of the first normalization method, and use for the chromosomal sequence label of the first normalization method number and determine for chromosomal second normalized value of the first normalization method for the number of a chromosomal sequence label of the second normalization method; And (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for chromosomal second normalized value of the first normalization method and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in the sample to which.First and second threshold values can be identical, or they can be different.In the step (c) of this method, show that presence or absence is for described interested chromosomal a kind of dysploidy for described interested chromosomal first normalized value with comparing of threshold value, and for the comparatively validate presence or absence of chromosomal second normalized value of described first normalization method and threshold value for the determination of interested chromosomal a kind of dysploidy.In some embodiments, the first normalized value is a first chromosome dosage, and it is the number for described interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method; And the second normalized value is a second karyomit(e) dosage, it is the number for the chromosomal sequence label of the first normalization method and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as according to normalized karyomit(e) value (NCV) following calculating, as above
NCV ij = x ij - μ ^ j σ ^ j
Step 110,150,160 and 140 according to method as shown in Figure 1 describes the second embodiment.The number (110) providing sequence label is checked order to the fetus obtained from maternal sample and maternal nucleic acids.To the sequence label being mapped to an interested karyomit(e) (such as karyomit(e) 21), and the sequence label being mapped to a normalization method karyomit(e) (such as karyomit(e) 9) carry out counting and for calculate for interested chromosomal corresponding first normalized value (such as karyomit(e) dosage), and as the sequence label being mapped to the first normalization method karyomit(e) (such as karyomit(e) 9) and the ratio of number of sequence label being mapped to a second normalization method karyomit(e) (such as karyomit(e) 11), calculate for chromosomal second normalized value (such as karyomit(e) dosage) (150) of the first normalization method.First and second normalized values (that is, karyomit(e) dosage) separately accordingly compared with the first and second threshold values (160), and carry out determination and the checking (140) of a kind of chromosomal aneuploidy of presence or absence.Alternately, two normalized values (namely, two karyomit(e) dosage) be expressed as the first and second normalized karyomit(e) values (NCV), the average of the first chromosome dosage that the one NCV makes the first chromosome dosage corresponding in one group of qualified samples is associated, and the 2nd NCV makes the average of the second karyomit(e) dosage karyomit(e) dosage corresponding in same group of qualified samples be associated, as:
NCV ij = x ij - μ ^ j σ ^ j
wherein with the estimation average for the karyomit(e) dosage of jth in one group of qualified samples and standard deviation accordingly, and x ijfor the viewed jth of a test sample i karyomit(e) dosage.First and second normalized values (that is, NCV) separately compared with predetermined threshold (160), and carry out determination and the checking (140) of a kind of chromosomal aneuploidy of presence or absence.
As discussed previously, the method can identify rare dysploidy (such as 9 trisomys) and common dysploidy (such as trisomy 21), chromosomal aneuploidy, and can identify the multiple chromosomal aneuploidies from order-checking information, this order-checking information obtains from the single order-checking batch about test sample nucleic (such as cfDNA).In some embodiments, single or multiple chromosomal aneuploidy is identified in any one in karyomit(e) 1-22, chromosome x and karyomit(e) Y.Chromosomal aneuploidy can be identified in interested karyomit(e) and/or the first or second normalization method karyomit(e).In some embodiments, the method identifies the single or multiple chromosomal aneuploidies being selected from trisomy 21,13 trisomys, 18 trisomys, 9 trisomys and X monosomy.
Can concentrate in one or more independently qualified samples and determine normalization method karyomit(e).In some embodiments, can concentrate in one or more qualified samples all chromosomal normalization method karyomit(e) determined in genome.Determine for all chromosomal normalization method karyomit(e) in genome, permission use order-checking information determines the chromosomal aneuploidy in genomic each karyomit(e), and this order-checking information is that the single order-checking batch of the nucleic acid of always test sample obtains.
In all embodiments, normalization method karyomit(e) can be selected as follows.
Normalization method karyomit(e) for karyomit(e) 1 is selected from karyomit(e) 10,11,9 and 15.In one embodiment, the first and second normalization method karyomit(e)s for karyomit(e) 1 are karyomit(e) 10 and karyomit(e) 11.
Normalization method karyomit(e) for karyomit(e) 2 is selected from karyomit(e) 8,7,12 and 14.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 2 are karyomit(e) 8 and karyomit(e) 7.
Normalization method karyomit(e) for karyomit(e) 3 is selected from karyomit(e) 6,5,8 and 18.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 3 are karyomit(e) 6 and karyomit(e) 5.
Normalization method karyomit(e) for karyomit(e) 4 is selected from 3,5,6 and 13.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 4 are karyomit(e) 13 and karyomit(e) 5.
Normalization method karyomit(e) for karyomit(e) 5 is selected from 6,3,8 and 18.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 5 are karyomit(e) 6 and karyomit(e) 3.
Normalization method karyomit(e) for karyomit(e) 6 is selected from 5,3,8 and 18.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 6 are karyomit(e) 5 and karyomit(e) 3.
Normalization method karyomit(e) for karyomit(e) 7 is selected from 12,2,14 and 8.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 7 are karyomit(e) 12 and karyomit(e) 2.
Normalization method karyomit(e) for karyomit(e) 8 is selected from 2,7,12 and 3.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 8 are karyomit(e) 2 and karyomit(e) 3.
Normalization method karyomit(e) for karyomit(e) 9 is selected from 11,10,1 and 14.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 9 are karyomit(e) 11 and karyomit(e) 10.
Normalization method karyomit(e) for karyomit(e) 10 is selected from 1,11,9 and 15.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 10 are karyomit(e) 1 and karyomit(e) 11.
Be selected from 1,10,9 and 15 for the normalization method karyomit(e) as interested chromosomal karyomit(e) 11.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 11 are karyomit(e) 1 and karyomit(e) 10.
Normalization method karyomit(e) for karyomit(e) 12 is selected from 7,14,2 and 8.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 12 are karyomit(e) 7 and karyomit(e) 14.
Normalization method karyomit(e) for karyomit(e) 13 is selected from karyomit(e) 4, the group of karyomit(e) 2-6, karyomit(e) 5 and karyomit(e) 6.In one embodiment, for the first and second karyomit(e) normalization method karyomit(e)s of karyomit(e) 13 be the group of karyomit(e) 4 and karyomit(e) 2-6 accordingly.The group of karyomit(e) 2-6 can be used as the first or second normalization method karyomit(e) for interested karyomit(e) 13, and can be used as the chromosomal normalization method karyomit(e) of the first normalization method for karyomit(e) 13.In some embodiments, can perform all chromosomal checking in group.Liang Ge karyomit(e) group can be used as the first and second normalization method karyomit(e)s for karyomit(e) 13, and wherein the karyomit(e) of the first group is different from the karyomit(e) of the second group.
Normalization method karyomit(e) for karyomit(e) 14 is selected from 12,7,2 and 9.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 14 are karyomit(e) 12 and karyomit(e) 7.
Normalization method karyomit(e) for karyomit(e) 15 is selected from 1,10,11 and 9.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 2 are karyomit(e) 1 and karyomit(e) 10.
Normalization method karyomit(e) for karyomit(e) 16 is selected from 20,17,15 and 1.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 16 are karyomit(e) 20 and karyomit(e) 17.
Normalization method karyomit(e) for karyomit(e) 17 is selected from 16,20,19 and 22.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 17 are karyomit(e) 16 and karyomit(e) 20.
Normalization method karyomit(e) for karyomit(e) 18 is selected from 8,3,2 and 6.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 18 are karyomit(e) 8 and karyomit(e) 3.
Normalization method karyomit(e) for karyomit(e) 19 is selected from 22,17,16 and 20.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 19 are chromosome 22 and karyomit(e) 17.
Normalization method karyomit(e) for karyomit(e) 20 is selected from 16,17,15 and 1.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 20 are karyomit(e) 16 and karyomit(e) 17.
Normalization method karyomit(e) for karyomit(e) 21 is selected from 9,11,14 and 1.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for karyomit(e) 21 are karyomit(e) 9 and karyomit(e) 11.
Normalization method karyomit(e) for chromosome 22 is selected from 19,17,16 and 20.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for chromosome 22 are karyomit(e) 19 and karyomit(e) 17.
Normalization method karyomit(e) for chromosome x is selected from 6,5,13 and 3.In one embodiment, the first and second karyomit(e) normalization method karyomit(e)s for chromosome x are karyomit(e) 6 and karyomit(e) 5.
Normalization method karyomit(e) for karyomit(e) Y is selected from the group of karyomit(e) 2-6, karyomit(e) 3, karyomit(e) 4 and karyomit(e) 5.In another embodiment, for the first and second karyomit(e) normalization method karyomit(e)s of karyomit(e) Y be the group of karyomit(e) 3 and karyomit(e) 2-6 accordingly.The group of karyomit(e) 2-6 can be used as the first or second normalization method karyomit(e) for karyomit(e) Y, or is used as the normalization method karyomit(e) for the first normalization method karyomit(e) (such as karyomit(e) 3) for karyomit(e) Y.In some embodiments, not existing of all chromosomal dysploidy in the group of 2-6 is verified.Liang Ge karyomit(e) group can be used as the first and second normalization method karyomit(e)s for karyomit(e) 13, and wherein the karyomit(e) of the first group is different from the karyomit(e) of the second group.Exemplified by for karyomit(e) 13 and karyomit(e) Y, normalization method karyomit(e) can be a karyomit(e) or a karyomit(e) group.
In some embodiments, these methods may relate to the analysis for the chromosomal sequence label of the normalization method of 3 or 4 except interested karyomit(e).
Therefore, in some embodiments, the method determines a kind of fetal chromosomal aneuploidy of presence or absence by following steps in the parent test sample comprising fetus and maternal nucleic acids: (a) obtains the sequence information for fetus in maternal sample and maternal nucleic acids, to identify for the number of an interested chromosomal sequence label and the number for three chromosomal sequence labels of normalization method; B () uses the number of sequence label to calculate for interested chromosomal first, second and the 3rd normalized value; And (c) will compare for interested chromosomal first, second and the 3rd normalized value and one or more threshold value, to determine a kind of fetus dysploidy of presence or absence in maternal sample.In some embodiments, be a first chromosome dosage for interested chromosomal first normalized value, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method; And be a second karyomit(e) dosage for interested chromosomal second normalized value, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the second normalization method; And be a trisome dosage for interested chromosomal 3rd normalized value, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the 3rd normalization method.Optionally, first, second and the 3rd normalized value can be expressed as the normalized karyomit(e) value (NCV) herein as described in other places.
In addition, in some embodiments, the method verifies the determination of presence or absence for interested chromosomal a kind of dysploidy by following steps in the parent test sample comprising fetus and maternal nucleic acids molecule: (a) obtains the sequence information for fetus in maternal sample and maternal nucleic acids, to identify for the number of an interested chromosomal sequence label and the number for three chromosomal sequence labels of normalization method; (b) use for the label of interested chromosomal mapping number and determine for interested chromosomal first normalized value for the number of a chromosomal label of the first normalization method; (c) use for the chromosomal label of the first normalization method number and determine for chromosomal second normalized value of the first normalization method for the number of a chromosomal label of the second normalization method; (d) use for the chromosomal label of the second normalization method number and determine for the second normalization method chromosomal 3rd normalized value for the number of a chromosomal label of the 3rd normalization method; And (e) will compare for interested chromosomal first, second and the 3rd normalized value and one or more threshold value, to determine a kind of fetus dysploidy of presence or absence in maternal sample.In some embodiments, the first normalized value is a first chromosome dosage, and it is the number for described interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method; And the second normalized value is a second karyomit(e) dosage, it is the number for the chromosomal sequence label of the first normalization method and the ratio for the number of a chromosomal sequence label of the second normalization method; And the 3rd normalized value is a trisome dosage, it is the number for the chromosomal sequence label of the second normalization method and the ratio for the number of a chromosomal sequence label of the 3rd normalization method.Optionally, first, second and the 3rd normalized value can be expressed as the normalized karyomit(e) value (NCV) herein as described in other places.
In some embodiments, the method determines a kind of fetal chromosomal aneuploidy of presence or absence by following steps in the parent test sample comprising fetus and maternal nucleic acids: (a) obtains the sequence information for fetus in maternal sample and maternal nucleic acids, to identify for the number of an interested chromosomal sequence label and the number for four chromosomal sequence labels of normalization method; B () uses the number of sequence label to calculate for interested chromosomal first, second, third and the 4th normalized value; And (c) will for interested chromosomal first, second, third and the 4th normalized value and one or more threshold value compare, to determine a kind of fetus dysploidy of presence or absence in maternal sample.In some embodiments, be a first chromosome dosage for interested chromosomal first normalized value, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method; And be a second karyomit(e) dosage for interested chromosomal second normalized value, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the second normalization method; And be a trisome dosage for interested chromosomal 3rd normalized value, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the 3rd normalization method; And be a tetrasome dosage for interested chromosomal 4th normalized value, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the 4th normalization method.Optionally, first, second, third and the 4th normalized value can be expressed as the normalized karyomit(e) value (NCV) herein as described in other places.
In some embodiments, the method is determined by following steps and is verified that presence or absence is for interested chromosomal a kind of dysploidy in the parent test sample comprising fetus and maternal nucleic acids molecule: (a) obtains the sequence information for fetus in maternal sample and maternal nucleic acids, to identify for the number of an interested chromosomal sequence label and the number for four chromosomal sequence labels of normalization method; (b) use for interested chromosomal mapping number of tags and determine for interested chromosomal first normalized value for a chromosomal number of tags of the first normalization method; (c) use for the chromosomal label of the first normalization method number and determine for chromosomal second normalized value of the first normalization method for the number of a chromosomal label of the second normalization method; And (d) use for the chromosomal label of the second normalization method number and determine for the second normalization method chromosomal 3rd normalized value for the number of a chromosomal label of the 3rd normalization method; (e) use for the chromosomal label of the 3rd normalization method number and determine for the 3rd normalization method chromosomal 4th normalized value for the number of a chromosomal label of the 4th normalization method; And (f) will for interested chromosomal first, second, third and the 4th normalized value and one or more threshold value compare, to determine a kind of fetus dysploidy of presence or absence in maternal sample.In some embodiments, the first normalized value is a first chromosome dosage, and it is the number for described interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method; And the second normalized value is a second karyomit(e) dosage, it is the number for the chromosomal sequence label of the first normalization method and the ratio for the number of a chromosomal sequence label of the second normalization method; And the 3rd normalized value is a trisome dosage, it is the number for the chromosomal sequence label of the second normalization method and the ratio for the number of a chromosomal sequence label of the 3rd normalization method; And the 4th normalized value is a tetrasome dosage, it is the number for the chromosomal sequence label of the 3rd normalization method and the ratio for the number of a chromosomal sequence label of the 4th normalization method.Optionally, first, second, third and the 4th normalized value can be expressed as the normalized karyomit(e) value (NCV) herein as described in other places.
In these embodiments, first, second, third and the 4th normalization method karyomit(e) can be selected from the normalization method karyomit(e) of above elaboration.For example, for karyomit(e) 1 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 10,11,9 and 15; For karyomit(e) 2 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 8,7,12 and 14; For karyomit(e) 3 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 6,5,8 and 18; For karyomit(e) 4 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 3,5,6 and 13; For karyomit(e) 5 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 6,3,8 and 18; For karyomit(e) 6 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 5,3,8 and 18.For karyomit(e) 7 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 12,2,14 and 8; For karyomit(e) 8 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 2,7,12 and 3; For karyomit(e) 9 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 11,10,1 and 14; For karyomit(e) 10 first, second, third and the 4th normalization method karyomit(e) can be selected from 1,11,9 and 15; For karyomit(e) 11 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 1,10,9 and 15; For karyomit(e) 12 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 7,14,2 and 8; For karyomit(e) 13 first, second, third and the 4th normalization method karyomit(e) can be selected from the group, 5 and 6 of karyomit(e) 4, karyomit(e) 2-6; For karyomit(e) 14 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 12,7,2 and 9; For karyomit(e) 15 first, second, third and the 4th normalization method karyomit(e) can be selected from 1,10,11 and 9; For karyomit(e) 16 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 20,17,15 and 1; For karyomit(e) 17 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 16,20,19 and 22; For karyomit(e) 18 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 8,3,2 and 6; For karyomit(e) 19 first, second, third and the 4th normalization method karyomit(e) can be selected from chromosome 22,17,16 and 20; For karyomit(e) 20 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 16,17,15 and 1; For karyomit(e) 21 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 9,11,14 and 1; For chromosome 22 first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 19,17,16 and 20; For chromosome x first, second, third and the 4th normalization method karyomit(e) can be selected from karyomit(e) 6,5,13 and 3; And for karyomit(e) Y first, second, third and the 4th normalization method karyomit(e) can be selected from group, the karyomit(e) 3,4 and 5 of karyomit(e) 2-6.
