WO2014101024A1 - Method, system and computer readable medium for determining fetal sexes of twins - Google Patents

Method, system and computer readable medium for determining fetal sexes of twins Download PDF

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Publication number
WO2014101024A1
WO2014101024A1 PCT/CN2012/087582 CN2012087582W WO2014101024A1 WO 2014101024 A1 WO2014101024 A1 WO 2014101024A1 CN 2012087582 W CN2012087582 W CN 2012087582W WO 2014101024 A1 WO2014101024 A1 WO 2014101024A1
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sequencing data
twins
chromosome
fetuses
determined
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PCT/CN2012/087582
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French (fr)
Chinese (zh)
Inventor
郑晶
张春雷
谢伟伟
蒋浩君
郭靖
杨焕明
张秀清
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深圳华大基因医学有限公司
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Priority to PCT/CN2012/087582 priority Critical patent/WO2014101024A1/en
Publication of WO2014101024A1 publication Critical patent/WO2014101024A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination

Definitions

  • This invention relates to the field of biomedicine and, in particular, to the field of genetic variation testing, and more particularly to methods, systems and computer readable shields for determining the sex of a fetus in a fetus. Background technique
  • Twins can be divided into identical twins and fraternal twins.
  • the development of two embryos from one fertilized egg is called an identical twin.
  • the twins developed from a single fertilized egg carry the same genetic background, and their gender, appearance, etc. are identical. If one of the fetuses has a genetic disease, the other fetus must be the same.
  • single-oval twins may be monochorionic mono-amnion, mono-chorionic double amnion, and double-chorioamnion amnion depending on the time of separation.
  • a fraternal twin is a discharge of two egg cells at a time, both of which develop into two embryos after fertilization.
  • fraternal twins randomly inherit the parent's genome according to Mendelian inheritance, and the types of genes carried are not identical. Therefore, they may have different genders and appearances, and the carrying status of genetic diseases may also be different.
  • the fraternal twins may also be double chorionic double amnion or monochorionic double amnion.
  • the detection techniques commonly used in prenatal and postnatal testing for twins are divided into non-invasive prenatal techniques and invasive prenatal techniques.
  • non-invasive prenatal technical damage is small, no trauma sampling is needed, but the current detection methods have disadvantages: 1) The risk factor of Down's child is calculated by checking the concentration of serum metabolites, the age of pregnant women, and the gestational age of blood stasis. However, the false positive rate and false negative rate of this Down's screening method are both high; 2) Ultrasound examination depends on the equipment and doctor's personal experience, can not form a unified standard, can not judge the developmental abnormalities of unclear or controversial ultrasound indicators .
  • the prenatal antenatal detection technology collects fetal samples with high accuracy, but it is easy to cause abortion, amniocentesis and other problems, which brings certain abortion risks to pregnant women and fetuses. It also has time limitations and invasive sampling. In a specific time zone. Moreover, the current methods are technically demanding and require lengthy experimental procedures and high costs.
  • the invention proposes a method of determining the sex of a fetus in a twin.
  • UR% determining the sex of the fetus in the twins, wherein at least one of the twins is determined to be a male fetus when at least one of the following conditions is satisfied, when none of the following conditions are satisfied.
  • Y-UR% is greater than the first threshold; and -1111% (-1; 11% is greater than the second threshold.
  • the invention proposes a computer readable medium.
  • the computer readable medium stores instructions adapted to be executed by the processor to determine the sex of the fetus in the twins by: obtaining a plurality of sequencing data of blood DNA of the twin pregnant women; Aligning the sequencing data with a human reference genomic sequence to obtain a unique aligned sequencing data set consisting of a plurality of unique aligned sequencing data; determining unique unique alignment sequencing data contained in the unique alignment sequencing data set a number M; determining the number N y of unique alignment sequencing data derived from the Y chromosome in the unique alignment sequencing data set, and at least one of the number N x of unique alignment sequencing data derived from the X chromosome; determining the chromosome
  • the sequencing data content of Y-UR% and the X-UR% sequencing data content of at least one of X-UR%, wherein, according to the formula Y-UR% N y /M, the Y-
  • X-UR% N X / M, to determine the content of the sequencing data of the X chromosome -UR%; and determining the sex of a fetus in twins, wherein, when the following conditions to When one of determining at least one of the twin male fetuses, when the following conditions are not satisfied, determining whether the twins are female fetuses: Y-UR 0 /. Greater than the first threshold; and ⁇ -13 ⁇ 4% «-13 ⁇ 4% is greater than the second threshold.
  • the computer-readable shield it is possible to effectively determine whether a male fetus is present in the twins, and then according to the fetal DNA concentration in the maternal plasma, the two fetus sexes can be effectively inferred.
  • the invention proposes a system for determining the sex of a fetus in a twin.
  • the system comprises: a sequencing device for sequencing blood DNA of a twin pregnant woman to obtain a plurality of sequencing data; a comparison device, the comparison device and the sequencing device Connected, the sequencing data is aligned with the reference genomic sequence of the organism to obtain a unique aligned sequencing data set consisting of a plurality of unique aligned sequencing data; an analysis device, the analysis device and the ratio Connected to the device and used to determine The fetal sex in the twins, wherein the analyzing device further comprises: a first calculating module, wherein the first calculating module is configured to determine a number M of unique aligned sequencing data included in the unique aligned sequencing data set; a calculation module, the second calculation module is configured to determine a number ⁇ N y of the unique alignment sequencing data derived from the ⁇ chromosome in the unique alignment sequencing data set, and
  • the invention also proposes a system for determining the sex of a fetus in a twin.
  • the system comprises: a sequencing device for sequencing blood DNA of the twin pregnant women to obtain a plurality of sequencing data; and the computer readable medium of the present invention as described above , wherein the computer readable shield stores instructions for determining the sex of the fetus in the twins.
  • the total number of sequencing data located on a chromosome is proportional to the length of the chromosome and the content of the chromosome in the biological sample, the system is used to determine the blood DNA of the pregnant woman of the twins.
  • the number of unique alignments of the X and Y chromosomes in the sample and the amount of sequencing data can effectively determine whether there are male fetuses in the twins, and based on the estimated fetal DNA concentration in the maternal plasma, the two fetus genders can be effectively inferred.
  • FIG. 1 shows a schematic flow chart of a method for determining the sex of a fetus in a twin, in accordance with one embodiment of the present invention
  • FIG. 2 shows a schematic structural view of a system for determining the sex of a fetus in a twin according to an embodiment of the present invention
  • Figure 3 shows the chromosome number of sample S 1 obtained by the method of the present invention, in accordance with one embodiment of the present invention.
  • Figure 4 shows the chromosomal digital karyotype results of sample S2 obtained using the method of the present invention, in accordance with one embodiment of the present invention. Detailed description of the invention
  • first and second are used for descriptive purposes only, and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated.
  • the features of the first and second features may include one or more of the features, either explicitly or implicitly. Further, in the description of the present invention, unless otherwise stated, the meaning of "multiple" is two or More than two.
  • the invention proposes a method for determining the presence or absence of a male fetus in a twin.
  • the method can include:
  • the blood DNA of the twin pregnant women who need to be tested is first sequenced to obtain a plurality of sequencing data.
  • the method of extracting blood DNA of a pregnant woman of a twin is not particularly limited.
  • the blood DNA of the twin pregnant women can be extracted by at least one selected from the group consisting of magnetic bead method and column chromatography, preferably magnetic bead method.
  • the presence or absence of a male fetus in the twins can be effectively determined by the method of the present invention.
  • the principle of magnetic extraction of DNA by magnetic beads is as described above, and will not be described herein.
  • sequencing device that can be employed according to an embodiment of the present invention is not particularly limited.
  • sequencing is performed by at least one selected from the group consisting of Roche/454 GS Junior. Illumina/MiSeq and Life Tecnologies/Ion Torrent PGM, taking into account the advantages of instrument portability and high throughput performance. Thereby, it is possible to further improve the efficiency of determining whether or not a male fetus is present in the twins by utilizing the characteristics of high-throughput and deep sequencing of these sequencing devices.
  • the sequencing type can be single-end (one-way) sequencing or pair-end (two-way) sequencing. According to some embodiments of the invention, the sequenced data is 36 to 100 bp in length.
  • the sequencing platform employed is Ilhmiina/Solexa and the sequencing type is SE36 sequencing.
  • the efficiency of the subsequent analysis can be further improved.
  • the sequencing amount is a 5M sequencing short fragment.
  • the method for constructing the sequencing library can include: First, the DNA fragment is blunt-ended and the base A is added at the end, and the linker is ligated to obtain a DNA fragment having a linker;
  • the DNA having the adaptor is amplified to obtain an amplification product, that is, a sequencing library.
  • a tag sequence Index can be introduced into the sequencing library during the process of constructing the sequencing library, for example, an index can be introduced in the linker, or a tag sequence Index can be introduced during the amplification process.
  • simultaneous sampling of multiple test samples can be achieved by using different tag sequences for different samples.
  • the obtained sequencing data is aligned with a human reference genome sequence to obtain a unique alignment sequencing data set composed of a plurality of unique alignment sequencing data; and then, the unique alignment sequencing data is determined.
  • the number of unique aligned sequencing data contained in the set is M; then, the number N x and N y of unique aligned sequencing data derived from the X and Y chromosomes, respectively, in the unique aligned sequencing data set are determined.
  • the human reference genomic sequence used is a reference sequence obtained by masking the repeat sequence of the human genome sequence, such as the NCBI database.
  • the latest version of the Human Genome Reference Sequence is the human genome reference sequence of version 36 (hgl8; NCBI Build 36) in the NCBI database.
  • sequence alignment can be performed by any sequence alignment program, such as Short Oligonucleotide Analysis Package (SOAP) and BWA alignment (available by those skilled in the art) At least one of Burrows-Wheeler Aligner is performed to align the sequencing data with a reference genomic sequence to obtain the position of the sequencing data on the reference genome. Sequence alignment can be performed using default parameters provided by the program, or can be selected by those skilled in the art as needed.
  • the comparison software employed is SOAP aligner/soap2
  • the term "unique alignment sequencing data” refers to a sequence that has only a unique position on a reference genomic sequence when the sequencing data is aligned with a reference genomic sequence, as Unique reads.
  • Unique reads In an embodiment of the invention, in order to avoid interference from repetitive sequences, it is necessary to remove those DNA sequences that are located in tandem repeats and transposition repeat positions in the human genome reference sequence, and only those DNA sequences that can be mapped to unique positions in the genome are counted. That is, the only alignment of the sequencing data.
  • the unique alignment sequencing data maps the DNA sequences from the DNA of twin pregnant women to the specific chromosomes after being interrupted and sequenced.
  • the twins of the twin pregnant women can be determined based on the values of ⁇ -111% «-1; 1% and Y-UR%. Whether there is a male fetus. Specifically, when at least one of the following conditions is satisfied, it can be determined that at least one of the twins is a male fetus, and when none of the following conditions are satisfied, it can be determined that the twins are female fetuses: ⁇ -1!1% is greater than the first threshold ; and Y-UR%/X-UR% are greater than the second threshold.
  • the first threshold value and the second threshold value are determined by the following method: 100 samples of maternal fetal plasma samples are taken, and Y-UR is calculated separately. Y-UR%/X-U %, and draw the Y-UR of each sample separately. /. And the normal distribution curve of Y-UR%/X-UR%, according to the central limit theorem, the Y-UR% and Y-UR%/X-UR% of the 100 samples are in accordance with the normal distribution, taking Y-UR The right interval point of the °/W 99% confidence interval is taken as the first threshold, taking Y-UR%/X-UR°/. The right interval point of the 99% confidence interval is used as the second threshold.
  • the first threshold is determined to be 0.0004, and the second threshold is 0.0075.
  • Y-UR% is greater than 0.0004 or Y-UR%/X-UR% is greater than 0.0075, it can be determined that a male tire is present in the twin.
  • the method when it is determined that at least one of the twins is a male fetus, the method further includes: first, estimating the fetal DNA concentration based on the obtained N x and N Y respectively, so as to obtain the first fetus respectively L DNA M%chrX, and second fetal DNA concentration %chrY.
  • fetal DNA concentration estimation can be performed by the following steps: selecting normal male and normal female plasma sample sets as controls, respectively calculating ⁇ -111% and -UR% of each plasma sample; respectively calculating each male plasma Sample Y-UR 0 /.
  • the arithmetic mean of X-UR% was calculated for each female plasma sample as Y-UR°/ ⁇ n X-UR 0 /.
  • the arithmetic mean value denoted as Y F and X F ; then, the first fetal DNA M%chrX estimated using the X chromosome is calculated according to the formula (X-UR%-X F )/(X M -X F ), according to The formula (Y-UR%-Y F )/(Y M -Y F ) calculates the second fetal DNA concentration 3 ⁇ 4%chrY estimated using the Y chromosome, where -111% of the formula is the X of the plasma sample of the pregnant woman to be tested
  • the -UR% value, Y-UR% is the Y-UR% value of the plasma sample of the pregnant woman to be tested.
  • the significant difference p-values of the % ⁇ 13 ⁇ 4 and %(;1« ⁇ are determined. Further, the sex of the two fetuses in the twins can be determined based on the p-value. Specifically, according to an embodiment of the present invention, P-value>0.1, it can be determined that the fetus in the twins is a male fetus. If p-value ⁇ 0.1, and /chrX is greater than %chrY, it can be determined that there is one male fetus and one female fetus in the twin.
  • the concentration of DNA on the X and Y chromosomes contributed by the two fetuses will be equal (because the two fetuses provide two X chromosomes and two Y chromosomes); if the two fetuses are a male fetus and a female
  • the concentration of DNA on the X and Y chromosomes contributed by the two fetuses is that the DNA concentration on the X chromosome is higher than the DNA concentration on the Y chromosome (because of this When the two fetuses provide three X chromosomes and one Y chromosome).
  • the sex of the twin fetus can also be inferred by using the fetal DNA concentration estimated by the variant chromosome. Specifically, if the estimated fetal DNA concentration using the variant chromosome is about using the ⁇ chromosome Estimating the 1/2 of the fetal DNA concentration obtained, it is concluded that the two fetuses are two male fetuses.
  • the gender of the fetus is estimated by using the estimated fetal DNA concentration of the mutant chromosome, which is premised on the assumption that only one fetus is abnormal (because the probability of abnormality of both fetuses is small), and further, The fetal concentration estimated using abnormal chromosomes is only the DNA concentration provided by one of the fetuses. If both fetuses are male, then the fetal concentration estimated using the sputum chromosome is the DNA concentration provided by the two fetuses, theoretically twice the fetal concentration estimated using the mutated chromosome. Thus, when the fetal DNA concentration estimated using the mutant chromosome is about 1/2 of the fetal DNA concentration estimated using the sputum chromosome, it can be inferred that both fetuses are male fetuses.