Sequence measurement
In certain methods of the present invention, the sequence information obtained for fetus and maternal nucleic acids in the sample to which carrys out the number of recognition sequence label, comprises and checking order to the fetus in sample and maternal nucleic acids molecule.
Sequence information is by using any one checked order in (NGS) method to the next generation that the DNA profiling increased with clonal fashion or single DNA molecules check order in mode parallel on a large scale, genomic dna (such as Cell-free DNA) in maternal sample is checked order obtain (such as people such as Wo Keerding (Volkerding), clinical chemistry (Clin Chem) 55:641-658 [2009]; Maze can M (Metzker M), naturally comments on described in (Nature Rev) 11:31-46 [2010]).Except high through-put sequence information, NGS provides quantitative information, and wherein each sequence reads is computable " sequence label ", and these sequence labels represent individual cloned DNA template or single DNA molecular.The synthesis method order-checking that the sequencing technologies of NGS includes but not limited to Manganic pyrophosphate complex initiation, use multiple reversible dye-terminators to carry out, connects by oligonucleotide probe the order-checking carried out and ionic semiconductor checks order.Can check order from the DNA (i.e. singleplex order-checking) of independent sample individually, or when single order-checking runs, as index genome molecules, DNA from multiple sample can be pooled together and carry out checking order (namely, multiple order-checking), to produce the reading of DNA sequence dna up to some hundred million.Following illustrate the example of sequencing technologies, these technology may be used for obtaining the sequence information according to method of the present invention.
Some sequencing technologies are commercially available, such as from Ang Fei company of the U.S. (Affymetrix Inc.) (Sani Wei Er (Sunnyvale), CA) sequencing by hybridization platform, with from 454 Life Sciences Corp. (454Life Sciences) (Bradford (Bradford), CT), California Hayward hundred million is sensible/Suo Liesha company (Illumina/Solexa) (Hayward (Hayward), CA) with spiral Biological Science Co., Ltd (Helicos Biosciences) (Cambridge (Cambridge), MA) synthesis method order-checking platform, and from Applied biosystems (Applied Biosystems) (Foster city (Foster City), CA) connection method order-checking platform, as described below.Except using the synthesis method of spiral Biological Science Co., Ltd (HelicosBiosciences) to check order the single-molecule sequencing carried out, other single-molecule sequencing technology comprise the SMRT of Pacific Ocean Biological Science Co., Ltd (Pacific Biosciences) tMtechnology, ion Torrent tMtechnology, and the nanoporous order-checking of just developing, such as, by Oxford nanoporous technology.Although the Sang Geerfa of automatization (Sanger method) is regarded as ' first-generation ' technology, the inventive method can be applied to the biological detection using Sang Geer order-checking (comprising the Sang Geer order-checking of automatization).In addition, the inventive method can be applied to the biological assay using nucleic acid imaging technique (such as atomic force microscope (AFM) or transmission electron microscopy (TEM)).Following illustrated example sequencing technologies.
In one embodiment, method of the present invention comprises use single-molecule sequencing technology, single-molecule sequencing (the Helicos True Single Molecule Sequencing that spiral is real; TSMS) technology obtains sequence information people such as (, described in science (Science) 320:106-109 [2008]) such as Harris T.D. (HarrisT.D.) for genomic dna (such as fetus and parent cfDNA).In tSMS technology, DNA sample is cut into the chain of about 100 to 200 Nucleotide, and polyA sequence is added to 3 ' end of each DNA chain.Each chain is marked by adding fluorescently-labeled adenylic acid (AMP).Then these DNA chains are hybridized to the groove that flows, and it contains millions of the few T (oligo-T) being fixed to flowing rooved face and catches site.Template can be at about 100,000,000 template/cm 2density.Then flowing groove is loaded in an instrument, such as HeliScope tMsequenator, and illuminated with laser light flowing rooved face, disclose the position of each template.CCD camera can draw the position of the template on flowing rooved face.Then cut and wash template fluorescent marker off.Sequencing reaction is started by introducing archaeal dna polymerase and fluorescently-labeled Nucleotide.Few T nucleic acid is used as primer.The Nucleotide of mark, in the mode of template-directed, is attached on this primer by polysaccharase.Removing polysaccharase and unconjugated Nucleotide.By making the imaging of flowing rooved face, distinguish the template that the guiding with fluorescently-labeled Nucleotide combines.After imaging, cutting step eliminates fluorescent marker, and repeats this process with other fluorescently-labeled Nucleotide, until reach the reading length of hope.Collection step sequence information is added with each Nucleotide.Got rid of by the whole gene order-checking of single-molecule sequencing technology and preparing the amplification of the PCR base in sequencing library, and the substantivity of sample preparation allows the direct measurement of sample, instead of the measurement of the copy of sample.
In another embodiment, method of the present invention comprise use 454 check order (Roche Holding Ag (Roche)) obtain sequence information (described in the people such as such as Margules M (Margulies, M.) nature (Nature) 437:376-380 [2005]) for genomic dna (such as fetus and parent cfDNA).454 order-checkings relate to two steps.In the first step, DNA is cut into the fragment of an about 300-800 base pair, and these fragments terminate with flush end.Then oligonucleotide aptamer is connected to the end of these fragments.Aptamer is used as the primer of amplification and these fragments that check order.Such as use aptamer B, it contains 5 ' biotin label, and these fragments can be attached to DNA and catch on pearl, such as, on the pearl of Streptavidin coating.Pcr amplification is attached to the fragment on pearl in many oil drippings water emulsion.Result is multiple copies of the DNA fragmentation of clonal expansion on each pearl.In second step, in hole (picoliter size), catch these pearls.Manganic pyrophosphate complex initiation is carried out to each DNA fragmentation is parallel.Add one or more Nucleotide to produce by an optical signal of the CCD camera record in sequenator.The few nucleotide of strength of signal and combination is proportional.Manganic pyrophosphate complex initiation make use of the pyrophosphate (PPi) of the release when Nucleotide adds.Under the existence of adenylic acid (AMP) 5 ' phosphosulfate salt, PPi is converted into ATP by ATP sulfurylase.Luciferase uses ATP that fluorescein is changed into oxyluciferin, and this reaction creates light, and this light is distinguished and analyzes.
In another embodiment, the inventive method comprises use SOLiD tMtechnology (Applied Biosystems, Inc. (Applied Biosystems)) obtains the sequence information for genomic dna (such as fetus and parent cfDNA).At SOLiD tMin connection method order-checking, genomic dna is cut into fragment, and aptamer is attached to 5 ' and 3 ' end of these fragments, to produce fragment library.Alternately, by being connected to by aptamer on 5 ' and 3 ' end of these fragments, these fragments can be distributed, digesting the fragment of these distributions to produce inner aptamer, and 5 ' and the 3 ' end of fragment aptamer being attached to generation is upper matches storehouse to produce, introduce inner aptamer.Next, preparation clone pearl group in the microreactor containing pearl, primer, template and PCR component.After PCR, denature template and concentrated pearl, to be separated the pearl having and extend template.Make the template on selected pearl stand to allow to be attached to 3 ' on slide glass to modify.By the base (or base pair) that order hybridization and connection portion random oligonucleotide are determined with the central authorities of the identification of being rolled into a ball by specific fluorescent, this sequence can be determined.After record color, cut and remove the oligonucleotide connected, and then repeating this process.
In another embodiment, method of the present invention comprises the unit molecule (SMRT in real time using Pacific Ocean Biological Science Co., Ltd (Pacific Biosciences) tM) sequencing technologies obtains for the sequence information of genomic dna (such as fetus and parent cfDNA).In SMRT order-checking, in DNA building-up process, image is carried out to the continuous combination of the Nucleotide of dye marker.Make single DNA polymerase molecule be attached on the basal surface of independent null mode wavelength recognition device (ZMW recognizer), these recognizers obtain sequence information be incorporated in the primer strand in growth at the Nucleotide connecting phosphorus while.ZMW is an enclosed construction, and it allows to the archaeal dna polymerase of the background of observing by the fluorescent nucleotide for diffusion turnover ZMW (by microsecond meter) rapidly, the combination of mononucleotide.Nucleotide is attached in the chain grown with some milliseconds.During this period, fluorescence excitation marker and produce fluorescent signal, and cut away this fluorescence labels.It is combined which base the identification of the corresponding fluorescence of dyestuff indicates.Repeat this process.
In another embodiment, method of the present invention comprises the sequence information (such as at Sony (Soni) GV and Mei Le A. (Meller A.), described in clinical chemistry (Clin Chem) 53:1996-2001 [2007]) using nanoporous order-checking acquisition for genomic dna (such as fetus and parent cfDNA).By multiple company just industrial develop nanoporous sequenced dna analytical technology, comprise Oxford nanoporous company (Oxford Nanopore Technologies) (Oxford, Britain).Nanoporous order-checking is a kind of single-molecule sequencing technology, and thus along with it is by a nanoporous, a monomolecular DNA is by direct Sequencing.Nanoporous is an aperture, and its rank is diameter 1 nanometer.Nanoporous is immersed in conductive fluid, and applies an electromotive force (voltage) across it, create because ionic conduction is through a slight electric current of nanoporous.For the size and shape of nanoporous, the amount of the electric current of flowing is responsive.Along with DNA molecular is through nanoporous, each Nucleotide on DNA molecular to block nanoporous in various degree, to change the magnitude of the electric current through nanoporous in various degree.Therefore, along with DNA molecular is through the reading of the change representation DNA sequence of the electric current of nanoporous.
In another embodiment, method of the present invention comprises the sequence information (such as described in U.S. Patent Application Publication No. 20090026082) using effect electric crystal (chemFET) the array acquisition of chemosensitivity field for genomic dna (such as fetus and parent cfDNA).In an example of this technology, DNA molecular can be placed in reaction chamber, and template molecule can be hybridized to and be attached on the sequencing primer of polysaccharase.Can to be attached in new nucleic acid chain at 3 ' the one or more triphosphate in end place of sequencing primer by distinguishing with the change in the electric current of chemFET.An array can have multiple chemFET sensor.In another example, mononucleotide can be attached on pearl, and these nucleic acid can increase on this pearl, and independent pearl can be transferred in the independent reaction chamber on chemFET array, wherein each room has chemFET sensor, and nucleic acid can be sequenced.
In another embodiment, method of the present invention comprises the sequence information of technology acquisition for genomic dna (such as fetus and parent cfDNA) of use kingfisher branch and subsidiaries (HalcyonMolecular), and this technology employs transmission electron microscopy (TEM).The method, be called as individual molecule and place rapid nano transfer (Individual Molecule Placement Rapid Nano Transfer, IMPRNT), comprise and utilizing by the monatomic resolution transmission electron microscope imaging of high molecular (150kb or the larger) DNA of heavy atom marker selected marker, and to the ultrathin membrane of base spacing in ultra dense degree (3nm chain is to chain) parallel array, arrange these molecules with consistent base.Use electron microscope carrys out the molecule on imaging film, to determine the position of heavy atom marker, and extracts the base sequence information from DNA.The method is further illustrated in the open WO 2009/046445 of PCT patent.The method allows to check order complete human genome within the time being less than ten minutes.
In another embodiment, DNA sequencing technology is ion torrent company (Ion Torrent) single-molecule sequencing, it makes semiconductor technology match with the chemistry that simply checks order, on a semiconductor die the information (A, C, G, T) of chemical code is directly translated as numerical information (0,1).In essence, when being attached in a DNA chain by Nucleotide by polysaccharase, discharge a hydrogen ion as by product.Ion torrent company (Ion Torrent) uses the hole array of a highdensity micromachined, carries out these Biochemical processes with extensive parallel mode.Each pore volume receives a different DNA molecular.Be an ion-sensitive layer under this some holes, and be an ionization sensor under it.Such as, at Nucleotide, a C, when being added to a DNA profiling and being then attached in a DNA chain, by release hydrogen ion.Electric charge from this ion will change the pH value of solution, this change can identify by the ionization sensor of ion torrent.This sequenator---solid-state pH meter minimum in the world in essence---judges base, directly chemically information to numerical information.A this ion human genome machine (Ion personal GenomeMachine, PGM tM) then sequenator sequentially flood this chip with Nucleotide one by one.If the next Nucleotide flooding this chip does not mate, so by the change of record less than voltage, and base can not be judged.If there are two bases equally on DNA chain, so voltage will double, and this chip will be recorded to through two of judgement the same bases.Direct Recognition allows by the combination of recording Nucleotide second.
In another embodiment, method of the present invention comprises and obtains sequence information (such as people such as Bentley (Bentley), described in Nature (nature) 6:53-59 [2009]) for genomic dna (such as fetus and parent cfDNA) by using the synthesis method of hundred million sensible companies (Illumina) to check order and carrying out extensive parallel order-checking based on the order-checking chemistry of reversible terminator to millions of DNA fragmentations.Template DNA can be genomic dna, such as cfDNA.In some embodiments, the genomic dna from isolated cell is used as template, and is split into the length of hundreds of base pairs.In other embodiments, cfDNA is used as template, and does not need segmentation, because cfDNA exists with short-movie section.For example, fetus cfDNA circulates with the pieces being less than 300bp in blood flow, and parent cfDNA is with the pieces circulation (people such as Lee (Li) about between 0.5 and 1Kb according to estimates, clinical chemistry (ClinChem), 50:1002-1011 [2004]).The genomic dna that the sequencing technologies of hundred million sensible companies relies on segmentation is to the attachment of a plane (oligonucleotide anchor point be attached to above optionally transparent surface).Template DNA is produced the flush end of 5 ' phosphorylation by end reparation, and the polymerase activity of Ke Lienuo (Klenow) fragment is used to add the 3 ' end of single A base to flat phosphorylated cdna fragment.The DNA fragmentation for being connected on oligonucleotide aptamer has been prepared in this interpolation, and has a single T base hung, to increase joint efficiency at their 3 ' end.These aptamer oligonucleotides are complementary with flowing groove anchor point.Under restriction diluting condition, aptamer is modified, single-stranded template DNA is added to flowing groove, and is fixed to anchor point by hybridization.The DNA fragmentation of attachment is extended and bridge amplification, produces the super-high density order-checking flowing groove with several hundred million bunches, each about 1000 copies all containing same template.In one embodiment, the genomic dna of random division, such as cfDNA, front in experience cluster amplification (cluster amplification), use PCR to increase to it.Alternately, use without amplification gene group storehouse goods, and use cluster amplification to carry out the genomic dna of enrichment random division (namely individually, cfDNA) (the people such as Ke Zharewa (Kozarewa), natural method (Nature Methods), 6:291-295 [2009]).Use a kind of four color DNA synthesis method sequencing technologies of robust to check order these templates, this technology have employed the reversible terminator having and can remove fluorescence dye.Laser excitation and total internal reflection optical is used to obtain hypersensitivity fluorescence identifying.The short data records reading with about 20-40bp (such as 36bp) is compared relative to the reference gene group of covering repetition, and uses specifically developed data analysis pipeline software to judge hereditary difference.After first time, reading completed, can these templates of in-situ regeneration, so that second reading that can obtain from the end opposite of these fragments.Therefore, the order-checking of the also or in pairs end of single end of these DNA fragmentations can be used.Carry out the DNA fragmentation existed in sample part order-checking, and by the sequence label comprising the reading of predetermined length (such as 36bp) reflect known reference gene group.The label penetrated can be mapped count.