  • the tag sequence Index can be introduced into the sequencing library during the process of constructing the sequencing library, thereby implementing different tag sequences for different samples.
  • multiple test samples are sequenced, and the sequencing data of each sample is effectively distinguished by the label, and then the values of M, N x and N y of each sample can be respectively obtained, so that whether there are male tires in the subsequent twins respectively Judgment.
  • the method of determining whether a male fetus is present in a twin may further comprise: statistically comparing the number of short sequences sequenced to the unique alignment on the X and Y chromosomes (N x , N y Calculating the ratio of the number of short sequences sequenced by the unique alignment to the short sequence number (M) of the whole genome unique alignment sequence, respectively, X-UR% and Y-UR%, when ⁇ -13 ⁇ 4% is greater than the first threshold When Y-UR% X-UR% is greater than the second threshold, it is determined that there is a male fetus in the twins.
  • the gender of the twins is determined by combining the fetal DNA concentration in the plasma of the pregnant woman. The specific method has been described in detail above and will not be described again.
  • the first threshold is 0.0004 (i.e., greater than 0.0004 is determined to be the presence of a male fetus) and the second threshold is 0.0075 (i.e., greater than 0.0075 is determined to be the presence of a male fetus).
  • the total number of sequencing data located on a chromosome is proportional to the length of the chromosome and the content of the chromosome in the biological sample
  • the sequencing data content of the Y chromosome in the sample is Y-UR%, or Y-UR% X-UR. / value, can effectively determine whether there are male fetuses in the twins. Further, based on the fetal DNA concentration in the plasma of the pregnant woman, the sex of the two fetuses in the twins can be further determined.
  • the present invention provides a computer readable medium.
  • the computer readable medium stores instructions thereon, the instructions being adapted to be executed by the processor to determine in the twins by the following steps Fetal gender:
  • the sequencing data is aligned with a human reference genomic sequence to obtain a unique aligned sequencing data set consisting of multiple unique aligned sequencing data.
  • the reference genomic sequence is a human genome reference sequence of version 36 of the NCBI database.
  • the sequencing data is aligned with the reference genomic sequence of the organism using at least one of SOAP and BWA.
  • the sequencing data has a length of 36 100 bp.
  • the number M of unique aligned sequencing data contained in the unique aligned sequencing data set is determined.
  • the unique alignment sequencing data sets at least one of the number N of unique alignment sequencing data derived from the Y chromosome and the number N x of unique alignment sequencing data derived from the X chromosome.
  • the sequencing data content of the Y chromosome is determined to be Y-UR 0 /.
  • determining the sex of the fetus in the twins wherein, when at least one of the following conditions is satisfied, determining that at least one of the twins is a male fetus, and determining that the twins are female Y when none of the following conditions are satisfied -UR% is greater than the first threshold; and ⁇ -1; 1% «-13 ⁇ 4% is greater than the second threshold.
  • the first threshold is 0.0004 and the second threshold is 0.0075.
  • the method when it is determined that at least one of the twins is a male fetus, the method further includes: performing fetal DNA concentration estimation based on the N x and ⁇ ⁇ , respectively, to obtain the first fetus separately DNA concentration. /. chrX, and second fetal DNA concentration % ⁇ 1; and determining the %chrX and %.
  • the computer-readable shield is used to determine the twin
  • the Y-UR%, or Y-UR%/X-UR% value of the Y chromosome in the blood DNA sample of the pregnant woman can effectively determine whether there is a male fetus in the twin. Further, based on the fetal DNA concentration in the maternal plasma, the sex of the two fetuses in the twins can be further determined.
  • the invention proposes a system for determining the sex of a fetus in a twin.
  • the system includes: a sequencing device A100, a comparison device A200, and an analysis device A300.
  • the sequencing device A100 is configured to sequence blood DNA of a twin pregnant woman to obtain a plurality of sequencing data
  • the comparison device A200 is connected to the sequencing device A100 for using the sequencing data with a human reference genome
  • the sequences are aligned to obtain a unique aligned sequencing data set consisting of a plurality of unique aligned sequencing data
  • analytical device A300 is coupled to comparison device A200 and is used to determine fetal gender in the twins.
  • the reference genomic sequence is the human genome reference sequence of version 36 of the NCBI database.
  • the sequencing data is aligned with the reference genomic sequence of the organism using at least one of SOAP and BWA.
  • the analyzing device A300 further includes: a first calculating module A310, configured to determine the number of unique aligned sequencing data included in the unique aligned sequencing data set! a second calculation module A320, configured to determine the number N y of unique alignment sequencing data derived from the Y chromosome in the unique alignment sequencing data set, and the number of unique alignment sequencing data derived from the X chromosome N x At least one of; a third calculating module A330, configured to determine a sequencing data content Y-UR of the Y chromosome.
  • the first threshold is 0.0004 and the second threshold is 0.0075.
  • Y-UR% is greater than 0.0004 or Y-UR%/X-UR% is greater than 0.0075, it can be determined that a male fetus is present in the twin.
  • the judging module A340 may further include a verification unit adapted to perform the following operations when determining that at least one of the twins is a male fetus: performing the fetus separately based on the N x and N Y
  • the DNA concentration is estimated to obtain a first fetal DNA concentration %chrX, and a second fetal DNA concentration %chrY, respectively; and to determine the % 1 «3 ⁇ 4 sum. /.
  • p-value of chrY wherein, if p-value>0.1, it is determined that the fetus in the twins is a male fetus, if p-value ⁇ 0.1, and %(;1 ⁇ is greater than %chrY, then determining There is one male and one female in the twins.
  • the system is used to determine the blood DNA of the pregnant woman of the twins.
  • the Y-UR%, or the value of Y-UR%/X-UR%, of the Y chromosome in the sample can be effectively confirmed. Whether there are male fetuses in twins. The sex of the two fetuses in the twins can then be effectively determined based on the fetal DNA concentration in the maternal plasma.
  • the present invention proposes another system for determining the sex of a fetus in a twin.
  • the system may include: a sequencing device for sequencing genomic DNA of the biological sample to obtain a plurality of sequencing data; and the computer readable medium described above.
  • the system is used to determine the Y chromosome in the blood DNA sample of the twin pregnant woman.
  • the sequencing data content of Y-UR%, or the value of Y-UR%/X-UR%, can effectively determine whether a male fetus is present in the twins. Further, based on the fetal DNA concentration in maternal plasma, the sex of the two fetuses in the twins can be effectively determined.
  • the method, system and computer readable medium of the present invention for determining the sex of a fetus in a twin have the following advantages:
  • the following steps are performed to verify the chromosome number of the patient.
  • Abnormal and twin gender conditions :
  • the DNA of the above 2 plasma samples (ie, 1 twin T21 positive sample and 1 twin fetal T18 positive sample, hereinafter referred to as Sl, S2) was extracted according to the Tiangen DP327-02 Kit protocol.
  • the extracted DNA was according to the modified Illumina/Solexa standard.
  • the library construction process is carried out, and the linker used for sequencing is added to both ends of the 200 bp DNA molecule.