In one embodiment, reference gene group sequence is NCBI36/hg18 sequence, is it in World Wide Web, at genome.ucsc.edu/cgi-bin/hgGateway? org=Human & db=hg18 & hgsid=166260105 can obtain.In another embodiment, reference gene group sequence is GRCh37/hg19, and it can obtain at genome.ucsc.edu/cgi-bin/hgGateway in World Wide Web.Sequence from other reference gene groups of multiple species can obtain at ncbi.nlm.nih.gov/genomes/leuks.cgi in NCBI website.Other sources of open sequence information comprise GenBank, dbEST, dbSTS, EMBL (European Molecular Bioglogy Laboratory) and DDBJ (DNA Data Bank of Japan).Multiple computerized algorithm can be used for aligned sequences, comprise instead of limit: the restriction BLAST (people such as A Erqiuer (Altschul), 1990), BLITZ (MPsrch) (Sturrock (Sturrock) & Collins (Collins), 1993), FASTA (amber gloomy (Person) & Li Puman (Lipman), 1988), BOWTIE (the people such as glug plum moral (Langmead), genome biology (Genome Biology) 10:R25.1-R25.10 [2009]), or the ELAND (sensible company (Illumina of San Diego, CA, USA hundred million, Inc.), San Diego (San Diego), CA, USA).In one embodiment, checked order in one end of the copy of the clonal expansion of blood plasma cfDNA molecule, and it processed by the information biology compare of analysis being used for Illumina gene element analyzer, this analyser employs effectively extensive comparison (ELAND) software of RiboaptDB.
In some embodiments of method described herein, the sequence label of mapping comprises the sequence reads for about 20bp, about 25bp, about 30bp, about 35bp, about 40bp, about 45bp, about 50bp, about 55bp, about 60bp, about 65bp, about 70bp, about 75bp, about 80bp, about 85bp, about 90bp, about 95bp, about 100bp, about 110bp, about 120bp, about 130, about 140bp, about 150bp, about 200bp, about 250bp, about 300bp, about 350bp, about 400bp, about 450bp or about 500bp.Expect that technical superiority will make the single-ended reading that can carry out being greater than 500bp, when producing pairing end reading, this reading allows to the reading for being greater than about 1000bp.In one embodiment, the sequence label mapped comprises sequence reads for 36bp and compares by sequence label and reference sequences the mapping realizing sequence label, to determine the chromosomal origin of nucleic acid (such as cfDNA) molecule checked order, and do not need specific genetic sequences information.The mispairing of little degree (each sequence label 0-2 mispairing) can be allowed, to explain the little polymorphism between the genome that may reside in reference gene group and biased sample.
Each sample obtains multiple sequence label.In some embodiments, obtain from reference gene group reading being mapped to each sample be included in (such as 36bp) between 20 and the reading of 40bp at least about 3x10 6individual sequence label, at least about 5x10 6individual sequence label, at least about 8x10 6individual sequence label, at least about 10x10 6individual sequence label, at least about 15x10 6individual sequence label, at least about 20x10 6individual sequence label, at least about 30x10 6individual sequence label, at least about 40x10 6individual sequence label or at least about 50x10 6individual sequence label.In one embodiment, the mapped all regions being mapped to reference gene group of all sequences reading.In one embodiment, the label in all regions (such as all karyomit(e)) being mapped to reference gene group is counted, and determine in hybrid dna sample, the CNV (that is, overexpression or expression deficiency) of interested sequence (such as karyomit(e) or its part).The method does not need the differentiation between two genomes.
In some embodiments, the method determines a kind of fetal chromosomal aneuploidy of presence or absence by following steps in the parent test sample comprising fetus and maternal nucleic acids molecule: (a) obtains the sequence information for fetus in maternal sample and maternal nucleic acids, to identify for the number of an interested chromosomal sequence label and the number at least two chromosomal sequence labels of normalization method, wherein sequence information comprises order-checking (NGS) of future generation, comprise the synthesis method order-checking using multiple reversible dye-terminators to carry out, comprise connection method order-checking, or comprise single-molecule sequencing, b () uses the number of sequence label to calculate for interested chromosomal first normalized value and second normalized value, and (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for interested chromosomal second normalized value and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in the sample to which.First and second threshold values can be identical, or they can be different.In the step (c) of this method, show that presence or absence is for described interested chromosomal a kind of dysploidy for described interested chromosomal first normalized value with comparing of threshold value, and for the comparatively validate presence or absence of described interested chromosomal second normalized value and threshold value for the determination of interested chromosomal a kind of dysploidy.In some embodiments, the first normalized value is a first chromosome dosage, and it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method; And the second normalized value is a second karyomit(e) dosage, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as normalized karyomit(e) value (NCV) as described herein.
In some other embodiments, the method verifies the determination of presence or absence for interested chromosomal a kind of dysploidy by following steps in the parent test sample comprising fetus and maternal nucleic acids molecule: (a) obtains the sequence information for fetus and maternal nucleic acids in the sample to which, to identify for the number of the sequence label of an interested chromosomal mapping and the number at least two chromosomal sequence labels of normalization method, wherein obtain sequence information and comprise order-checking (NGS) of future generation, comprise the synthesis method order-checking using multiple reversible dye-terminators to carry out, comprise connection method order-checking, or comprise single-molecule sequencing, (b) use for interested chromosomal label number and determine for interested chromosomal first normalized value for the number of a chromosomal label of the first normalization method, and use for the chromosomal sequence label of the first normalization method number and determine for chromosomal second normalized value of the first normalization method for the number of a chromosomal sequence label of the second normalization method, and (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for chromosomal second normalized value of the first normalization method and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in the sample to which.First and second threshold values can be identical, or they can be different.In the step (c) of this method, show that presence or absence is for described interested chromosomal a kind of dysploidy for described interested chromosomal first normalized value with comparing of threshold value, and for the comparatively validate presence or absence of chromosomal second normalized value of described first normalization method and threshold value for the determination of interested chromosomal a kind of dysploidy.In some embodiments, the first normalized value is a first chromosome dosage, and it is the number for described interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method; And the second normalized value is a second karyomit(e) dosage, it is the number for the chromosomal sequence label of the first normalization method and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as the normalized karyomit(e) value (NCV) calculated as described herein.
In some embodiments, the first normalized value is a first chromosome dosage, and it is the number for described interested chromosome sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method; And the second normalized value is a second karyomit(e) dosage, it is the number for the chromosomal sequence label of the first normalization method and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as normalized karyomit(e) value (NCV) as described herein.
Biological fluid comprises, as limiting examples, and blood, blood plasma, serum, sweat, tears, phlegm, urine, phlegm, ear effluent (ear flow), lymph liquid, saliva, cerebrospinal fluid, irrigating solution (ravages), bone marrow floater liquid (bone marrow suspension), vaginal discharge (vaginal flow), through the irrigating solution of uterine neck, brain liquid, ascites, milk, breathes, secretory product, amniotic fluid and the leukapheresis sample of intestines and genitourinary tract.In some embodiments, this sample is such as, by non-invasive process easily obtainable sample, blood, blood plasma, serum, sweat, tears, phlegm, urine, phlegm, ear effluent and saliva.Preferably, this biological sample is peripheral blood sample, or blood plasma or sera components.In other embodiments, this biological sample is cotton swab or smear, examination of living tissue sample, or cell cultures.In another embodiment, this sample is the mixture of two or more biological samples, and the biological example product of imitating can comprise two or more biological fluid sample, tissue sample and cell culture samples.As used in this, term " blood ", " blood plasma " and " serum " clearly contain their separated part or the part of processing.Similarly, when a sample be take from a kind of examination of living tissue, cotton swab, smear, etc. time, should " sample " contain clearly derived from this examination of living tissue, cotton swab, smear, etc. the separated part of processing or part.
In some embodiments, sample can derive from multiple source, include but not limited to, from Different Individual, the different stages of development of identical or different individuality, different diseased individuals (such as suffer from cancer or suspect the individuality with genetic block), the sample of normal individual, at the sample that the different steps of the disease of individuality obtains, derive from the sample of experience to the individuality that the difference of disease is treated, from the sample of the individuality of experience varying environment factor, or the individuality to a kind of state of an illness susceptible, or be exposed to a kind of individuality of transmissible disease factor (such as HIV), and be donorcells, the sample of the individuality of the recipient of tissue and/or organ.In some embodiments, sample is the sample of the mixture comprising the different sources sample deriving from identical or different experimenter.For example, sample can comprise the mixture of the cell deriving from two or more individualities, as usual in crime scene find.In one embodiment, this sample is the maternal sample deriving from pregnant female (such as pregnant woman).In this case, this sample can use method described herein to analyze, to provide the antenatal diagnosis of potential chromosome abnormalty in fetus.This maternal sample can be tissue sample, biological fluid sample or cell sample.Biological fluid comprises (as limiting examples): blood, blood plasma, serum, sweat, tears, phlegm, urine, phlegm, ear effluent, lymph, saliva, cerebrospinal fluid, irrigating solution (ravages), bone marrow floater liquid, vaginal discharge, through the irrigating solution of uterine neck, brain liquid, ascites, milk, breathes, the secretory product of intestines and genitourinary tract, and leukapheresis sample.In some embodiments, this sample is by non-invasive process easily obtainable sample, such as, and blood, blood plasma, serum, sweat, tears, phlegm, urine, phlegm, ear effluent and saliva.In some embodiments, this biological sample is peripheral blood sample, or blood plasma or serum separated part.In other embodiments, this biological sample is cotton swab or smear, examination of living tissue sample or cell cultures.In another embodiment, maternal sample is the mixture of two or more biological samples, and such as, a kind of biological sample can comprise two or more biological fluid sample, tissue sample and cell culture samples.As disclosed above, their separated part or the part of processing clearly contained in term " blood ", " blood plasma " and " serum ".Similarly, when a sample take from examination of living tissue, cotton swab, smear, etc. time, this " sample " clearly contain derived from examination of living tissue, cotton swab, smear, etc. the separated part of processing or part.
Sample can also be derive from the tissue of vitro culture, cell or other sources containing polynucleotide.These samples cultivated can take from multiple source, include but not limited to, maintain the culture (such as tissue or cell) under different culture media and condition (such as pH value, pressure or temperature), maintain the culture (such as tissue or cell) of the period of different lengths, by biological factors or reagent (such as drug candidate, or conditioning agent) culture (such as tissue or cell) that processes, or the culture of dissimilar tissue or cell.
Be that people know from the method for biological origin isolating nucleic acid, and depend on that the character in source is by difference.Those of ordinary skill in the art can easily isolate as the nucleic acid required for method described herein from a source.In some cases, it can be favourable for being ruptured by the nucleic acid molecule in nucleic acid samples.Fracture can be random, or it can be special, such as, use the situation that digestion with restriction enzyme reaches.Be known for the method for random fracture in this area, and comprise such as restricted dnase digestion, alkaline purification and physical shear.In one embodiment, sample nucleic obtains as cfDNA, and it does not experience fracture.In other embodiments, sample nucleic obtains as genomic dna, and its experience is broken into the fragment of about 500 or more base pairs, and can easily use NGS method to it.
For determining that the sample of CNV (such as karyomit(e) and part dysploidy) comprises the genomic nucleic acids being present in (i.e. cell) genomic nucleic acids in cell or " acellular ".Genomic nucleic acids comprises DNA and RNA.Preferably, genomic nucleic acids is genomic nucleic acids and/or the cfDNA of cell.In some embodiments, the genomic nucleic acids of sample is cell DNA, and it can by extracting genomic dna with artificial or mechanical system and obtain from intact cell from the intact cell with identical or different genetic composition.Cell DNA can such as from derive from an experimenter the intact cell with identical genetic composition, from the mixture of the intact cell of different experimenter or obtain from the mixture of the intact cell different in genetic composition deriving from an experimenter.The method extracting genomic dna from intact cell is known in the art, and depends on the character in source and different.In some embodiments, it may be favourable for being ruptured by cell genomic dna.Fracture can be random, or it can be specific, digest as such as used restriction endonuclease realize.The method of random segment is known in the art, and comprises such as restrictive dnase digestion, alkaline purification and physical shear.In some embodiments, sample nucleic obtains with cell genomic dna form, cell genomic dna rupture, becomes the fragment with about 500 or more base pairs, and these fragments can be checked order by check order (NGS) of future generation.
In some embodiments, cell genomic dna is obtained to identify the chromosomal aneuploidy comprising the sample of individual gene group.For example, cell genomic dna can obtain from the cell sample only comprising pregnant female, and namely this sample is not containing Fetal genome sequence.Identify that chromosomal aneuploidy may be used for comparing, to identify fetal chromosomal aneuploidy with the chromosomal aneuploidy identified in the mixture of the fetus be present in Maternal plasma and maternal gene group and/or polymorphism from individual gene group (such as only maternal gene group).Similarly, cell genomic dna can obtain from the patient's (such as cancer patient) being in different treatment stage, so that by analyzing to the possible change of chromosomal aneuploidy in sample DNA and/or polymorphism the effect evaluating treatment plan.
In some embodiments, obtain acellular nucleic acid, such as Cell-free DNA (cfDNA) is favourable.Acellular nucleic acid (comprising Cell-free DNA) can be obtained from the biological sample including but not limited to blood plasma, serum and urine by diverse ways known in the art (people such as model (Fan), institute of NAS periodical (Proc Natl Acad Sci) 105:16266-16271 [2008]; Little go out the people such as (Koide), antenatal diagnosis (Prenatal Diagnosis) 25:604-607 [2005]; People such as old (Chen), Natural medicine (Nature Med.) 2:1033-1035 [1996]; The people such as Lu (Lo), lancet (Lancet) 350:485-487 [1997]; The people such as Bo Taizhatu (Botezatu), clinical chemistry (Clin Chem.) 46:1078-1084,2000; And the people such as (Su) that revives, molecular diagnostics magazine (J Mol.Diagn.) 6:101-107 [2004]).In order to from cellular segregation cfDNA, part can be used to be separated (fractionation), centrifugal (such as density gradient centrifugation), DNA specificity precipitation or high-flux cell sorting and/or separation method.Commercially available test kit (Indianapolis, the state of Indiana Roche Diagnistics company (Roche Diagnostics, Indianapolis, IN) for being manually separated with automatization cfDNA can be obtained; Markon welfare Ya Zhou Valencia Kai Jie company (Qiagen, Valencia, CA); Delaware State Du Lun cot Lei-Nei Geer company (Macherey-Nagel, Duren, DE)).The biological sample comprising cfDNA has been used in analysis, so that by determining that the sequencing analysis of chromosomal aneuploidy and/or different polymorphisms determines the multiple chromosome abnormalty of presence or absence (such as trisomy 21).
Before preparing sequencing library, specifically or non-specifically enrichment can be present in the cfDNA in sample.The unspecific enrichment of sample DNA refers to the whole genome amplification of the genomic DNA fragment of sample, and it may be used for increasing sample DNA level before preparation cfDNA sequencing library.Unspecific enrichment can be the selective enrichment being present in one of two genomes comprised in more than one genomic sample.For example, unspecific enrichment can be the selective enrichment of the Fetal genome in maternal sample (it can be obtained by currently known methods), to improve the relative proportion of fetus and mother body D NA in sample.Alternately, unspecific enrichment can be two the genomic non-selective amplification be present in sample.For example, non-specific amplification can be the non-specific amplification of fetus and mother body D NA in the sample of the mixture of the DNA comprised from fetus and maternal gene group.The method of whole genome amplification is known in the art.The example that the PCR (DOP) of primer, primer extension round pcr (PEP) and multiple displacement amplification (MDA) are whole genome amplification methods made by degeneracy oligonucleotide.In some embodiments, the sample of the mixture of the cfDNA from different genes group is comprised not for the genomic cfDNA enrichment be present in mixture.In other embodiments, the sample comprising the mixture of the cfDNA from different genes group carries out unspecific enrichment for any one genome be present in sample.
Application
The acellular foetal DNA circulated in maternal blood and RNA can be used to the early stage Non-invasive Prenatal Diagnosis (NIPD) of the ever-increasing hereditary conditions of number, both can be used for management and also can help reproduction decision-making.The existence of the Cell-free DNA circulated in blood flow is known more than 50 years.Recently, in the parent blood flow of gestation time, to have found to exist foetal DNA people such as (, lancet (Lancet) 350:485-487 [1997]) sieve (Lo) of circulation in a small amount.Be considered to be derived from dying placenta cells, the short-movie section that acellular foetal DNA (cfDNA) has been proved to be by length being typically less than 200bp forms, (people such as old (Chan)), clinical chemistry, 50:88-92 [2004]), early to only have 4 weeks pregnant in can be distinguished that (she draws the people such as Nice (Illanes), early stage human developmental (EarlyHuman Dev), 83:563-566 [2007]), and knownly within a few hours of childbirth, namely from maternal circulation, removed (the people such as sieve (Lo), American Journal of Human Genetics (Am J Hum Genet), 64:218-224 [1999]).Except cfDNA, can also distinguish the fragment of (cfRNA) of acellular fetal rna in parent blood flow, this is derived from gene transcribed in fetus or placenta.The new chance for NIPD is provided from the extraction of these fetus genetic elements of maternal blood sample and analysis subsequently.