  • Each sample is added with a different tag sequence (index), and then hybridized with the flowcell surface complementary link under certain conditions.
  • the nucleic acid molecules were clustered and then sequenced by single-end sequencing on IlluminaHiseq2000 to obtain a DNA fragment sequence of 36 bp in length.
  • DNA obtained from the above plasma sample was constructed according to the modified Illumina/Solexa standard procedure, and the specific procedure was referred to the prior art (see Illumina/ available at http://www.mumina.com7).
  • the Solexa standard library specification is incorporated by reference in its entirety.
  • the DNA library size and insert were determined to be about 200 bp by the 2100 Bioanalyzer (Agilent), and the QPCR was accurately quantified and used.
  • each sample was obtained with a data volume of about 0.25 G, wherein each sample was subjected to the aforementioned index label.
  • the districts are separated.
  • the obtained sequencing data of each sample is compared with the human genome reference sequence of version 36 (hgl8; NCBIBuild36) in the NCBI database, so as to obtain multiple unique comparisons of each sample respectively.
  • the unique alignment of the sequencing data consists of sequencing data sets.
  • the ratio of the average number of sequencing sequences ( n ' ) of the color bodies is 2, which is the base pair value, that is,
  • Threshold and Filtering - To obtain reliable results, filter the average normalized value of each chromosome. If the average normalized value is less than -1.28 or greater than 1.28 (95% CI), it will be output as a positive result.
  • Table 1 lists the karyotype results of the samples S1 and S2, Figure 3 shows the chromosomal digital karyotype results of the sample S1 obtained by the method of the present invention; and Figure 4 shows the sample S2 obtained by the method of the present invention. Chromosome digital karyotype results.
  • ratio of unique sequencing data were determined from the concentration of each sample X chromosome unique sequencing data than the number N x; statistics were unique to the ratio of the X chromosome and ⁇ of each sample For sequencing short sequence numbers N x , N y , where ⁇ is the number of unique alignment sequencing data derived from the X chromosome, and N y is the number of unique alignment sequencing data derived from the Y chromosome. Then, the ratio of the above-mentioned unique alignment sequencing short sequence number to the whole genome unique alignment sequencing short sequence number (M) was calculated separately, and respectively recorded as X-UR 0 /.
  • Y-UR% is greater than 0.0004, it is determined that there is a male fetus, Y-UR%/X-UR°/.
  • 0.0075 it is determined that there is a male fetus.
  • the sex of the two fetuses in the twins is determined. Specifically, %chrX with%. When 1 « ⁇ significant difference p-value>0.1, it can be inferred that the two fetuses are two male fetuses, when %chrX and %. 111" ⁇ significant difference - ⁇ 111 ⁇ 2 ⁇ 0.1) and %(;111 ratio. /. When chrY is high, it can be inferred that the two fetuses are a male fetus and a fetus.
  • the body of the twin fetus can be inferred by reference to the estimated fetal DNA concentration of the variant chromosome. Specifically, if the estimated fetal DNA concentration of the variant chromosome is about 1/2 of the estimated fetal DNA concentration of the Y chromosome, then the two fetuses are inferred. For the two male tires.
  • chromosomal variation eg, aneuploid
  • the body of the twin fetus can be inferred by reference to the estimated fetal DNA concentration of the variant chromosome. Specifically, if the estimated fetal DNA concentration of the variant chromosome is about 1/2 of the estimated fetal DNA concentration of the Y chromosome, then the two fetuses are inferred. For the two male tires.
  • the inventor concludes that at least one of the twins of the sample S1 is a male tire, and further, according to the front
  • the inference method because %chrX and %(;11 ⁇ estimated fetal DNA concentration difference is significant, the p-value is 0.408, so it can be determined that the twins are male and female.
  • the %chrX and % ⁇ differential significance p-value is 0.051
  • the estimated fetal DNA concentration (%chrY) based on the Y chromosome is 2.55 times the estimated fetal DNA concentration of the 18th variant chromosome, thereby determining that the S2 sample is two Male tires.
  • %chrX represents the fetal DNA concentration estimated by the relatively unique alignment of the short sequence number on the X chromosome
  • %chrY represents the fetal DNA concentration estimated by the relatively unique alignment of the short sequence number on the Y chromosome
  • %clirl8/%chr21 The fetal DNA concentration estimated by the relatively unique alignment of the sequenced short sequences on the chromosome that is mutated.
  • the method, system and computer readable medium of the present invention for determining the sex of a fetus in a twin can be effectively used to determine the sex of a fetus in a twin of a twin pregnant woman.

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Abstract

Provided are a method, system and computer readable medium for determining fetal sexes of twins. The method for determining fetal sexes of the twins comprises: sequencing aiming at DNA in blood of a woman pregnant with twins; comparing sequencing data, so as to obtain a unique comparison sequencing data set; determining the number M of unique comparison sequencing data contained in the unique comparison sequencing data set; determining at least one of the numbers Nx and Ny of unique comparison sequencing data from an X chromosome and a Y chromosome respectively; determining at least one of the sequencing data content Y-UR% of the Y chromosome and the sequencing data content X-UR% of the X chromosome; and judging fetal sexes of the twins, wherein at least one of the twins is determined to be a male fetus when at least one of the following conditions is met, and the twins are determined to be female fetuses when the following conditions are not met: Y-UR% is greater than a first threshold value; and Y-UR%/X-UR% is greater than a second threshold value.

Description

确定双胞胎中胎儿性别的方法、 ***和计算机可读介质  Method, system and computer readable medium for determining the sex of a fetus in a twin
优先权信息 Priority information
无 扶术领域  No field of aid
本发明涉及生物医学领域， 具体地， 涉及遗传变异检验领域， 更具体地， 本发明涉 及确定 胞胎中胎儿性别的方法、 ***和计算机可读介盾。 背景技术  Field of the Invention This invention relates to the field of biomedicine and, in particular, to the field of genetic variation testing, and more particularly to methods, systems and computer readable shields for determining the sex of a fetus in a fetus. Background technique
双胞胎可以分为同卵双胎和异卵双胎。 由一个受精卵发育成两个胚胎称为同卵双胎。 这种由单个受精卵发育而来的双胞胎携带有同样的遗传背景， 其性别、 相貌等完全相同， 若其中一个胎儿患有某种遗传疾病， 另一个胎儿也必然是同样的情况。 一般情况下， 单卵 双胞胎根据分离的时间不同， 其胎盘可以是单绒毛膜单羊膜类、 单绒毛膜双羊膜类及双绒 毛膜双羊膜类。 异卵双胎是指一次排出两个***， 都受精后发育成两个胚胎。 异卵双胎 的胎儿会按照孟德尔遗传规律随机遗传父母的基因组， 携带的基因类型并不完全相同， 因 此可能具有不同性别及相貌， 对于遗传疾病的携带情况也可能会不同。 异卵双胎同样可能 是双绒毛膜双羊膜类或单绒毛膜双羊膜类。  Twins can be divided into identical twins and fraternal twins. The development of two embryos from one fertilized egg is called an identical twin. The twins developed from a single fertilized egg carry the same genetic background, and their gender, appearance, etc. are identical. If one of the fetuses has a genetic disease, the other fetus must be the same. In general, single-oval twins may be monochorionic mono-amnion, mono-chorionic double amnion, and double-chorioamnion amnion depending on the time of separation. A fraternal twin is a discharge of two egg cells at a time, both of which develop into two embryos after fertilization. Fetuses of fraternal twins randomly inherit the parent's genome according to Mendelian inheritance, and the types of genes carried are not identical. Therefore, they may have different genders and appearances, and the carrying status of genetic diseases may also be different. The fraternal twins may also be double chorionic double amnion or monochorionic double amnion.
Θ前国内外产前对于双胞胎检测诊断常用的检测技术分为无创产前技术和有创产 前技术。 其中， 无创产前技术损伤小， 无需创伤取样， 但目前的检测方法有不足地方： 1 )通过血清代谢物检查浓度以及孕妇年龄， 釆血孕周等， 计算出的唐氏儿的危险系数。 但这种唐氏筛查法的假阳性率和假阴性率都偏高； 2 ) 超声检查依赖设备和医生个人经 验， 不能形成统一的标准， 对超声指标不明确或有争议的发育异常无法判断。 而有创产 前检测技术对胎儿样本进行采集， 准确率高， 但是容易导致流产， 羊膜腔炎等问题， 给 孕妇和胎儿带来一定的流产风险， 同时也具有时间局限性， 有创取样需在特定的时间区 段进行。 并且， 目前的方法， 对技术要求高， 且均需要冗长的实验程序和高额的费用。  The detection techniques commonly used in prenatal and postnatal testing for twins are divided into non-invasive prenatal techniques and invasive prenatal techniques. Among them, non-invasive prenatal technical damage is small, no trauma sampling is needed, but the current detection methods have disadvantages: 1) The risk factor of Down's child is calculated by checking the concentration of serum metabolites, the age of pregnant women, and the gestational age of blood stasis. However, the false positive rate and false negative rate of this Down's screening method are both high; 2) Ultrasound examination depends on the equipment and doctor's personal experience, can not form a unified standard, can not judge the developmental abnormalities of unclear or controversial ultrasound indicators . The prenatal antenatal detection technology collects fetal samples with high accuracy, but it is easy to cause abortion, amniocentesis and other problems, which brings certain abortion risks to pregnant women and fetuses. It also has time limitations and invasive sampling. In a specific time zone. Moreover, the current methods are technically demanding and require lengthy experimental procedures and high costs.
因此， 目前的产前检测尤其是双胞胎性别检测仍有待改进。 发明内容  Therefore, current prenatal testing, especially twin sex testing, remains to be improved. Summary of the invention
本发明旨在至少解决现有技术中存在的技术问题之一。 在本发明的一个方面， 本发明提出了一种确定双胞胎中胎儿性别的方法。 根据本发明 的实施例， 该方法包括： 针对双胞胎孕妇的血液 DNA进行测序， 以便获得多个测序数据； 将所述测序数据与人类的参照基因组序列进行比对， 以便获得由多个唯一比对测序数据构 成的唯一比对测序数据集； 确定所述唯一比对测序数据集中所包含的唯一比对测序数据的 数目 M; 确定所述唯一比对测序数据集中来源于 Y染色体的唯一比对测序数据的数目 Ny, 以及来源于 X染色体的唯一比对测序数据的数目 Nx的至少一种；确定 Y染色体的测序数据 含量丫-1；1 %和 X染色体的测序数据含量 X-UR%的至少一种，其中，按照公式 Y-UR%=Ny/M , 确定 Y染色体的测序数据含量 Y-U % , 按照公式 X-UR%=NX/M, 确定 X染色体的测序数 据含量 X-UR%; 以及判断所述双胞胎中胎儿的性别，其中， 当满足下列条件的至少之一时， 确定所述双胞胎中的至少一个为男胎， 当下列条件均不满足时， 确定所述双胞胎均为女胎： Y-UR%大于第一阈值； 和 -1111% (-1；11%大于第二阈值。 利用该方法， 能够有效地确定双 胞胎中是否存在男胎，进而根据孕妇血浆中胎儿 DNA浓度，可以有效确定两个胎儿的性别。 The present invention aims to solve at least one of the technical problems existing in the prior art. In one aspect of the invention, the invention proposes a method of determining the sex of a fetus in a twin. According to an embodiment of the invention, the method comprises: sequencing blood DNA of a twin pregnant woman to obtain a plurality of sequencing data; comparing the sequencing data with a human reference genome sequence to obtain a plurality of unique alignments A unique alignment sequencing data set consisting of sequencing data; determining a number M of unique alignment sequencing data contained in the unique alignment sequencing data set; determining a unique alignment sequencing of the unique alignment sequencing data set derived from the Y chromosome The number of data N y , and at least one of the number N x of unique alignment sequencing data derived from the X chromosome; determining the sequencing data content of the Y chromosome 丫-1; 1% and the X-UR% of the X chromosome At least one of which, according to the formula Y-UR%=N y /M , determines the YU % of the Y chromosome sequence, and determines the X-chromosome sequencing data content according to the formula X-UR%=N X /M. UR%; and determining the sex of the fetus in the twins, wherein at least one of the twins is determined to be a male fetus when at least one of the following conditions is satisfied, when none of the following conditions are satisfied Determining that the twins are female fetuses: Y-UR% is greater than the first threshold; and -1111% (-1; 11% is greater than the second threshold. With this method, it is possible to effectively determine whether there are male fetuses in the twins, and further According to the fetal DNA concentration in the plasma of pregnant women, the sex of the two fetuses can be effectively determined.
在本发明的另一方面， 本发明提出了一种计算机可读介廣。根据本发明的实施例， 该 计算机可读介质上存储有指令， 该指令适于被处理器执行以便通过下列步骤确定双胞胎中 的胎儿性别： 获取双胞胎孕妇的血液 DNA的多个测序数据； 将所述测序数据与人类的参照 基因组序列进行比对， 以便获得由多个唯一比对测序数据构成的唯一比对测序数据集； 确 定所述唯一比对测序数据集中所包含的唯一比对测序数据的数目 M; 确定所述唯一比对测 序数据集中来源于 Y染色体的唯一比对测序数据的数目 Ny, 以及来源于 X染色体的唯一比 对测序数据的数目 Nx的至少一种； 确定 Ύ染色体的测序数据含量 Y-UR%和 X染色体的测 序数据含量 X-UR%的至少一种， 其中， 按照公式 Y-UR%=Ny/M , 确定 Y染色体的测序数 据含量 Y-UR% , 按照公式 X-UR%=NX/M, 确定 X染色体的测序数据含量 -UR%; 以及判 断所述双胞胎中胎儿的性别， 其中， 当满足下列条件的至少之一时， 确定所述双胞胎中的 至少一个为男胎， 当下列条件均不满足时， 确定所述双胞胎均为女胎： Y-UR0/。大于第一阈 值； 和 丫-1¾%«-1¾%大于第二阈值。 如前所述， 利用该计算机可读介盾， 能够有效地确 定双胞胎中是否存在男胎， 进而根据孕妇血浆中胎儿的 DNA浓度， 可以有效推断两胎儿性 别。 In another aspect of the invention, the invention proposes a computer readable medium. According to an embodiment of the invention, the computer readable medium stores instructions adapted to be executed by the processor to determine the sex of the fetus in the twins by: obtaining a plurality of sequencing data of blood DNA of the twin pregnant women; Aligning the sequencing data with a human reference genomic sequence to obtain a unique aligned sequencing data set consisting of a plurality of unique aligned sequencing data; determining unique unique alignment sequencing data contained in the unique alignment sequencing data set a number M; determining the number N y of unique alignment sequencing data derived from the Y chromosome in the unique alignment sequencing data set, and at least one of the number N x of unique alignment sequencing data derived from the X chromosome; determining the chromosome The sequencing data content of Y-UR% and the X-UR% sequencing data content of at least one of X-UR%, wherein, according to the formula Y-UR%=N y /M, the Y-UR% of the Y chromosome is determined. according to the equation X-UR% = N X / M, to determine the content of the sequencing data of the X chromosome -UR%; and determining the sex of a fetus in twins, wherein, when the following conditions to When one of determining at least one of the twin male fetuses, when the following conditions are not satisfied, determining whether the twins are female fetuses: Y-UR 0 /. Greater than the first threshold; and 丫-13⁄4%«-13⁄4% is greater than the second threshold. As described above, using the computer-readable shield, it is possible to effectively determine whether a male fetus is present in the twins, and then according to the fetal DNA concentration in the maternal plasma, the two fetus sexes can be effectively inferred.