The method may be used in the maternal sample comprising fetus and maternal nucleic acids molecule (such as cfDNA), determine a kind of fetal chromosomal aneuploidy of presence or absence.Present method is a kind of method independent of polymorphism being applicable to NIPD, and does not need fetus cfDNA and parent cfDNA phase to distinguish the determination that can realize a kind of fetus dysploidy.
In some embodiments, sample is a kind of biological fluid sample, such as blood sample or its part.Preferably, biological sample is selected from blood plasma, serum and urine.In some embodiments, maternal source sample is a kind of peripheral blood sample.In other embodiments, maternal source sample is a plasma sample.Fetus can be realized by the extensive parallel NGS sequence measurement of any one with the order-checking of maternal nucleic acids.In one embodiment, order-checking is the extensive parallel order-checking to the cfDNA molecule increased with clonal fashion or multiple independently cfDNA molecule.In another embodiment, order-checking is the described extensive parallel order-checking of the extensive parallel synthesis order-checking using multiple reversible dye-terminators to carry out.In another embodiment, order-checking is the extensive parallel order-checking using extensive parallel connection method order-checking to perform.
In some embodiments, the chromosomal aneuploidy that presence or absence at least two is different can be determined or verify to the method.In one embodiment, the method is by determining at least two interested karyomit(e) repeating step (a)-(c) fetal chromosomal aneuploidy that presence or absence at least two kinds is different, wherein these steps comprise (a) and obtain sequence information for fetus in maternal sample and maternal nucleic acids, to identify for a number of an interested chromosomal multiple sequence label and a number at least two chromosomal multiple sequence labels of normalization method; B () uses the number of sequence label to calculate for interested chromosomal one first and second normalized value; And (c) will compare for interested chromosomal first normalized value and a first threshold and will compare for interested chromosomal second normalized value and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in the sample to which.First and second threshold values can be identical, or they can be different.In the step (c) of this method, show that presence or absence is for described interested chromosomal a kind of dysploidy for described interested chromosomal first normalized value with comparing of threshold value, and for the comparatively validate presence or absence of described interested chromosomal second normalized value and threshold value for the determination of interested chromosomal a kind of dysploidy.In some embodiments, first normalized value is a first chromosome dosage, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and the second normalized value is a second karyomit(e) dosage, it is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as normalized karyomit(e) value (NCV) as described herein.
Alternately, the method is by determining at least two interested karyomit(e) repeating step (a)-(c) fetal chromosomal aneuploidy that presence or absence at least two kinds is different, wherein these steps comprise (a) and obtain sequence information for fetus and maternal nucleic acids in the sample to which, to identify for a number of the sequence label of an interested chromosomal multiple mapping and a number at least two chromosomal multiple sequence labels of normalization method; (b) use for interested chromosomal label number and determine for interested chromosomal first normalized value for the number of a chromosomal label of the first normalization method, and use for the chromosomal sequence label of the first normalization method number and determine for chromosomal second normalized value of the first normalization method for the number of a chromosomal sequence label of the second normalization method; And (c) will compare with a first threshold for interested chromosomal first normalized value and will compare with a Second Threshold for chromosomal second normalized value of the first normalization method, to determine a kind of fetus dysploidy of presence or absence in the sample to which.First and second threshold values can be identical, or they can be different.In the step (c) of this method, show that presence or absence is for described interested chromosomal a kind of dysploidy for described interested chromosomal first normalized value with comparing of threshold value, and for the comparatively validate presence or absence of chromosomal second normalized value of described first normalization method and threshold value for the determination of interested chromosomal a kind of dysploidy.In some embodiments, first normalized value is a first chromosome dosage, it is the number for described interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, light and the second normalized value is a second karyomit(e) dosage, and it is the number for the chromosomal sequence label of the first normalization method and the ratio for the number of a chromosomal sequence label of the second normalization method.Optionally, the first and second normalized values can be expressed as normalized karyomit(e) value (NCV) as described herein.
In these embodiments, the method can be repeated to determine a kind of fetal chromosomal aneuploidy of presence or absence for all karyomit(e).
Confirmable example that is a kind of or at least two kinds of different chromosomal aneuploidies comprises T21, T13, T18, T2, T9 and X monosomy.In some embodiments, maternal sample obtains from a pregnant woman.In some embodiments, maternal sample is a kind of biological fluid sample, such as a blood sample or the blood plasma fractions that obtains from blood sample.In some embodiments, maternal sample is a plasma sample.In some embodiments, the nucleic acid in maternal sample is cfDNA molecule.
The example of fetal chromosomal aneuploidy includes but not limited to complete Trisomy or monosomy or partial trisomy or monosomy.The example of complete fetal trisomic comprises trisomy 21 (T21; Mongolism), 18 trisomy (T18; Edward's syndrome (Edward ' s Syndrome)), trisomy 16 (T16), 22 trisomy (T22; Cat's eye syndrome (Cat Eye Syndrome)), 15 trisomys (T15), 13 trisomy (T13; Handkerchief tower syndrome (Patau Syndrome)), 8 trisomy (T8; Wa Keni syndrome (Warkany Syndrome)), 9 trisomys (T9), 2 trisomys and XXY (Klinefelter syndrome (Kleinefelter Syndrome)), XYY or XXX trisomy.The example of partial trisomy comprises 1q32-44, has the 9p trisomy of trisomy, 4 trisomy mosaics, 17p trisomy, part 4q26-qter trisomy, 9 trisomys, part 2p trisomy, part 1q trisomy and/or part 6p trisomy/6q monosomy.The example of fetus monosomy comprises chromosome x monosomy; And the partial monoploidy of karyomit(e) 13, karyomit(e) 15, karyomit(e) 16, karyomit(e) 18, karyomit(e) 21 and chromosome 22, these monosomy are known relevant with abortion.The chromosomal partial monoploidy of the dysploidy typically related to can also be determined by method of the present invention.Monosomy 18p is rare chromosomal disorders, wherein all or part of galianconism (p) (monosomic) of deletion 18.This disease is typically characterised in that of short and small stature, the mental retardation of variable degrees, language retardation, the deformity in skull and face (cranium face) region, and/or extra body abnormality.For different case, relevant craniofacial defect can alter a great deal in scope and seriousness.The patient's condition caused by the change in the structure of karyomit(e) 15 and number comprises peace lattice Mann syndrome and Pu Ruide-Willie Cotard, and they relate to the loss of the gene activity in the same part (15q11-q13 region) of karyomit(e) 15.Should be appreciated that in father and mother carrier, some transpositions and micro-deleted can be asymptomatic, but still the central genetic disease in offspring can be caused.Such as, carry the micro-deleted healthy mother of 15q11-q13 and can bear the child suffering from peace lattice Mann syndrome (a kind of serious neurodegenerative disease).Therefore, the present invention may be used for identifying this type of excalation in fetus.Partial monoploidy 13q is a kind of rare chromosomal disorders, when it occurs in one section of karyomit(e) 13 long-armed (q) disappearance (monomer).The baby suffering from partial monoploidy 13q during birth can show low birthweight, the deformity of head and face (craniofacial area), skeletal abnormality (especially hand and pin), and other body abnormalities.Mental retardation is the feature of this patient's condition.Suffer from the individuality of this disease in birth, the mortality ratio between infancy is very high.The case of nearly all partial monoploidy 13q does not all have obvious cause and occurs at random (sporadic).22q11.2 deletion syndrome, also referred to as DiGeorge syndrome, is the syndrome caused by the disappearance of a bit of chromosome 22.Disappearance (22q11.2) occur in this to one of karyomit(e) long-armed on karyomit(e) near middle.This syndromic feature even also can change very wide in the member of same family, and affects a lot of parts of health.Characteristic sign and symptom can comprise inborn defect, as congenital heart disease, and the defect of jaw, commonly the most relevant with closedown neuromuscular problem (velopharyngeal insufficiency), learning disorder, the Light Difference in facial characteristics, and recurrent infection.Micro-deleted in chromosomal region 22q11.2 is associated with the risk of the increase of schizoid 20 to 30 times.In one embodiment, method of the present invention is used to determining section monosomy, including, but not limited to: monosomy 18p, the partial monoploidy (15q11-q13) of karyomit(e) 15, partial monoploidy 13q, and the partial monoploidy that can use method determination chromosome 22 of the present invention.
1. in some embodiments, chromosomal aneuploidy is the chromosomal aneuploidy completely occurred with chimeric state.For example, in some embodiments, chromosomal aneuploidy is the dysploidy existed with real chromosomal mosaic form, and wherein fetal cell can comprise two kinds of different caryogram.In other embodiments, chromosomal aneuploidy is relevant with the mosaic being mainly limited to placenta tissue.The placenta mosaic (CPM) of being limited to represent the karyomit(e) of cell in cell and baby in placenta form between difference.When finding CPM, it represents the trisomy cell line in placenta and the normally diploid karyomit(e) complement in baby the most commonly.But the case of about 10% is relevant with fetus.Think that the abnormal cells of the suitable high number existed in placenta disturbs normal placental function.Impaired placenta cannot support gestation, and this may cause losing the normal baby of karyomit(e) (Tyson (Tyson) and Ka Laosaike (Kalousek), 1992).For many euchromosome trisomys, chimeric case is only had just to survive to mature.For example, 2 complete trisomys obviously impel the abortion of first three months, occur in the gestation of generally acknowledge clinically 0.16%.2 trisomys seem only to have under chimeric state and when trisomy is mainly limited in placenta tissue just compatible with life.Although authenticated the case of the antenatal 2 trisomy mosaics determined of certain number, 2 chimeric trisomys have presented one of more difficult consulting situation.Result from normal to neonatal death not etc.Oligohydramnios (low amniotic fluid) and bad endometrial growth are modal features.Abnormal the possibility of result mainly in placenta trisomic level high and the result of low-level trisomy may be there is in baby self.Some uncommon trisomys (such as 9 trisomys) chimeric or occur under non-chimeric state, and can present different clinical manifestations.Find that when chorionic villus is sampled mosaic 9 trisomy presents a kind of consulting situation of difficulty.After diagnosing out 9 trisomys during CVS, should get rid of the trisomy in fetus with amniocentesis and a series of ultrasonic wave, this trisomy causes the symptom comprising skull, neural system abnormity and mental retardation.Also may there is the abnormity of heart, kidney and musculoskeletal system.When CVS but not amniocentesis time find trisomy most of cases in, result is normal.But, also may there is abnormal results.Although authenticated the case of the antenatal 9 trisomy mosaics determined of certain number, result from normal to neonatal death not etc.Some trisomys are rare and fatal, and other trisomys can be survived when being confined to placenta cells.In the latter cases, can get rid of this trisomy with other test (such as amniocentesis) after determining trisomy is fetal trisomic.
2. the method is also applicable to determine any chromosome abnormalty when one of parents are the known carrier of this exception.These including, but not limited to: chromosomal chimeric for little additional markers thing; T (11; 14) (p15; P13) transposition; Unbalanced transposition t (8; 11) (p23.2; P15.5); 11q23 is micro-deleted; The lucky syndrome 17p11.2 disappearance of Smith-Ma; 22q13.3 lacks; Xp22.3 is micro-deleted; 10p14 lacks; 20p is micro-deleted; DiGeorge syndrome [del (22) (q11.2q11.23)]; WILLIAMS-DARLING Ton syndrome (7q11.23 and 7q36 disappearance); 1p36 lacks; 2p is micro-deleted; Neurofibroma Class1 (17q11.2 is micro-deleted), Yq lacks; Wolf-Hirschhorn syndrome (WHS, 4p16.3 are micro-deleted); 1p36.2 micro-deleted; 11q14 lacks; 19q13.2 is micro-deleted; Rubinstein-Taybi syndrome (16p13.3 is micro-deleted); 7p21 is micro-deleted; Miller-Di Ke syndrome (17p13.3), 17p11.2 lacks; And 2q37 is micro-deleted.
The method can also with for determining that the analysis of the antenatal symptom that other and mother and/or fetus are relevant is combined.The method is also applicable to copy number variation (CNV) determining any interested sequence in multiple sample, these samples comprise the mixture of the genomic nucleic acids deriving from least two different genes groups, and known or suspect that these two different genes groups are different in the amount of one or more interested sequence.In some embodiments, the method may be used for determining a kind of chromosomal aneuploidy (see example 1) of presence or absence in twin fetus gestation.In hetero-ovular twins gestation, the method can to determine in twin pregnancy a kind of chromosomal aneuploidy of presence or absence, and determines that one or two twin fetuses carry dysploidy by setting up for the respective fetus mark of twins and being compared by its fetus mark relevant with dysploidy.Can by the first and second fetus marks determined accordingly for the first and second twin fetuses that check order to polymorphic sequence (SNP in such as Maternal plasma cfDNA).Each fetus mark can calculate as the main allelotrope part contributed by mother and the ratio of the secondary equipotential Gene Partial to be contributed by fetus.For determining that the method for fetus mark in Maternal plasma cfDNA is described in the following: unsettled U.S. Patent application 12/958,347 (name is called " for determining the method (Methods for Determining Fraction of Fetal Nucleic Acids inMaternal Samples) of the mark of fetal nucleic acid in maternal sample "), 12/958,356 (name is called " determining dysploidy and fetus mark (Simultaneous determination of Aneuploidy and Fetal Fraction) " simultaneously) (two are all filed on December 1st, 2010), and 13/009,718 (name is called " being identified in the polymorphic sequence (Identification of polymorphic sequences inmixtures of genomic DNA by whole genome sequencing) in the mixture of genomic dna by genome sequencing ", be filed on January 19th, 2011), these patents are combined in this in full with it all by reference.Because hetero-ovular twins will be at least different at some SNP site places, therefore two independently fetus marks (first and second) can be determined.The known NCV for karyomit(e) 21 of sample for having twin pregnancy, the fetus mark be then associated with dysploidy can be estimated as the difference percentage between the karyomit(e) dosage for aneuploid twins sample and the mean value of karyomit(e) 21 dosage in the qualified samples of training set, i.e. NCV karyomit(e) 21 dosage of the NCV of NCV karyomit(e) 21 dosage in qualified samples average karyomit(e) 21 dosage in the test sample/in the test sample.Be associated with dysploidy and the mark using the NCV for karyomit(e) 21 to calculate one first or the second fetus mark will determining corresponding to the difference be used in SNP sequence, identify it is that one or two twin fetuses carry dysploidy thus.
Except the suitability that the method is used for determining the chromosomal aneuploidy showing hereditary conditions in fetus, can determine to apply the method presence or absence show disease (such as cancer) and/or morbid state chromosome abnormalty, determine presence or absence pathogenic agent (such as virus) nucleic acid, determine the chromosome abnormalty relevant with graft versus host disease (GVHD) and determine individual contribution in forensic analysis.
CNV in human genome obviously affects mankind's diversity and vulnerability (strangles the people such as east (Redon), nature (Nature) 23:444-454 [2006]; Human genome research (Genome Res) 19:1682-1690 [2009] such as Sha Yihe (Shaikh)).Known CNV facilitates genetic diseases by different mechanisms, causes gene dosage imbalance or gene disruption in most of case.Except CNV is directly related with genetic block, known they mediated the character mutation that may be harmful to.Recently, several research has been reported, compared with normal control, in complicated illness (as autism, ADHD and schizophrenia), the load of the CNV of rare or new life increases, highlight potential pathogenicity bo (people such as Sai Bate (Sebat), the 316:445-449 [2007] of rare or unique CNV; The people such as Walsh (Walsh), science (Science) 320:539-543 [2008]).CNV is produced by genome rearrangement, mainly owing to lacking, copying, insert and unbalanced translocation events.
Multiple embodiment of the present invention provides a kind of method, for evaluating the copy number variation of the interested sequence (such as relevant clinically sequence) in a test sample, this test sample comprises the mixture of the nucleic acid derived from two different genes groups, and these nucleic acid known or under a cloud be different in the amount of one or more interested sequence.The mixture of nucleic acid is the cell derived from two or more types.In one embodiment, this nucleic acid mixture is the cell derived from normal and cancer, these cell-derived experimenters from suffering from a kind of medical condition (such as cancer).
It is believed that a lot of solid tumor, as breast cancer, proceeding to transfer by the accumulation of some genetic aberrations from opening the beginning.[help the people such as rattan (Sato), cancer research (Cancer Res.), 50:7184-7189 [1990]; The people such as Jones's agate (Jongsma), clinical pathology magazine (J Clin Pathol): molecular pathology (Mol Path) 55:305-309 [2002])].This type of genetic aberrations can cause proliferation advantage, genetic instability and adjoint drilling rapidly to bear drug-fast ability along with their are accumulated, and the vasculogenesis strengthened, proteolysis and metabolism.These genetic aberrations can or affect recessive " tumor suppressor gene " or affect active oncogene.The tumor suppression allelotrope causing the disappearance of loss of heterozygosity，LOH (LOH) and restructuring to be considered to by exposing sudden change plays Main Function in tumour progression.
Suffer from the circulation of the patient of malignant tumour in diagnosis and had been found that cfDNA, these malignant tumours are including, but not limited to the lung cancer (people such as Pa Sake (Pathak), clinical chemistry (Clin Chem), 52:1833-1842 [2006]), prostate cancer (is permitted the people such as watt minister Bach (Schwartzenbach), Clinical Cancer Research (ClinCancer Res), 15:1032-8 [2009]), and breast cancer (is permitted the people such as watt minister Bach (Schwartzenbach), can obtain online at breast-cancer-research.com/content/11/5/R71, [2009]).In the circulation cfDNA of cancer patients, the identification of the confirmable genomic instability relevant to cancer is potential diagnosis and forecasting tool.In one embodiment, method of the present invention has evaluated the CNV of interested sequence in the sample to which, this sample comprises the mixture of the nucleic acid derived from an experimenter, known or suspect that this experimenter suffers from cancer, such as cancer, sarcoma, lymphoma, leukemia, gonioma and blastoma.In one embodiment, this sample is derivative (processing) plasma sample from peripheral blood, and it comprises the mixture of the cfDNA of the cell derived from normal and cancer.In another embodiment, the biological sample determining whether there is CNV is needed to be mixture derived from cancer and non-cancerous cells, these cells are from other biological fluid, these biological fluids are including, but not limited to serum, sweat, tears, phlegm, urine, phlegm, ear effluent, lymph liquid, saliva, cerebrospinal fluid, irrigating solution (ravages), bone marrow floater liquid, vaginal discharge, through the irrigating solution of uterine neck, brain liquid, ascites, milk, breathe, the secretory product of intestines and genitourinary tract, and leukapheresis sample, or at biopsy, cotton swab, or in smear.
Interested sequence is a kind of nucleotide sequence, known or suspect this sequence the development of cancer and/or progress in work.The example of interested sequence comprises nucleotide sequence, and as described below, these sequences are amplified or delete in cancer cells.
The dominant acting gene be associated with human entity knurl typically via the expression of process LAN or change to play their effect.Gene amplification is a kind of common mechanism causing genetic expression to be raised.Evidence from cytogenetical study shows, in the people's breast cancer more than 50%, there occurs remarkable amplification.It should be noted that most, the amplification being positioned at the proto-oncogene human epidermal growth factor receptor 2 (HER2) on karyomit(e) 17 causes the process LAN of the HER2 acceptor on cell surface, thus causing excessive in breast cancer and other malignant tumours and the signal of the dysregulation (people such as Piao (Park), clinical breast cancer (Clinical Breast Cancer), 8:392-401 [2008]).In other human malignancies, had been found that multiple oncogene is amplified.In human tumor, the example of cellular oncogene amplification comprises the amplification of the following: promyelocytic leukemia clone HL60, and the c-myc in small cell lung cancer, former neuroblastoma (stage III and IV), neuroblastoma cell line, Retinoblastoma Cells system and primary tumo(u)r, and the N-myc in small cell lung cancer cell system and tumour, L-myc in small cell lung cancer cell system and tumour, c-myb in acute myelocytic leukemia and in colon carcinoma cell line, epidermoid carcinoma cell, and the former c-erbb gone crazy in glioma, lung, colon, bladder, and the c-K-ras-2 in the primary carcinoma of rectum, N-ras (Wa Musi H. (Varmus H.) in breast cancer cell line, genetics yearbook (Ann Rev Genetics), 18:553-612 (1984), [quote people such as fertile gloomy (Watson), the molecular biology (MolecularBiology of the Gene) (the 4th edition of gene, Benjamin/healthy and free from worry publishing company (Benjamin/CummingsPublishing Co.) 1987)].
The chromosome deletion relating to tumor suppressor gene can play a kind of vital role in the development of solid tumor and progress.Retinoblastoma tumor suppressor gene (Rb-1) (being positioned at chromosome 13q14) is the tumor suppressor gene of characterization the most widely.Rb-1 gene product (nuclear phosphoprotein of a kind of 105kDa) obviously plays an important role in cell cycle regulating, and (person of outstanding talent is according to people such as (Howe), institute of NAS periodical (Proc Natl Acad Sci) (U.S.), 87:5883-5887 [1990]).By by a point mutation also or the allelic inactivation of these two genes of chromosome deletion cause the change of Rb albumen or lose expression.Have been found that Rb-i gene alteration does not exist only in retinoblastoma, but also be present in other malignant tumours, as osteosarcoma, the small cell lung cancer (people such as Rui Gede (Rygaard), cancer research (Cancer Res), 50:5312-5317 [1990)]) and breast cancer.Restriction fragment length polymorphism (RFLP) research shows, this type of tumor type lost heterozygosity through the 13q that is everlasting, this prompting is due to total chromosome deletion, one of allelotrope of Rb-1 gene the is lost (people such as Bai Kaoke (Bowcock), American Journal of Human Genetics (Am J Hum Genet), 46:12 [1990]).Comprise and relate to karyomit(e) 6 and other are with x linkedly copying, lacking and the abnormal region showing karyomit(e) 1 of karyomit(e) 1 of unbalanced translocation, particularly q21-1q32 and 1p11-13, may hold fall ill with the chronic of myeloproliferative tumour and advanced stage goes up relevant oncogene or the tumor suppressor gene (people such as OK a karaoke club horse Sa (Caramazza), Europe hematology magazine (Eur J Hematol), 84:191-200 [2010]).Myeloproliferative tumour is also associated with the disappearance of karyomit(e) 5.The complete loss of karyomit(e) 5 or intercalary deletion are modal chromosome abnormalities in myelodysplastic syndrome (MDS).Del (the 5q)/5q-MDS patient be separated has the prognosis more favourable than those patients suffering from extra caryogram defect, and they tend to development myeloproliferative tumour (MPN) and acute myelocytic leukemia.The frequency that unbalanced karyomit(e) 5 lacks has drawn an idea, that is: 5q holds one or more tumor suppressor gene, and these genes play basic effect in the growth control of hemopoietic stem cell/hemopoietic progenitor cell (HSCsHPC).The cytogenetics in the region (CDR) of usual disappearance maps the candidate tumor suppressor gene concentrating on 5q31 and 5q32 and identify, comprise ribosomal subunit RPS14, transcription factor Egr1/Krox20 and cytoskeleton remodeling proteins, α-Lian albumen (Ai Siman (Eisenmann), oncogene (Oncogene), 28:3429-3441 [2009]).Cytogenetics and the allelotype research of fresh and tumor cell line are verified, from the allelic loss in the some clear and definite region (comprising 3p25,3p21-22,3p21.3,3p12-13 and 3p14) on chromosome 3p be in the main epithelial cancer of the wide spectrum of cancer at lung cancer, breast cancer, kidney, head and neck cancer, ovarian cancer, cervical cancer, colorectal carcinoma, carcinoma of the pancreas, esophagus cancer, bladder cancer and other organs involved the earliest with modal genomic abnormality.Some tumor suppressor genes have been mapped to chromosome 3p region, and think that intercalary deletion or promotor high methylation are prior at the developing 3p of cancer or loss ((the An Geluoni D. (Angeloni D.) of complete chromosome 3, functional genomics bulletin (Briefings FunctionalGenomics), 6:19-39 [2007]).
The newborn infant and the children that suffer from mongolism (DS) usually present inborn symptomatic leukemia and have the risk of the increase of acute myelocytic leukemia and Acute Lymphoblastic Leukemia.Karyomit(e) 21 (holding about 300 genes) can involve various structures distortion, such as, transposition in leukemia, lymphoma and solid tumor, disappearance and amplification.In addition, identified be arranged in gene on karyomit(e) 21 tumour occur the vital role that rises.The isostructural distortion of company of the number of entities of karyomit(e) 21 is associated with leukemia, and specific gene comprises RUNX1, TMPRSS2 and TFF, they are positioned at 21q, work (Feng Nacike C (Fonatsch C) gene, karyomit(e) and cancer C in tumour occurs, (GeneChromosomes Cancer), 49:497-508 [2010]).
In one embodiment, means are this method provided to the cognation between the degree evaluated gene amplification and tumour and develop.Amplification and/or disappearance and the association between carcinoma stage or grade can be important for prognosis, because this type of information can form the definition of hereditary tumor grade, this prediction can have the following course of disease of the more late tumor of the worst prognosis better.In addition, the information about earlier amplifications and/or deletion events can be useful when these events being associated in the predictive factors of progression of disease subsequently.Can by by the gene amplification of present method identification and disappearance and other known parameters (as tumor grade, medical history, Brd/Urd marker index, Hormonal States, nodus lymphoideus transferring rate, tumor size, survival time with from epidemiology and biostatistics research other tumor characteristics obtainable) associate.Such as, need can comprise atypical hyperplasia, the carcinoma in situ of conduit, the cancer of stage I-III and lymphnode metastatic by the Tumour DNA that present method carries out testing, to allow to be identified in amplification and the cognation between disappearance and stage.The association made can make effective therapeutic intervention become possibility.Such as, the gene of a process LAN can be contained in the region of consistent amplification, and perhaps its product can accept therapeutic attachment (such as, growth factor receptor tyrosine kinase p185 hER2).
By determining that the method may be used for identifying the amplification relevant to resistance and/or deletion events from primary cancer to the copy number variation of those nucleic acid of cell transferring to other positions.If gene amplification and/or disappearance are the one performances of the karyotype instability allowing resistance to develop rapidly, so compared with the tumour of the patient from chemosensitivity, the more amplifications in the primary tumo(u)r of the patient from chemoresistant and/or disappearance will be expected.Such as, if the amplification of specific gene causes drug-fast development, so will expect to obtain consistent amplification around the region of those genes in the tumour cell of the patient from chemoresistant instead of in primary tumo(u)r.Can allow to identify the patient that or can not benefit from adjuvant therapy in the discovery of gene amplification and/or the cognation between disappearance and development of drug resistance.
For determining device and the system of CNV
The analysis of sequencing data and the determination drawn thus typically use different computer hardwares, computerized algorithm and computer program to perform.Therefore, the inventive method computer-implemented or computer assisted method typically.
In one embodiment, the invention provides a kind of computer program for generation of an output, this output shows a kind of fetus dysploidy of presence or absence in a test sample.This computer product comprises a computer-readable medium, this medium has a kind of record executable logic of computer thereon, presence or absence fetus dysploidy is determined for enabling treater, this logic comprises: a kind of reception program, for receiving the sequencing data of the nucleic acid molecule at least partially from maternal biological sample, wherein said sequencing data comprises sequence reads; The logic of computer-aided, for analyzing the fetus dysploidy of the data from described reception; And a kind of written-out program, for generation of exporting with the existence showing described fetus dysploidy, not existing or kind.Use the computer-readable medium with stored thereon computer-readable instruction can to perform the methods of the present invention, to carry out a kind of method for identifying any CNV (dysploidy of such as chromosomal or part).In one embodiment, the invention provides a kind of computer-readable medium, this medium has storage computer-readable instruction thereon and suspects at least one karyomit(e) relevant with a kind of chromosomal aneuploidy (such as trisomy 21,13 trisomys, 18 trisomys or X monosomy) for identifying.
In one embodiment, the invention provides a kind of computer-readable medium, this medium has storage computer-readable instruction thereon for performing the method comprised the following steps: (a) uses the sequence information obtained from fetus in the sample to which and maternal nucleic acids to identify for a number of an interested chromosomal multiple sequence label and a number at least two chromosomal multiple sequence labels of normalization method; B () uses the number of sequence label to calculate for interested chromosomal first normalized value and second normalized value; And (c) will compare with a first threshold for interested chromosomal first normalized value and will compare with a Second Threshold for interested chromosomal second normalized value, to determine a kind of fetus dysploidy of presence or absence in the sample to which.Computer-readable medium can have the storage computer-readable instruction for performing a kind of method thereon, a first chromosome dosage for interested chromosomal first normalized value in the method, this the first chromosome dosage is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and be a second karyomit(e) dosage for interested chromosomal second normalized value in the method, this the second karyomit(e) dosage is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the second normalization method.
In one embodiment, the invention provides a kind of computer-readable medium, this medium has storage computer-readable instruction thereon for performing the method comprised the following steps: (a) uses the sequence information obtained from fetus in the sample to which and maternal nucleic acids to identify for a number of an interested chromosomal multiple sequence label and a number at least two chromosomal multiple sequence labels of normalization method; (b) use for interested chromosomal sequence label number and determine for interested chromosomal first normalized value for the number of a chromosomal sequence label of the first normalization method, and use for the chromosomal sequence label of the first normalization method number and determine for chromosomal second normalized value of the first normalization method for the number of a chromosomal sequence label of the second normalization method; C () will compare with a first threshold for interested chromosomal first normalized value and will compare with a Second Threshold for chromosomal second normalized value of the first normalization method, to determine a kind of fetus dysploidy of presence or absence in the sample to which.Computer-readable medium can have the storage computer-readable instruction for performing a kind of method thereon, a first chromosome dosage for interested chromosomal first normalized value in the method, this the first chromosome dosage is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and be a second karyomit(e) dosage for interested chromosomal second normalized value in the method, this the second karyomit(e) dosage is the number for the chromosomal sequence label of the first normalization method and the ratio for the number of a chromosomal sequence label of the second normalization method.
In one embodiment, the invention provides a kind of computer processing system, it is adjusted or is configured and is performed according to the inventive method.For example, the invention provides a kind of computer processing system, it is adapted and configures to perform the method comprised the following steps: (a) uses and identify for a number of an interested chromosomal multiple sequence label and a number at least two chromosomal multiple sequence labels of normalization method from the sequence information that fetus and maternal nucleic acids obtain in the sample to which; B () uses the number of sequence label to calculate for interested chromosomal first normalized value and second normalized value; And (c) will compare with a first threshold for interested chromosomal first normalized value and will compare with a Second Threshold for interested chromosomal second normalized value, to determine a kind of fetus dysploidy of presence or absence in the sample to which.Computer processing system can be adapted and configure to perform a kind of method, a first chromosome dosage for interested chromosomal first normalized value in the method, this the first chromosome dosage is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and be a second karyomit(e) dosage for interested chromosomal second normalized value in the method, this the second karyomit(e) dosage is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the second normalization method.
In one embodiment, the invention provides a kind of computer processing system, it is adapted and configures to perform the method comprised the following steps: (a) uses the sequence information obtained from fetus in the sample to which and maternal nucleic acids to identify for a number of interested chromosomal multiple sequence label and a number at least two chromosomal multiple sequence labels of normalization method; (b) use for interested chromosomal sequence label number and determine for interested chromosomal first normalized value for the number of a chromosomal sequence label of the first normalization method, and use for the chromosomal sequence label of the first normalization method number and determine for chromosomal second normalized value of the first normalization method for the number of a chromosomal sequence label of the second normalization method; C () will compare with a first threshold for interested chromosomal first normalized value and will compare with a Second Threshold for chromosomal second normalized value of the first normalization method, to determine a kind of fetus dysploidy of presence or absence in the sample to which.Computer processing system can be adjusted and be configured to perform a kind of method, a first chromosome dosage for interested chromosomal first normalized value in the method, this the first chromosome dosage is the number for interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and be a second karyomit(e) dosage for interested chromosomal second normalized value in the method, this the second karyomit(e) dosage is the number for the chromosomal sequence label of the first normalization method and the ratio for the number of a chromosomal sequence label of the second normalization method.
Present invention also offers and be adapted or configure to perform the device according to method of the present invention, wherein this device optionally comprises the order-checking device being adapted or configuring and check order to fetus in the sample to which and maternal nucleic acids molecule.For example,: (a) checks order device to the invention provides the device comprising the following, it is adapted or is disposed for use sequence measurement as described herein to check order to fetus in the sample to which and maternal nucleic acids molecule, thus formation sequence information; And (b) computer processing system, it is adapted or is disposed for be used in the sequence information by order-checking device generation in method as described herein, wherein this computer processing system is optionally directly connected to order-checking device, make like this sequence information can automatically from order-checking device transmission to computer processing system.This device may further include transferring device, and it is adapted or configures transfers to order-checking device for order-checking by sample.