在本发明的再一方面， 本发明提出了一种用于确定双胞胎中胎儿性别的***。根据本 发明的实施例， 该***包括： 测序装置， 所述测序装置用于针对双胞胎孕妇的血液 DNA进 行测序， 以便获得多个测序数据； 比对装置， 所述比对装置与所述测序装置相连， 用于所 述测序数据与所述生物的参照基因组序列进行比对， 以便获得由多个唯一比对测序数据构 成的唯一比对测序数据集； 分析装置， 所述分析装置与所述比对装置相连， 并且用于确定 双胞胎中胎儿性别， 其中， 所述分析装置进一步包括： 第一计算模块， 所述第一计算模块 用于确定所述唯一比对测序数据集中所包含的唯一比对测序数据的数目 M; 第二计算模块， 所述第二计算模块用于确定所述唯一比对测序数据集中来源于 Ύ染色体的唯一比对测序数 据的数曰 Ny, 以及来源于 X染色体的唯一比对测序数据的数目 Nx的至少一种； 第三计算 模块，所述第三计算模块用于确定 Y染色体的测序数据含量 Y-UR%和 X染色体的测序数据 含量 X-UR%的至少一种， 其中， 按照公式 Y-UR%=Ny/M， 确定 Y染色体的测序数据含量 Y-UR% , 按照公式 X-i %=Nx/M , 确定 X染色体的测序数据含量 X-UR%; 以及判断模块， 所述判断模块用于判断所述双胞胎中胎儿的性別， 其中， 当满足下列条件的至少之一时， 确定所述双胞胎中的至少一个为男胎， 当下列条件均不满足时， 确定所述双胞胎均为女胎： Y-UR%大于第一阈值； 和 丫-1；1 %«"1；1 %大于第二阈值。 根据本发明的实施例， 利用该系 统， 能够有效地确定双胞胎中胎儿的性别。 In yet another aspect of the invention, the invention proposes a system for determining the sex of a fetus in a twin. According to an embodiment of the invention, the system comprises: a sequencing device for sequencing blood DNA of a twin pregnant woman to obtain a plurality of sequencing data; a comparison device, the comparison device and the sequencing device Connected, the sequencing data is aligned with the reference genomic sequence of the organism to obtain a unique aligned sequencing data set consisting of a plurality of unique aligned sequencing data; an analysis device, the analysis device and the ratio Connected to the device and used to determine The fetal sex in the twins, wherein the analyzing device further comprises: a first calculating module, wherein the first calculating module is configured to determine a number M of unique aligned sequencing data included in the unique aligned sequencing data set; a calculation module, the second calculation module is configured to determine a number 曰N y of the unique alignment sequencing data derived from the Ύ chromosome in the unique alignment sequencing data set, and a number N of unique alignment sequencing data derived from the X chromosome At least one of x ; a third calculation module, configured to determine at least one of a sequencing data content Y-UR% of the Y chromosome and a sequencing data content X-UR% of the X chromosome, wherein, according to the formula Y-UR%=N y /M, determining the sequencing data content Y-UR% of the Y chromosome, determining the X-UR% of the X chromosome by the formula Xi%=N x /M; and the judgment module, The judging module is configured to determine the sex of the fetus in the twins, wherein, when at least one of the following conditions is met, determining that at least one of the twins is a male fetus, and when the following conditions are not satisfied, determining The twins are all female: Y-UR% is greater than the first threshold; and 丫-1; 1% «"1; 1% is greater than the second threshold. According to an embodiment of the present invention, the system can effectively determine the twins The sex of the fetus.
在本发明的又一方面， 本发明还提出了一种用于确定双胞胎中胎儿性别的***。 根 据本发明的实施例， 该***包括： 测序装置， 所述测序装置用于针对所述双胞胎孕妇的血 液 DNA进行测序， 以便获得多个测序数据； 以及前面所述的本发明的计算机可读介质， 其 中该计算机可读介盾上存储有能够确定双胞胎中胎儿性别的指令。 如前所述， 由于理论上， 定位到某条染色体上的测序数据的总数与该染色体的长度以及染色体在生物样本中的含量 成比例， 因而， 利用该***， 通过确定该双胞胎孕妇的血液 DNA样本中 X、 Y染色体的唯 一比对测序数据的数目以及测序数据含量， 可以有效地确定双胞胎中是否存在男胎， 进而 基于估算的孕妇血浆中的胎儿 DNA浓度， 可以有效推断两胎儿性别。 本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得 明显， 或通过本发明的实践了解到。 附图说明  In yet another aspect of the invention, the invention also proposes a system for determining the sex of a fetus in a twin. According to an embodiment of the invention, the system comprises: a sequencing device for sequencing blood DNA of the twin pregnant women to obtain a plurality of sequencing data; and the computer readable medium of the present invention as described above , wherein the computer readable shield stores instructions for determining the sex of the fetus in the twins. As mentioned above, since in theory, the total number of sequencing data located on a chromosome is proportional to the length of the chromosome and the content of the chromosome in the biological sample, the system is used to determine the blood DNA of the pregnant woman of the twins. The number of unique alignments of the X and Y chromosomes in the sample and the amount of sequencing data can effectively determine whether there are male fetuses in the twins, and based on the estimated fetal DNA concentration in the maternal plasma, the two fetus genders can be effectively inferred. The additional aspects and advantages of the invention will be set forth in part in the description which follows. DRAWINGS
本发明的上述和 /或附加的方面和优点从结合下面附图对实施例的描述中将变得明 显和容易理解， 其中：  The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from
图 1 显示了根据本发明一个实施例的用于确定双胞胎中胎儿性别的方法的流程示意 图；  1 shows a schematic flow chart of a method for determining the sex of a fetus in a twin, in accordance with one embodiment of the present invention;
图 2 显示了根据本发明一个实施例的用于确定双胞胎中胎儿性别的***的结构示意 图；  2 shows a schematic structural view of a system for determining the sex of a fetus in a twin according to an embodiment of the present invention;
图 3 示了根据本发明一个实施例的利用本发明的方法获得的样品 S 1 的染色体数字 核型结果； Figure 3 shows the chromosome number of sample S 1 obtained by the method of the present invention, in accordance with one embodiment of the present invention. Karyotype result
图 4显示了根据本发明一个实施例的利用本发明的方法获得的样品 S2的染色体数字核 型结果。 发明详细描述  Figure 4 shows the chromosomal digital karyotype results of sample S2 obtained using the method of the present invention, in accordance with one embodiment of the present invention. Detailed description of the invention
下面祥细描述本发明的实施例， 所述实施例的示例在附图中示出， 其中自始至终相 同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附 图描述的实施例是示例性的， 仅用于解释本发明， 而不能理解为对本发明的限制。  The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the drawings, wherein the same or similar reference numerals are used to refer to the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the drawings are intended to be illustrative only and not to limit the invention.