To illustrate in greater detail the present invention in the following example, these examples limit scope of the present invention as requested unintentionally by any way.Accompanying drawing is the integral part being intended to be considered to specification sheets of the present invention and explanation.Provide following instance to be described, and and unrestricted required invention.
Example
Example 1
The best to the extensive parallel DNA sequencing of the acellular foetal DNA from maternal blood carries out fetal chromosomal abnormalities is used to determine: independent of the test set 1 of training set 1
This research is carried out according to the human experimenter's scientific experimentation plan got permission by the Institutional Review Board (IRB) of each mechanism between in April, 2009 and in October, 2010 in 13 U.S. clinical areas by qualified fixed point clinical research personnel.Written consent book was obtained from every experimenter before participation research.This scientific experimentation plan is designed to provide blood sample and clinical data to support the development of non-invasive PGD method.18 years old or the age larger qualified participation of gravid woman.The patient pierced through for chorionic villi sampling (CVS) or the amnion of experience clinical indication collected blood before carrying out this program, and same result of collecting fetal karyotype.Extract peripheral blood sample (two pipes or altogether about 20mL) from all experimenters and be placed in acid citrate dextrose (ACD) pipe (Becton Dickinson Co., Ltd (Becton Dickinson)).All samples is all removed identity and specifies an anonymous patient No. ID.Blood sample is transported to laboratory in the temperature control type conveying containers provided for institute all through the night.Blood drawing and the time being subject to spending between sample are registered as the part that sample is ascended the throne.
Case study coordination personnel uses anonymous patient No. ID Pregnancy current with patient and history-sensitive clinical data typing to be studied in case report form (CRF).Sample from non-invasive antenatal program carried out to the cytogenetic of fetal karyotype in each laboratory and result be recorded in equally in research CRF.In the clinical database in all data that CRF obtains all typing laboratory.After the venipuncture sampling of 24 to 48 hours, utilize two step centrifuging to obtain acellular blood plasma from independent blood tube.Blood plasma from single blood tube enough carries out sequencing analysis.Cell-free DNA is extracted from cell-free plasma according to the explanation of manufacturers by using QIAamp DNA Blood Mini kit (Kai Jie company) (QIAamp DNA Blood Mini kit (Qiagen)).Because these acellular DNA fragmentations known are about 170 base pairs (bp) (Fan et al., Clin Chem 56:1279-1286 [2010]) in length, cracked without the need to making DNA before order-checking.
For the sample of this training group, cfDNA is delivered to Prognosys Biosciences, Inc. (LaJolla, CA) uses standard to manufacture business test plan Illumina Genome Analyzer IIx instrument (http://www.illumina.com/) to check order for sequencing library preparation (blunting and the cfDNA be connected on common aptamer).Obtain the single-ended reading of 36 base pairs.After completing order-checking, collect all bases and judge file and analyze.For test group sample, prepare sequencing library and check order on Illumina Genome Analyzer IIx instrument.Being prepared as follows of sequencing library is carried out.Illustrated total length scientific experimentation plan is the standard science test plan that provides of Illumina mainly, and only different from Illumina scientific experimentation plan on the purifying in the library of amplification.Illumina scientific experimentation plan indicates: the library of amplification uses gel electrophoresis to carry out purifying, and scientific experimentation plan described herein uses magnetic bead to carry out identical purification step.Use the cfDNA of the about 2ng purifying extracted from Maternal plasma to prepare an elementary sequencing library, this mainly uses nEBNext tMdNA sample prepares DNA reagent collection 1 (NEBNext tMdNA Sample Prep DNA Reagent Set 1) (Item Number: E6000L; New England Biolabs, Ipswich, MA) carry out according to the explanation of manufacturers.Except using Agencourt magnetic bead to replace purification column to carry out except final purifying to the product that aptamer is connected with reagent, be all according to the adjoint NEBNext for the sample preparation of genome dna library of scientific experimentation plan in steps tMreagent (uses gAII checks order) carry out.NEBNext tMstipulations have followed the stipulations that Illumina provides in essence, and this can obtain at grcf.jhml.edu/hts/protocols/11257047_ChIP_Sample_Prep.pd f place.
By the overhang of the cfDNA fragment of about 2ng purifying that comprises in 40 μ l by cfDNA being used in NEBNext in 1.5ml Eppendorf tube tMdNA sample prepares DNA reagent collection 1 (NEBNext tMdNA Sample Prep DNA Reagent Set 1) in provide the damping fluid of the phosphorylation of 5 μ l 10X, 2 μ l deoxynucleotide solution mixtures (every part of dNTP has 10mM), 1 μ l 1: 5 the diluent of DNA polymerase i, 1 μ l T4 archaeal dna polymerase and 1 μ l T4 polynucleotide kinase at 20 DEG C, hatch 15 minutes, according to end is repaired module and changes into the blunt end of phosphorylation.This sample is cooled to 4 DEG C, and use one at QIAQuick PCR purification kit (QIAQuick PCR Purification Kit) (markon welfare Ya Zhou Valencia Kai Jie company (QIAGENInc., Valenicia, CA)) in the quick post of QIA that provides carry out purifying.50 μ l reaction solutions are transferred in 1.5ml centrifuge tube, and adds the Qiagen Buffer PB of 250 μ l.The 300 μ l obtained are transferred in a quick post of QIA, by its in an Eppendorf centrifuge under 13,000RPM centrifugal 1 minute.The Qiagen Buffer PE of this post with 750 μ l is washed, and centrifugal again.Remaining ethanol by removing under 13,000RPM for centrifugal 5 minutes again.By DNA in the triumphant outstanding buffer reagent EB (QiagenBuffer EB) of 39 μ l by centrifugal come wash-out.Use 16 μ l containing Klenow fragment (3 ' to 5 ' exo minus) (NEBNext tMdNA sample prepares DNA reagent collection 1 (NEBNext tMdNA SamplePrep DNA Reagent Set 1)) the main mixed solution of dA tailing complete the dA tailing of the DNA of 34 μ l blunt ends, and according to manufacturers dA-Tailing Module hatches 15 minutes at 37 DEG C.This sample is cooled to 4 DEG C, and the post provided in MinElute PCR Purification Kit (QIAGEN Inc., Valencia, CA) is provided carries out purifying.50 μ l reaction solutions are transferred in 1.5ml centrifuge tube, and adds the Qiagen Buffer PB of 250 μ l.300 μ l are transferred in a MinElute post, by its in an Eppendorf centrifuge under 13,000RPM centrifugal 1 minute.The Qiagen Buffer PE of this post with 750 μ l is washed, and centrifugal again.Remaining ethanol by removing under 13,000RPM for centrifugal 5 minutes again.By DNA in the Qiagen Buffer EB of 15 μ l by centrifugal come wash-out according to quick Ligation Module, by the DNA elutriant of ten microlitres with 1: 5 of 1 μ l Illumina Genomic Adapter Oligo Mix (Item Number: 1000521) the 2X Quick Ligation Reaction Buffer of diluent, 15 μ l and the quick T4 DNA ligase of 4 μ l hatch 15 minutes at 25 DEG C.Sample is cooled to 4 DEG C, and uses a following MinElute post.150 microlitre Qiagen Buffer PE are added in 30 μ l reaction solutions, and whole volume is transferred in a MinElute post, by its in an Eppendorf centrifuge under 13,000RPM centrifugal 1 minute.The Qiagen Buffer PE of this post with 750 μ l is washed, and centrifugal again.By within centrifugal 5 minutes, removing remaining ethanol under 13,000RPM again.By DNA in the Qiagen Buffer EB of 28 μ l by centrifugal come wash-out.Use Illumina Genomic PCR primer (Item Number: 100537 and 1000538) and at NEBNext tMdNA sample prepares DNA reagent collection 1 (NEBNext tMdNA SamplePrep DNA Reagent Set 1) in the Phusion HF PCR premixed liquid (explanation according to manufacturers) that provides, the DNA elutriant that the aptamer of 23 microlitres is connected stands 18 PCR circulations (at 98 DEG C 30 seconds; 18 circulation continuous 10 seconds at 98 DEG C, at 65 DEG C 30 seconds, and at 72 DEG C 30 seconds; At finally extending in 72 DEG C 5 minutes, and at remaining on 4 DEG C).Use Agencourt AMPure XPPCR purification system (Agencourt Bioscience Corporation, Beverly, MA) according to the explanation (can obtain at www.beckmangenomics.com/products/AMPureXPProtocol_000387 v001.pdf place) of manufacturers, the product of amplification is carried out purifying.Agencourt AMPure XP PCR purification system eliminates unassembled dNTP, primer, primer dimer, salt and other pollutents, and has reclaimed the amplicon being greater than 100bp.By Qiagen EB damping fluid from the Agencourt pearl wash-out of the amplification product after purifying at 40 μ l, and use 2100 Bioanalyzer (Agilent technologies Inc., SantaClara, CA) Agilent DNA 1000 Kit to the distribution of sizes analysing these libraries.
For training and test both sample sets, the single-ended reading of 36 base pairs is checked order.
Data analysis and sample classification
Be that the sequence reads of 36 bases compares (http://hgdownload.cse.ucsc.edu/goldenPath/hg18/bigZips/) with the human genome assembly hg18 obtained from UCSC database by length.Be used in comparison process and allow the short gene fragment comparative device of the Bowtie of maximum two base mispairings (version 0.12.5) (Langmead et al., Genome Biol 10:R25 [2009]) to compare.The clear reading be mapped on a term single gene group position is only had just to be included.Carry out counting and being included in (see following content) in the calculating of karyomit(e) dosage to the genomic locus that reading maps.On Y chromosome from the sequence label of masculinity and femininity fetus without any differentiation map part region be excluded beyond analysis (exactly, from base 0 to base 2x10 6, base 10x10 6to base 13x10 6; And base 23x10 6to the end of Y chromosome.)
Same batch in the karyomit(e) distribution of sequence reads and between round order-checking variation the impact of distribution of fetus dysploidy on the sequence site mapped can be made not obvious.In order to correct this kind of variation, calculate karyomit(e) dosage when being normalized for the counting observed on predetermined normalization method karyomit(e) or one group of normalization method karyomit(e) by the counting in the site for given interested chromosomal mapping.First unaffected sample (i.e. qualified samples) training samples concentrate sample subset in identify normalization method karyomit(e) or normalization method karyomit(e) collection, these samples have the diploid karyotype for interested karyomit(e) 21,18,13 and X, each euchromosome are considered as the potential denominator in interested chromosome counting ratio with us.Select the denominator karyomit(e) (i.e. normalization method karyomit(e)) making in order-checking round and the karyomit(e) ratio checked order between round makes a variation minimum.Each interested karyomit(e) is confirmed as having different denominators (table 1).
Overall number for the sequence label of each interested chromosomal mapping is provided relative to the measuring of variation of the overall number of the sequence label of all the other karyomit(e)s mapping separately for each interested chromosomal karyomit(e) dosage in qualified samples.Therefore, qualified karyomit(e) dosage can identify karyomit(e) or karyomit(e) group, i.e. normalization method karyomit(e), it has closest to the change in the sample of interested chromosomal variation, and will be used as the ideal sequence of normalized value to carry out further statistical evaluation.
In training group (namely qualified and affected) for the karyomit(e) dosage of all samples also as explained below as the basis being used for definite threshold during dysploidy in the test sample identified.
Table 1
For determining the normalization method chromosome sequence of karyomit(e) dosage
For interested karyomit(e) each in each sample of test group, determine a normalized value and be used to determine presence or absence dysploidy.This normalized value calculates as a karyomit(e) dosage, and this karyomit(e) dosage can be calculated to provide a normalized karyomit(e) value (NCV) further.
Karyomit(e) dosage
For test group, for each sample each interested karyomit(e) 21,18,13, X and Y calculate a karyomit(e) dosage.As provided in above table 10, the karyomit(e) dosage of karyomit(e) 21 calculates with the ratio of the number of tags be mapped in the test sample of the karyomit(e) 9 tested in sample as the number of tags in the test sample of the karyomit(e) 21 be mapped in test sample; The karyomit(e) dosage of karyomit(e) 18 calculates with the ratio of the number of tags be mapped in the test sample of the karyomit(e) 8 tested in sample as the number of tags in the test sample of the karyomit(e) 18 be mapped in test sample; The karyomit(e) dosage of karyomit(e) 13 calculates with the ratio of the number of tags be mapped in the test sample of the karyomit(e) 2 to 6 tested in sample as the number of tags in the test sample of the karyomit(e) 13 be mapped in test sample; The karyomit(e) dosage of chromosome x calculates with the ratio of the number of tags be mapped in the test sample of the karyomit(e) 6 tested in sample as the number of tags in the test sample of the chromosome x be mapped in test sample; The karyomit(e) dosage of karyomit(e) Y calculates with the ratio of the number of tags be mapped in the test sample of the karyomit(e) 2 to 6 tested in sample as the number of tags in the test sample of the karyomit(e) Y be mapped in test sample.
Normalized karyomit(e) value
To use in each test sample for each interested chromosomal karyomit(e) dosage and the corresponding karyomit(e) dosage determined in the qualified samples of training group, use the normalized karyomit(e) value (NCV) of following Equation for Calculating:
NCV ij = x ij - μ ^ j σ ^ j
Wherein with the estimation training set average for a jth karyomit(e) ratio and standard deviation accordingly, and x ijfor the viewed jth of a sample i karyomit(e) ratio.When karyomit(e) ratio is normal distribution, NCV equals the statistics z score value for ratio.Do not observe in the fractile-fractile of the NCV from unaffected sample is drawn and significantly the departing from of the linear lag.In addition, the standard testing of the normalizing degree of NCV is failed to veto the null hypothesis of normality.For Ke's Er Monuofu-Vladimir Smirnov (Kolmogrov-Smirnov) and Xia Piluo-Weir gram (Shapiro-Wilk) two inspections, significance value is all greater than 0.05.
For test group, for each sample each interested karyomit(e) 21,18,13, X and Y calculate a NCV.In order to ensure a safe and efficient classification schemes, border conservative for dysploidy categorizing selection.In order to classify to autosomal aneuploid state, NCV > 4.0 is needed to be classified as by karyomit(e) affected (that is, be dysploidy for this karyomit(e)); And karyomit(e) classifies as unaffected by NCV < 2.5.The sample of the NCV that euchromosome has between 2.5 and 4.0 is classified as " judgement ".
In testing, heterosomal classification is by all being undertaken by following content sequential use NCV for X and Y:
1. if NCV Y is apart from mean value >-2.0 standard deviation of male sample, then this sample is classified as the male sex (XY).
If 2. NCV Y is apart from mean value <-2.0 standard deviation of male sample, and mean value >-2.0 standard deviation of NCV X distance women sample, then this sample is classified as women (XX).
If 3. NCV Y is apart from mean value <-2.0 standard deviation of male sample, and mean value <-3.0 standard deviation of NCV X distance women sample, then this sample is classified as monosomy X, i.e. Turner syndrome.
If 4. NCV does not meet any above standard, then this sample is classified as sex is " judgement ".
Result
Research demography
1 is registered altogether, 014 patient between in April, 2009 and in July, 2010.The demographics of patient, invasive Program Type and results of karyotype are summed up in table 2.The mean age of research participant be 35.6 years old (scope was at 17 to 47 years old) and pregnant age scope be 6 weeks 1 day to 38 weeks 1 day (average out to 15 weeks 4 days).The overall incidence of abnormal fetus karyotype is 6.8%, and wherein T21 sickness rate is 2.5%.In 946 experimenters with single pregnancy and caryogram, 906 (96%) presents the clinical generally acknowledged risk factors of at least one for the fetus dysploidy of prenatal course.Even if remove those only have the experimenter of high conceived age as its unique indication, data still illustrate for the very high false positive rate of current examination mode one.By ultrasonic result of ultrasonography of carrying out be: the nuchal translucency of increase, cystic hygroma or other structural birth defect, these are abnormal karyotypes that in this age group, foresight is the strongest.
Table 2
Patient demographics
* comprise the result of the fetus of polycyesis, * * is assessed by clinicist and reports
AMA=pregnant woman advanced age, NT=nuchal translucency
The distribution of the various ethnic background shown in this study population also illustrates in table 2.Generally, in this research, the patient of 63% is Caucasian, and 17% is Spaniard, and 6% is Aisa people, and 5% is multi-ethnic, and 4% is non-descendants American.Notice, the difference of race is changed significantly in different places.Such as, the three unities registers the Spain of 60% and the Caucasia experimenter of 26%, and three that are positioned at same state clinical points do not register Spain experimenter.As expected, in our not agnate result, recognizable difference is not observed.
Training data group 1
This training group research from collect between year December in April, 2009 to 2009, pick 71 samples 435 samples that the initial stage accumulates in succession.All experimenters in the experimenter of this First Series with affected fetus (abnormal karyotype) are included for order-checking, and have a random choose of suitable sample and data and the unaffected experimenter of random number.The Clinical symptoms of training group patient is consistent with the demographics of the overall study shown in table 11.The scope in pregnant age of the sample in training group is the scope from 10 weeks 0 day to 23 weeks 1 day.38 people experienced by CVS, and 32 people experienced by amniocentesis and 1 patient does not have the type (unaffected caryogram 46, XY) of the invasive program of specifying.The patient of 70% is Caucasian, and 8.5% is Spaniard, and 8.5% is Aisa people, and 8.5% is multi-ethnic.In order to the object of training, in this group, eliminate six samples checked order.4 samples are from experimenter's (discussing in detail below) of twin pregnancy, and 1 sample has T18, and this sample is contaminated in preparation process, and 1 sample has fetal karyotype 69, XXX, and remaining 65 samples are this training group.
The 13.7M (improvement due in time passing on sequencing technologies) of the number in unique sequence site (that is, in genome with the label that unique site identifies) from the 2.2M of the commitment of this training group research to later stage and changing.Exceed any potential change of these 6 times of scopes to monitor karyomit(e) ratio in the site of uniqueness, research beginning and at the end of run different, unaffected sample.For the round of front 15 unaffected samples, the average number in unique site is 3.8M and average karyomit(e) ratio for karyomit(e) 21 and karyomit(e) 18 is 0.314 and 0.528 respectively.For the round of rear 15 unaffected samples, the average number in unique site is 10.7M and average karyomit(e) ratio for karyomit(e) 21 and karyomit(e) 18 is 0.316 and 0.529 respectively.Along with the time lapse of training group research between karyomit(e) 21 and the karyomit(e) ratio of karyomit(e) 18, there is no statistically difference.
Fig. 2 illustrates the training group NCV for karyomit(e) 21,18 and 13.Result is consistent with a kind of hypothesis of normalization method degree shown in figure 2, and this hypothesis is: the diploid NCV of about 99% will fall into mean value ± 2.5 standard deviation.In 65 samples in this group, 8 have the NCV scope indicating the sample of the clinical caryogram of T21 to have is from 6 to 20.The NCV scope that the sample that four clinical caryogram had indicate fetus T18 has is from 3.3 to 12, and the NCV that the sample that two clinical caryogram had indicate fetal trisomic 13 (T13) has is 2.6 and 4.In affected sample, the distribution of NCV is because they are to the dependency of the per-cent of the fetus cfDNA in single sample.
Similar with euchromosome, in training group, determine heterosomal mean value and standard deviation.Heterosomal threshold value allows the masculinity and femininity fetus in 100% ground discriminating training group.
Test data set 1
Establish karyomit(e) ratio average and with the standard deviation from average of training group after, from the sample collected from 575 samples altogether between year June in January, 2010 to 2010, have selected a test group of 48 samples.One of them sample from twin pregnancy is removed from final analysis, so remaining 47 samples in test group.It is blind for making for the preparation of the sample of order-checking and the personnel of operating equipment clinical karyotype information.Pregnant age scope with similar (table 2) seen in training group.58% of invasive program is CVS, procedural demographic higher than overall, but also similar with training group.The experimenter of 50% is Caucasian, and 27% is Spaniard, and 10.4% is Aisa people and 6.3% is non-descendants American.
In test group, the number of unique sequence label is different from about 13M to 26M.For unaffected sample, for karyomit(e) 21 and karyomit(e) 18, karyomit(e) ratio is respectively 0.313 and 0.527.For karyomit(e) 21, karyomit(e) 18 and karyomit(e) 13, test group NCV shown in Figure 3 and classification provide in table 12.
Table 3
Test group classification data test group grouped data
* MX is the monosomy of X chromosome, and Y chromosome does not have sign
In test group, 13/13 experimenter with the caryogram being designated as fetus T21 is correctly identified as having the NCV of scope from 5 to 14.Eight/eight experimenters with the caryogram being designated as fetus T18 are correctly identified as having the NCV of scope from 8.5 to 22.In this test group, there is the simple sample classifying as T13 and be classified as wherein NCV and be approximately 3 do not judge.
For test data set, all male sample are correctly identified, and comprise the sample (table 3) with complex karyotype 46, XY+ marker chromosomes (can not be identified by cytogenetics).Have 19 to be correctly validated in 20 women's samples, and women's sample is classified as and does not judge.For three samples that caryogram in test group is 45, X, have two to be correctly validated as monosomy X in three, and 1 is classified as (table 3) that do not judge.
Twins
For having four in the sample that training group is selected at first and having one to be from twin pregnancy in test group.Threshold value may be subject to the puzzlement of the different values of the cfDNA expected in the environment of twin pregnancy as used herein.In training group, the caryogram from one of them twins sample is single chorion 47, XY+21.A second twins sample is different ovum and has carried out separately amniocentesis to each fetus.In this twin pregnancy, one of them fetus has the caryogram of 47, XY+21 and another has a normal caryogram 46, XX.In these two cases, be T21 based on the acellular classification of method discussed above by sample group.Other two twin pregnancies in training group are correctly classified as T21 unaffected (all twins all show the diploid karyotype for karyomit(e) 21).For the twin pregnancy in test group, only caryogram (46, XX) is established to twins B, and this algorithm correctly to be classified as T21 be unaffected.
Conclusion
These data show that extensive parallel sequencing can be used to from the blood of pregnant woman, determine multiple abnormal fetal karyotype.These data show, independently test group data can be used to identify to 100% of the sample with trisomy 21 and trisomy 18 correct classification.Even when having the fetus of abnormal sex chromosome caryogram, the algorithm of the method is utilized not have sample to be sorted out mistakenly.Importantly, this algorithm is being determined to show equally in presence or absence T21 in the group of two twin pregnancies well equally.In addition, this research checked the many continuous print samples from multiple center, not only represent the scope of the abnormal karyotype that people may see in commercial clinical environment, also illustrate the importance gestation do not affected by common trisomy accurately sorted out, to emphasize that the height existed in current Prenatal Screening is to unacceptable false positive rate.These data provide valuable opinion for utilizing the great potential of the method in future.The increase that analysis shows in the consistent Poisson counting statistics value of variance of the subset of unique gene locus.
These data are set up on the basis of the discovery of Fan and Quake, Fan with Quake confirms: use extensive parallel order-checking non-invasively to determine that the sensitivity of fetus dysploidy is only by restriction (Fan and Quake of counting statistics from Maternal plasma, PLos One 5, e10439 [2010]).Because order-checking information gathers throughout whole genome, institute in this way can determine any dysploidy or the variation of other copy numbers, comprises and inserting and disappearance.Caryogram from one of them sample has a little disappearance in karyomit(e) 11 between q21 and q23, when being analyzed in 500k base data box by sequencing data, observe the minimizing of the region interior label relative number about 10% of a 25Mb initial at q21 place.In addition, in training group, three in sample, are had to have complicated property caryogram due to the chimerism in cytogenetic.These caryogram are: i) 47, XXX [9]/45, X [6]; Ii) 45, X [3]/46, XY [17]; And iii) 47, XXX [13]/45, X [7].Show some sample ii containing the cell of XY and correctly classified as XY.By cytogenetic (consistent with mosaic Turner syndrome) all show the sample i (from CVS process) of the mixture of XXX and X cell and iii (from amniocentesis) be classified as respectively do not judge with monosomy X.
When testing this algorithm, for the karyomit(e) 21 of a sample (Fig. 3) from test group, another interesting data point is observed the NCV had between-5 and-6.Although this sample is diploid by cytogenetics on karyomit(e) 21, this caryogram illustrates triploid chimerism: 47, the XX+9 [9]/46, XX [6] with part for karyomit(e) 9.Because karyomit(e) 9 is used in the karyomit(e) dosage (table 1) determining karyomit(e) 21 in denominator, it reduce total NCV value.Result shockingly demonstrates the ability (see example 2) that the method determines fetal trisomic 9 in the case.Determine multiple karyomit(e) ratio to guarantee for interested chromosomal correct classification.In addition, establish for all autosomal normalization method karyomit(e) to improve the possibility (see example 5) that leap genome determines rare dysploidy.
The conclusion of the sensitivity of these methods of relating to persons such as Fan only at sequence measurement can be brought any random of used algorithm or systematic bias is taken into account time be only correct.If this sequencing data is not by suitably normalization method, then the analytical results of gained will be inferior to counting statistics.The people such as Zhao (Chiu) notice in the paper that they are recent, the measuring result of the karyomit(e) 18 and 13 that they use extensive parallel sequence measurement to obtain is coarse, and conclusion needs to carry out more studying determination people such as (, BMJ 342:c7401 [2011]) Chiu the method being applied to T18 and T13.The method used in the paper of the people such as Chiu simply employs the number of interested chromosomal sequence label in their case karyomit(e) 21, and this number has carried out normalization method by the overall number of the label in this order-checking round.The challenge part of this approach is: the distribution of label on each karyomit(e) can be different to order-checking round from order-checking round, and measure because this increasing dysploidy the entire change measured.In order to the result of Chiu algorithm and the chromosomal ratio used in this example be contrasted, the method test data of karyomit(e) 21 and 18 being used the people such as Chiu to recommend is analyzed again, as shown in Figure 4.Generally, compression in the scope of NCV be observed for each of karyomit(e) 21 and 18, and observed the reduction determining rate, the NCV threshold value 4.0 that wherein make use of for dysploidy classification correctly identifies the T18 sample of the T21 and 5/8 of 10/13 from our test group.
The people such as Ehrich equally only focus on T21 and employ the algorithm (Ehrichet al., Am J Obstet Gynecol 204:205 e1-e11 [2011]) identical with people such as Chiu.In addition, after the test group z score measures and outside reference data (i.e. training group) of observing them one offsets, they have carried out retraining to establish classification boundaries to test group.Although this method is feasible in principle, in practice by challenging be determine need how many samples carry out training and need how long once to carry out retraining to guarantee the correct of these grouped datas.A kind of method alleviating this problem comprises contrast in each order-checking round, and these are to amount of illumination baseline and calibrate for quantitative behavior.
The data using present method to obtain show, when the algorithm for chromosome counting data being normalized is optimised, extensive parallel order-checking can determine multiple fetal chromosomal abnormalities from the blood plasma of pregnant woman.Present method is used for quantitatively not only the Stochastic sum system change between order-checking round being reduced to minimum, and also allowing to classify to dysploidy throughout whole genome, is T21 and T18 the most significantly.Larger sample collection is needed to test algorithm for determining T13.For this purpose, a clinical study that is likely, blind, many places is being carried out to prove the diagnostic accuracy of present method further.
Example 2
Gemini ratio is used to verify the determination of dysploidy: to be normalized normalization method karyomit(e)
As described in previous case, present method be based on the number being mapped to interested chromosomal sequence label for being mapped to like displaying and interested chromosome races, the normalization method of sample room and the number of the sequence label of the sample of variability between round of checking order.In order to verify the classification of dysploidy and normalization method karyomit(e) used in Exclusion analysis itself is non-euploid chromosomal (namely existing with the copy number of deformity), determine the normalization method of the first normalization method karyomit(e) (namely for determining that karyomit(e) dosage is for the karyomit(e) of classifying to the common dysploidy relating to karyomit(e) 21,18 and X) as follows.
Use as described in example 1 from the qualified samples of training set 1 and the qualified samples from test set 1, analyze order-checking information to identify for the first normalization method at least one second normalization method karyomit(e) chromosomal, this first normalization method karyomit(e) is used for determining presence or absence T21, T18 or chromosome x dysploidy (accordingly see table 4,5 and 6).
A. for the second normalization method karyomit(e) of the first normalization method karyomit(e) 9:
The normal chromosomal 21 determined in order to the use first normalization method karyomit(e) 9 verified as determined in example 1 is genotypic to be determined, use each other karyomit(e) calculating for the karyomit(e) dosage of karyomit(e) 9, namely as label and the ratio of label being mapped to karyomit(e) 1-8 and 10-22 of the karyomit(e) 9 be mapped in each qualified samples (normal specimens) in training set 1 and each qualified samples in test set, and CV% (table 4) is calculated.As discussed previously, for identifying that the chromosomal CV% of normalization method is the CV value of the karyomit(e) dosage determined in diploid sample.
Table 4
The second normalization method for the first normalization method karyomit(e) 9 is chromosomal to be determined
The karyomit(e) with minimum variability is confirmed as from the karyomit(e) 11 in the qualified samples of training set and test set two collection.
Selective staining body 11 as the second normalization method karyomit(e) for after the determination of checking use first normalization method karyomit(e) 9 to the dysploidy (i.e. T21) for karyomit(e) 21, for the karyomit(e) dosage of each test sample calculating for karyomit(e) 9/ karyomit(e) 11.As described in example 1, using the average karyomit(e) dosage 0.834054 ± 0.005213 (mean value ± S.D) for karyomit(e) 9/ karyomit(e) 11 as determined in the qualified samples of training set, determining the NCV (Fig. 5) for each test sample.
The NCV that data show the exception that uses karyomit(e) 9 to calculate for karyomit(e) 21 low (tests the average 5-6 NCV of samples lower than all the other; Fig. 3) correspond to use karyomit(e) 11 as during the second normalization method karyomit(e) for the high NCV of the exception of karyomit(e) 9 (the average 5-6 NCV higher than all the other test samples).Data show that sample has karyomit(e) 9 dysploidy, and demonstrate the determination of diploid karyomit(e) 21 in sample.This result is consistent with the aneuploid caryogram for sample, and this aneuploid caryogram has been shown as trisomy 9 mosaic 47, XX+9 [9]/46, XX [6].The caryogram of trisomy 9 uses amniotic fluid samples to determine.In addition, these data show the method and can identify rare chromosomal aneuploidy (such as trisomy 9).
B. for the second normalization method karyomit(e) of the first normalization method karyomit(e) 8:
The karyomit(e) dosage (it is used to the normalization method karyomit(e) determining presence or absence T18 as described in example 1) for karyomit(e) 8 is calculated for other karyomit(e) each, namely as being mapped to the ratio of karyomit(e) 8 with the label of karyomit(e) 1-7 and 9-22 in each qualified samples (normal specimens) in training set 1 and each qualified samples in test set 1, and CV% (table 5) is calculated.
Table 5
The second normalization method for the first normalization method karyomit(e) 8 is chromosomal to be determined
The karyomit(e) with minimum variability is confirmed as from the karyomit(e) 11 in the qualified samples of training set and test set two collection.
Selective staining body 2 as the second normalization method karyomit(e) for after the determination of checking use first normalization method karyomit(e) 8 to the dysploidy (i.e. T18) for karyomit(e) 18, for the karyomit(e) dosage of each test sample calculating for karyomit(e) 8/ karyomit(e) 2.Using the average karyomit(e) dosage 0.60102532 ± 0.00318442 (mean value ± S.D) for karyomit(e) 8/ karyomit(e) 2 as determined in the qualified samples of training set, determining the NCV (Fig. 6) for each test sample.
Fig. 6 shows the dysploidy all do not existed in all test samples for the first normalization method karyomit(e) 8, demonstrates thus and uses karyomit(e) 8 as the determination of normalization method karyomit(e) to presence or absence T18 dysploidy.
C. for the second normalization method karyomit(e) of the first normalization method karyomit(e) 6:
The karyomit(e) dosage (it is used to the normalization method karyomit(e) determining the dysploidy of presence or absence chromosome x as described in example 1) for karyomit(e) 6 is calculated for other karyomit(e) each, namely as being mapped to the ratio of karyomit(e) 6 with the label of karyomit(e) 1-5 and 7-22 in each qualified samples (normal specimens) in training set and each qualified samples in test set, and CV% (table 6) is calculated.
Table 6
The second normalization method for the first normalization method karyomit(e) 6 is chromosomal to be determined
The karyomit(e) with minimum variability is determined to be in the karyomit(e) 5 in the qualified samples in training set and the karyomit(e) 3 in the qualified samples of test set.