需要说明的是， 术语 "第一，，、 "第二" 仅用于描述目的， 而不能理解为指示或暗 示相对重要性或者隐含指明所指示的技术特征的数量。 由此， 限定有 "第一"、 "第二" 的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中， 除非另有说明， "多个" 的含义是两个或两个以上。  It should be noted that the terms "first," and "second" are used for descriptive purposes only, and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. The features of the first and second features may include one or more of the features, either explicitly or implicitly. Further, in the description of the present invention, unless otherwise stated, the meaning of "multiple" is two or More than two.
用于确定双胞胎中是否存在男胎的方法  Method for determining the presence or absence of a male fetus in a twin
在本发明的一个方面， 本发明提出了一种用于确定双胞胎中是否存在男胎的方法。 参 考图 1 , 该方法可以包括：  In one aspect of the invention, the invention proposes a method for determining the presence or absence of a male fetus in a twin. Referring to Figure 1, the method can include:
S100: 基因组 DNA测序  S100: Genomic DNA sequencing
在该步驟中， 首先针对需要进行检测的双胞胎孕妇的血液 DNA进行测序， 以便获得多 个测序数据。 根据本发明的实施例， 双胞胎孕妇的血液 DNA的提取方法不受特别限制。 根 据本发明的实施例， 双胞胎孕妇的血液 DNA可以通过选自磁珠法和柱层析法的至少一种提 取， 优选磁珠法。 由此， 利用本发明的方法能够有效地确定双胞胎中是否存在男胎。 其中， 磁珠法提取 DNA的原理如前所述， 在此不再赘述。  In this step, the blood DNA of the twin pregnant women who need to be tested is first sequenced to obtain a plurality of sequencing data. According to an embodiment of the present invention, the method of extracting blood DNA of a pregnant woman of a twin is not particularly limited. According to an embodiment of the present invention, the blood DNA of the twin pregnant women can be extracted by at least one selected from the group consisting of magnetic bead method and column chromatography, preferably magnetic bead method. Thus, the presence or absence of a male fetus in the twins can be effectively determined by the method of the present invention. Among them, the principle of magnetic extraction of DNA by magnetic beads is as described above, and will not be described herein.
根据本发明的实施例， 可以采用的测序装置的类型并不受特别限制。 根据本发明的具 体实施例，考虑到仪器便携性的优势以及高通量性能，测序是通过选自 Roche/454 GS Junior. Illumina/MiSeq以及 Life Tecnologies/Ion Torrent PGM的至少之一进行的。 由此， 能够利用 这些测序装置的高通量、 深度测序的特点， 进一步提高确定确定双胞胎中是否存在男胎的 效率。 测序类型可以为 single-end (单向）测序或者 Pair-end (双向） 测序。 才艮据本发明的 一些实施例， 测序数据的长度为 36~100 bp。 在本发明的一个实施方案中， 所采用的测序平 台为 Ilhmiina/Solexa, 测序类型为 SE36测序。 由此， 可以进一步提高后续分析的效率。 此 夕卜， 在本发明的一个实施方案中， 测序量为 5M测序短片段。  The type of sequencing device that can be employed according to an embodiment of the present invention is not particularly limited. In accordance with a specific embodiment of the present invention, sequencing is performed by at least one selected from the group consisting of Roche/454 GS Junior. Illumina/MiSeq and Life Tecnologies/Ion Torrent PGM, taking into account the advantages of instrument portability and high throughput performance. Thereby, it is possible to further improve the efficiency of determining whether or not a male fetus is present in the twins by utilizing the characteristics of high-throughput and deep sequencing of these sequencing devices. The sequencing type can be single-end (one-way) sequencing or pair-end (two-way) sequencing. According to some embodiments of the invention, the sequenced data is 36 to 100 bp in length. In one embodiment of the invention, the sequencing platform employed is Ilhmiina/Solexa and the sequencing type is SE36 sequencing. Thereby, the efficiency of the subsequent analysis can be further improved. Further, in one embodiment of the invention, the sequencing amount is a 5M sequencing short fragment.
本领域技术人员可以根据所采用的测序平台来选择适当的测序文库构建方法， 筒言之， 构建测序文库的方法可以包括： 首先， 对 DNA片段进行平端化处理和末端添加碱基 A, 并连接接头， 以便得到具有接 头的 DNA片段； 以及 A person skilled in the art can select an appropriate sequencing library construction method according to the sequencing platform used. In other words, the method for constructing the sequencing library can include: First, the DNA fragment is blunt-ended and the base A is added at the end, and the linker is ligated to obtain a DNA fragment having a linker;
对具有接头的 DNA进行扩增， 得到扩增产物即测序文库。  The DNA having the adaptor is amplified to obtain an amplification product, that is, a sequencing library.
根据本发明的实施例， 可以在构建测序文库的过程中， 在测序文库中引入标签序列 Index, 例如可以在接头中引入 Index, 或者在扩增过程中引入标签序列 Index。 由此， 可以 通过针对不同的样本釆用不同的标签序列 , 从而实现同时对多个检测样本进行测序。  According to an embodiment of the present invention, a tag sequence Index can be introduced into the sequencing library during the process of constructing the sequencing library, for example, an index can be introduced in the linker, or a tag sequence Index can be introduced during the amplification process. Thus, simultaneous sampling of multiple test samples can be achieved by using different tag sequences for different samples.
S200: 确定 M、 Nx, Ny的值 S200: determining values of M, N x , N y
在该步骤中， 将所得到的测序数据与人类的参照基因组序列进行比对， 以便获得由多 个唯一比对测序数据构成的唯一比对测序数据集； 接着， 确定所述唯一比对测序数据集中 所包含的唯一比对测序数据的数目 M; 然后， 确定所述唯一比对测序数据集中分别来源于 X和 Y染色体的唯一比对测序数据的数目 Nx和 Ny。 In this step, the obtained sequencing data is aligned with a human reference genome sequence to obtain a unique alignment sequencing data set composed of a plurality of unique alignment sequencing data; and then, the unique alignment sequencing data is determined. The number of unique aligned sequencing data contained in the set is M; then, the number N x and N y of unique aligned sequencing data derived from the X and Y chromosomes, respectively, in the unique aligned sequencing data set are determined.
根据本发明的实施例， 本发明中， 在针对双胞胎孕妇的血液 DNA样品进行检测时， 所 采用的人类的参照基因组序列是人类基因组序列经过屏蔽掉重复序列后所得到的参考序 列， 例如 NCBI数据库中最新版本的人类基因组参考序列。 在本发明的具体实施例中， 参照 基因组序列是 NCBI数据库中版本 36 ( hgl8; NCBI Build 36 ) 的人类基因组参考序列。  According to an embodiment of the present invention, in the detection of a blood DNA sample for a pregnant woman of a twin, the human reference genomic sequence used is a reference sequence obtained by masking the repeat sequence of the human genome sequence, such as the NCBI database. The latest version of the Human Genome Reference Sequence. In a specific embodiment of the invention, the reference genome sequence is the human genome reference sequence of version 36 (hgl8; NCBI Build 36) in the NCBI database.