Selective staining body 5 for after the determination of checking use first normalization method karyomit(e) 6 to the dysploidy (such as monosomy X) for chromosome x, calculates karyomit(e) dosage for karyomit(e) 6/ karyomit(e) 5 for each test sample as the second normalization method karyomit(e).Using the average karyomit(e) dosage 0.954309 ± 0.003149 (mean value ± S.D) for karyomit(e) 6/ karyomit(e) 5 as determined in the qualified samples of training set 1, determining the NCV for each test sample.
Fig. 7 shows the dysploidy all do not existed in all test samples for the second normalization method karyomit(e) 5, demonstrates thus and uses karyomit(e) 6 as the first normalization method karyomit(e) to the determination of presence or absence chromosome x dysploidy.
These data show that the method may be used for determining rare dysploidy (such as trisomy 9), and the method may be used for verifying the determination result of presence or absence for interested chromosomal dysploidy by being normalized the first normalization method karyomit(e) with the second normalization method karyomit(e).The chromosomal normalization method of first normalization method is by alleged occurrence or do not exist for the chromosomal dysploidy of the first normalization method, and determines presence or absence dysploidy to verify the first result in the first or second normalization method karyomit(e).
Example 3
Use and determine for interested chromosomal at least two normalization method karyomit(e)s and verify chromosomal aneuploidy
In order to prove that the determination of chromosomal aneuploidy can be verified for interested chromosomal first and second normalization method karyomit(e)s by using, use karyomit(e) 10 and karyomit(e) 14 as second and the 3rd normalization method karyomit(e) for interested karyomit(e) 21, calculate the karyomit(e) dosage for karyomit(e) 21 in the example 1A using karyomit(e) 9 to calculate as the first normalization method karyomit(e).
Fig. 8 A shows the drawing of the NCV for the sample of 48 in test set 1, and these NCV are that the average of the corresponding karyomit(e) dosage be used in the qualified samples of training set 1 and S.D. calculate.The average CV% of the karyomit(e) dosage for karyomit(e) 21 in training set 1 is provided in table 7.
Table 7
The second normalization method for interested chromosome 21 is chromosomal to be determined
Show that the test sample identified in figure 3 has the low NCV of exception between-5 and-6 NCV with arrow in Fig. 8 A, and use karyomit(e) 9 as during the first normalization method karyomit(e) by the diploid correctly classified as karyomit(e) 21.Except use karyomit(e) 9 is as except the first normalization method karyomit(e), uses karyomit(e) 10 and use karyomit(e) 14 as other normalization method karyomit(e), in all test samples of test set 1, determine presence or absence trisomy 21.Use mean value 0.259070 ± 0.002823 S.D. for the second normalization method karyomit(e) 10, and use mean value 0.409420 ± 00.4965 S.D. to carry out the NCV shown in scaling system 8B and 8C accordingly for the second normalization method karyomit(e) 14.
Data shown in Fig. 8 B and C show the diplontic sample be previously classified as when karyomit(e) 9 is used as first normalization method karyomit(e) (Fig. 3 and 8A) for karyomit(e) 21 and are proved to be diploid for karyomit(e) 21 when karyomit(e) 10 (Fig. 8 B) or karyomit(e) 14 (Fig. 8 C) are used as normalization method karyomit(e).
Therefore, determine that presence or absence chromosomal aneuploidy can be verified as interested chromosomal normalization method karyomit(e) by using at least two coloured differently bodies.
Example 4
Chromosomal aneuploidy is determined in for the second normalization method karyomit(e) of the first normalization method karyomit(e) 8
In order to prove except the existence of rare chromosome abnormalty being different from trisomy 9 determined as determined in example 1 and 2, obtain sequence information from the second training set and the second test set, and calculate for the karyomit(e) 1-22 NCV for all karyomit(e) dosage separately as mentioned above.
Use karyomit(e) 8 as the first normalization method karyomit(e), carry out presence or absence in the sample from test set 2 and relate to the determination of the dysploidy of karyomit(e) 18.In order to verify the determination of presence or absence trisomy 18 in the test sample, the karyomit(e) dosage for karyomit(e) 8 is calculated for other karyomit(e) each, namely as being mapped to the ratio of karyomit(e) 8 with the label of karyomit(e) 1-7 and 9-22 in each qualified samples (normal specimens) in training set 2 and each qualified samples in test set 2, and CV% (table 8) is calculated.
Table 8
The second normalization method for the first normalization method karyomit(e) 8 is chromosomal to be determined
The karyomit(e) with minimum variability is determined to be in from the karyomit(e) 2 in the qualified samples of training set and test set two collection, and is used as the second normalization method karyomit(e) for verifying the determination of the dysploidy of presence or absence karyomit(e) 18.Use the first normalization method karyomit(e) 8, calculate the karyomit(e) dosage for karyomit(e) 8/ karyomit(e) 2 for each test sample.Using the average karyomit(e) dosage 0.601163 ± 0.002408 (mean value ± S.D) for karyomit(e) 8/ karyomit(e) 2 as determined in the qualified samples of training set 2, determining the NCV (Fig. 9 A) for each test sample.Fig. 9 A shows the dysploidy in the test sample that use first normalization method karyomit(e) 8 is analyzed for T18.Using karyomit(e) 2 as NCV exception low (about-10) for karyomit(e) 8 dosage during the second normalization method karyomit(e), show to there is the dysploidy for karyomit(e) 2 in the test sample.In order to verify that dysploidy is karyomit(e) 2 and non-chromosome 8, using the average karyomit(e) dosage 0.953953 ± 0.006302 (mean value ± S.D) for karyomit(e) 8/ karyomit(e) 7 as determined in the qualified samples of training set 2, determining the NCV (Fig. 9 B) for each test sample.Fig. 9 B show karyomit(e) 7 be used as the second normalization method karyomit(e) calculate for the dosage of the first normalization method karyomit(e) 8 and NCV time, test sample does not comprise aneuploid karyomit(e) 8.
These data acknowledgements the method may be used for determining rare dysploidy, and the method may be used for the determination result verifying presence or absence dysploidy, namely by determining that the first normalization method karyomit(e) (it is used as the molecule for calculating interested karyomit(e) dosage) does not exist with the copy number of deformity self, namely it is not non-euploid chromosomal.As shown in example 2 and 3, the determination of presence or absence dysploidy can be undertaken by the normalization method karyomit(e) that use at least two is different.Calculating for interested chromosomal karyomit(e) dosage and NCV, and comparative result to determine identical result time, different normalization method karyomit(e) can be used as independently molecule.Alternately, first in two different normalization method karyomit(e)s may be used for calculating for interested chromosomal dosage and NCV, and the second normalization method karyomit(e) may be used for the chromosomal dosage of calculating first normalization method and NCV, to verify that the first normalization method karyomit(e) does not have dysploidy.
Example 5
Determine that the first and second normalization method karyomit(e)s are for determining chromosomal aneuploidy
In order to identify for karyomit(e) 1-2, X and Y normalization method karyomit(e) separately, be used to use all karyomit(e)s as described in previous case to calculate for each chromosomal NCV per-cent by the order-checking information checking order obtained to all (the namely qualified and affected) samples from each in training set 1, test set 1 and training set 2.
The data of showing in table 9 provide four normalization method karyomit(e)s for each in all 1-22, X and Y chromosome, and these four normalization method karyomit(e)s are determined to be in 3 provided sample sets to be had for the minimum CV of matched doses.
The normalization method karyomit(e) with four minimum CV% is provided.The second minimum variability for karyomit(e) 13 is confirmed as being produced by the mean value of the summation of the karyomit(e) dosage for karyomit(e) 2-6.When using the mean value for the summation of the karyomit(e) dosage of karyomit(e) 2-6, the karyomit(e) dose variation for karyomit(e) Y is minimum.
Table 9
For all chromosomal normalization method karyomit(e)
Based on these results, no matter the second normalization method karyomit(e) is in interested chromosomal two selected normalization method karyomit(e)s, or second normalization method karyomit(e) be normalization method karyomit(e) for the first normalization method karyomit(e) (it is for interested chromosomal first normalization method karyomit(e)), normalization method karyomit(e) can be selected.
Although show at this and describe the preferred embodiments of the invention, it is evident that this type of embodiment only provides at this by way of example for those of ordinary skills.Those of ordinary skill in the art will expect this moment 1 numerous variants, change and substitute and without the need to deviating from the present invention.It should be understood that and can utilize multiple different replacement scheme to these embodiments of the present invention described here when implementing of the present invention.Scope of the present invention is defined by following claim and in method and structure in the scope of these claims and their equivalent cover thus being intended that of this.

Claims (16)

1., for determining a computer processing system for presence or absence fetal chromosomal aneuploidy in the parent test sample comprising fetus and maternal nucleic acids molecule, this computer processing system comprises:
A () device is for receiving sequence information for fetus and maternal nucleic acids molecule described in described sample and for identifying for a number of an interested chromosomal multiple sequence label and a number at least two chromosomal multiple sequence labels of normalization method;
B () device is for using the number of described sequence label to calculate for described interested chromosomal first normalized value and for described interested chromosomal second normalized value; And
C () device is used for comparing for described interested chromosomal described first normalized value and a first threshold and comparing for described interested chromosomal described second normalized value and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in described sample.
2. computer processing system as claimed in claim 1, wherein a first chromosome dosage for described interested chromosomal described first normalized value, described the first chromosome dosage is number for described interested chromosomal sequence label and a chromosomal ratio of the first normalization method, and be wherein a second karyomit(e) dosage for described interested chromosomal described second normalized value, described second karyomit(e) dosage is the number for described interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the second normalization method.
3., for determining a computer processing system for presence or absence fetal chromosomal aneuploidy in the parent test sample comprising fetus and maternal nucleic acids molecule, this computer processing system comprises:
(a) device for obtaining the sequence information for fetus and maternal nucleic acids described in described sample, to identify for a number of an interested chromosomal multiple sequence label and a number at least two chromosomal multiple sequence labels of normalization method;
(b) device for use for described interested chromosomal described sequence label number and determine for described interested chromosomal first normalized value for the number of a chromosomal sequence label of the first normalization method; And use for the chromosomal described sequence label of described first normalization method number and determine for chromosomal second normalized value of described first normalization method for the number of a chromosomal sequence label of the second normalization method;
C () device is used for comparing for described interested chromosomal described first normalized value and a first threshold and comparing for chromosomal described second normalized value of described first normalization method and a Second Threshold, to determine a kind of fetus dysploidy of presence or absence in described sample.
4. computer processing system as claimed in claim 3, wherein a first chromosome dosage for described interested chromosomal described first normalized value, described the first chromosome dosage is the number for described interested chromosomal sequence label and the ratio for the number of a chromosomal sequence label of the first normalization method, and be wherein a second karyomit(e) dosage for described interested chromosomal described second normalized value, described second karyomit(e) dosage is the number of the chromosomal sequence label of described first normalization method and the ratio for the number of a chromosomal sequence label of the second normalization method.
5. as the computer processing system in claim 1 to 4 as described in any one, comprise a device further for determining a first normalized karyomit(e) value and a second normalized karyomit(e) value (NCV), a wherein said NCV makes the average of the described the first chromosome dosage the first chromosome dosage corresponding in one group of qualified samples be associated, and wherein said 2nd NCV makes the average of the described second karyomit(e) dosage second karyomit(e) dosage corresponding in one group of qualified samples be associated, as:
NCV ij = x ij - &mu; ^ j &sigma; ^ j
wherein and δ jthe estimation average for the karyomit(e) dosage of jth in one group of qualified samples and standard deviation accordingly, and x ijfor the viewed jth of a test sample i karyomit(e) dosage.
6. as the computer processing system in claim 1 to 4 as described in any one, wherein:
Described normalization method karyomit(e) for karyomit(e) 21 is selected from karyomit(e) 9,11,14 and 1;
Described normalization method karyomit(e) for karyomit(e) 18 is selected from karyomit(e) 8,3,2 and 6;
Described normalization method karyomit(e) for karyomit(e) 13 is selected from karyomit(e) 4, the group of karyomit(e) 2-6, karyomit(e) 5 and karyomit(e) 6;
Described normalization method karyomit(e) for chromosome x is selected from karyomit(e) 6,5,13 and 3;
Described normalization method karyomit(e) for karyomit(e) 1 is selected from karyomit(e) 10,11,9 and 15;
Described normalization method karyomit(e) for karyomit(e) 2 is selected from karyomit(e) 8,7,12 and 14;
Described normalization method karyomit(e) for karyomit(e) 3 is selected from karyomit(e) 6,5,8 and 18;
Described normalization method karyomit(e) for karyomit(e) 4 is selected from karyomit(e) 3,5,6 and 13;
Described normalization method karyomit(e) for karyomit(e) 5 is selected from karyomit(e) 6,3,8 and 18;
Described normalization method karyomit(e) for karyomit(e) 6 is selected from karyomit(e) 5,3,8 and 18;
Described normalization method karyomit(e) for karyomit(e) 7 is selected from karyomit(e) 12,2,14 and 8;
Described normalization method karyomit(e) for karyomit(e) 8 is selected from karyomit(e) 2,7,12 and 3;
Described normalization method karyomit(e) for karyomit(e) 9 is selected from karyomit(e) 11,10,1 and 14;
Described normalization method karyomit(e) for karyomit(e) 10 is selected from karyomit(e) 1,11,9 and 15;
Described normalization method karyomit(e) for karyomit(e) 11 is selected from karyomit(e) 1,10,9 and 15;
Described normalization method karyomit(e) for karyomit(e) 12 is selected from karyomit(e) 7,14,2 and 8;
Described normalization method karyomit(e) for karyomit(e) 14 is selected from karyomit(e) 12,7,2 and 9;
Described normalization method karyomit(e) for karyomit(e) 15 is selected from karyomit(e) 1,10,11 and 9;
Described normalization method karyomit(e) for karyomit(e) 16 is selected from karyomit(e) 20,17,15 and 1;
Described normalization method karyomit(e) for karyomit(e) 17 is selected from karyomit(e) 16,20,19 and 22;
Described normalization method karyomit(e) for karyomit(e) 19 is selected from 22,17,16 and 20;
Described normalization method karyomit(e) for karyomit(e) 20 is selected from karyomit(e) 16,17,15 and 1; And
Described normalization method karyomit(e) for chromosome 22 is selected from karyomit(e) 19,17,16 and 20.
7., as the computer processing system in claim 1 to 4 as described in any one, wherein determine the presence or absence of at least two kinds of different fetal chromosomal aneuploidies.
8. computer processing system as claimed in claim 7, wherein determines the different fetal chromosomal aneuploidy of presence or absence for all karyomit(e).
9., as the computer processing system in claim 1 to 4 as described in any one, wherein said fetal chromosomal aneuploidy is selected from T21, T13, T18 and monosomy X; Or computer processing system as claimed in claim 8, wherein said different fetal chromosomal aneuploidy is selected from T21, T13, T18 and monosomy X.
10. as the computer processing system in claim 1-4 as described in any one, wherein:
Described maternal sample obtains from a pregnant woman;
Described maternal sample is a kind of biological fluid sample;
Described maternal sample is a plasma sample; And/or
Described nucleic acid molecule is cfDNA molecule.
11. computer processing systems as claimed in claim 10, wherein:
Receive described sequence information and comprise the sequence information receiving and obtained by next generation's order-checking (NGS);
Receive described sequence information and comprise the sequence information receiving and obtain by using multiple reversible dye-terminators to carry out synthesis method order-checking;
Receive described sequence information and comprise the sequence information receiving and obtained by connection method order-checking; Or
Receive described sequence information and comprise the sequence information receiving and obtained by single-molecule sequencing.
12. as the computer processing system in claim 1-4 as described in any one, and wherein said chromosomal aneuploidy is a kind of part or complete chromosomal aneuploidy.
13. as the computer processing system in claim 1-4 as described in any one, and wherein said parent test sample is the plasma sample obtained from a pregnant woman, and described nucleic acid molecule is cfDNA molecule.
14. 1 kinds are configured to the equipment determining presence or absence at least one fetus dysploidy in the parent test sample comprising fetus and maternal nucleic acids molecule, described equipment comprises, and: (a) checks order device, this order-checking device is configured to, to checking order at least partially in described fetus and maternal nucleic acids molecule, produce sequence information thus; And (b) computer processing system according to any one of claim 1-4.
15. equipment as claimed in claim 14, wherein said order-checking device is configured to carry out synthesis method order-checking.
16. equipment as claimed in claim 15, wherein said order-checking device is configured to use multiple reversible dye-terminators to carry out synthesis method order-checking.
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