根据本发明的实施例， 可以通过任何一种序列比对程序进行序列比对， 例如本领域技术 人员可获得的短寡核苷酸分析包 （ Short Oligo nucleotide Analysis Package , SOAP )和 BWA 比对（Burrows- Wheeler Aligner )的至少之一进行， 将测序数据与参考基因组序列进行比对， 得到测序数据在参考基因組上的位置。 进行序列比对可以使用程序提供的默认参数进行， 或者由本领域技术人员根据需要对参数进行选择。 在本发明的具体实施例中， 所采用的比 对软件是 SOAP aligner/soap2„  According to an embodiment of the present invention, sequence alignment can be performed by any sequence alignment program, such as Short Oligonucleotide Analysis Package (SOAP) and BWA alignment (available by those skilled in the art) At least one of Burrows-Wheeler Aligner is performed to align the sequencing data with a reference genomic sequence to obtain the position of the sequencing data on the reference genome. Sequence alignment can be performed using default parameters provided by the program, or can be selected by those skilled in the art as needed. In a particular embodiment of the invention, the comparison software employed is SOAP aligner/soap2
在本文中所使用的术语 "唯一比对测序数据"是指在将测序数据与参照基因组序列进行 比对时， 在参考基因组序列上仅有唯一位置的序列， 以 Unique reads表示。 在本发明的实施 例中， 为了避免重复序列的干扰， 需要去除那些定位于人类基因组参考序列中的串联重复 及转座重复位置的 DNA序列， 只统计那些可以定位到基因组唯一位置的 DNA序列， 即唯 一比对测序数据。唯一比对测序数据能够将来自双胞胎孕妇血液的 DNA分子经打断并测序 后的各 DNA序列定位于特定染色体。  As used herein, the term "unique alignment sequencing data" refers to a sequence that has only a unique position on a reference genomic sequence when the sequencing data is aligned with a reference genomic sequence, as Unique reads. In an embodiment of the invention, in order to avoid interference from repetitive sequences, it is necessary to remove those DNA sequences that are located in tandem repeats and transposition repeat positions in the human genome reference sequence, and only those DNA sequences that can be mapped to unique positions in the genome are counted. That is, the only alignment of the sequencing data. The unique alignment sequencing data maps the DNA sequences from the DNA of twin pregnant women to the specific chromosomes after being interrupted and sequenced.
S300: 确定 X-UR%和 Y-UR%  S300: Determine X-UR% and Y-UR%
在获取 M、 Nx、 Ny的值之后， 按照公式 X-UR%=NX/M和
Figure imgf000006_0001
, 分别确定 X 和 Y染色体的测序数据含量 X-UR%和 Y-UR%。 其中， 在本文中 "X染色体的测序数据含 量 X-UR%" 表示 X 染色体上的唯一比对测序数据的含量， "Y 染色体的测序数据含量 Y-U %" 表示 Y染色体上的唯一比对测序数据的含量。 After obtaining the values of M, N x , Ny , according to the formula X-UR%=N X /M and
Figure imgf000006_0001
, determine the X-UR% and Y-UR% of the sequencing data content of the X and Y chromosomes, respectively. In the text, "X-UR% of the X-chromosome sequence data" indicates the content of the unique alignment sequence data on the X chromosome, "Sequencing data content of the Y chromosome YU %" represents the content of the unique alignment sequencing data on the Y chromosome.
S400: 判断双胞胎中胎儿性别  S400: Judging the sex of the fetus in twins
在该步骤中， 在确定 X和 Υ染色体的测序数据含量 X-UR%和 Y-U %之后， 可以基于 丫-111 %«-1；1 %和 Y-UR%的值， 确定该双胞胎孕妇的双胞胎中是否存在男胎。 具体地， 当 满足下列条件的至少之一时， 可以确定双胞胎中的至少一个为男胎， 当下列条件均不满足 时， 可以确定该双胞胎均为女胎： 丫-1!1 %大于第一阈值； 和 Y-UR%/X-UR%大于第二阈值。  In this step, after determining the X-UR% and YU% of the X and Υ chromosome sequencing data, the twins of the twin pregnant women can be determined based on the values of 丫-111%«-1; 1% and Y-UR%. Whether there is a male fetus. Specifically, when at least one of the following conditions is satisfied, it can be determined that at least one of the twins is a male fetus, and when none of the following conditions are satisfied, it can be determined that the twins are female fetuses: 丫-1!1% is greater than the first threshold ; and Y-UR%/X-UR% are greater than the second threshold.
其中， 根据本发明的一个为实施例， 该第一阁值与第二阈值是通过以下方法确定的： 取女胎孕妇血浆样品 100 例， 分别计算 Y-UR。 Y-UR%/X-U %, 并分别绘制各样品的 Y-UR。/。和 Y-UR%/X-UR%的正态分布曲线， 根据中心极限定理， 该 100例样品的 Y-UR%和 Y-UR%/X-UR%均符合正态分布， 取 Y-UR°/W 99%置信区间的右区间点作为第一阀值， 取 Y-UR%/X-UR°/。的 99%置信区间的右区间点作为第二阈值。由此，确定该第一阈值为 0.0004, 该第二阈值为 0.0075。 从而， 当 Y- UR%大于 0.0004或 Y- UR%/X-UR%大于 0.0075时， 可以 确定该双胞胎中存在男胎。  Wherein, according to one embodiment of the present invention, the first threshold value and the second threshold value are determined by the following method: 100 samples of maternal fetal plasma samples are taken, and Y-UR is calculated separately. Y-UR%/X-U %, and draw the Y-UR of each sample separately. /. And the normal distribution curve of Y-UR%/X-UR%, according to the central limit theorem, the Y-UR% and Y-UR%/X-UR% of the 100 samples are in accordance with the normal distribution, taking Y-UR The right interval point of the °/W 99% confidence interval is taken as the first threshold, taking Y-UR%/X-UR°/. The right interval point of the 99% confidence interval is used as the second threshold. Thus, the first threshold is determined to be 0.0004, and the second threshold is 0.0075. Thus, when Y-UR% is greater than 0.0004 or Y-UR%/X-UR% is greater than 0.0075, it can be determined that a male tire is present in the twin.
此外， 根据本发明的实施例， 当确定双胞胎中的至少一个为男胎时， 可以进一步包括： 首先， 基于上述获得的 Nx和 NY, 分别进行胎儿 DNA浓度估算， 以便分别得到第一胎 L DNA M%chrX, 以及第二胎儿 DNA浓度％chrY。 根据本发明的实施例， 可以通过以 下步骤进行胎儿 DNA浓度估算： 分别选取正常男性和正常女性血浆样品集作为对照， 分别 计算各血浆样品的丫-111 %和 -UR%;分别计算各男性血浆样品的 Y-UR0/。和 X-UR%的算术 平均值， 记作 YM和 XM， 分别计算各女性血浆样品的 Y-UR°/^n X-UR0/。的算术平均值， 记 作 YF和 XF;然后，根据公式 (X-UR%-XF)/(XM-XF)计算出利用 X染色体估计的第一胎儿 DNA M%chrX,根据公式 (Y-UR%-YF)/(YM-YF)计算得出利用 Y染色体估计的第二胎儿 DNA浓¾%chrY, 其中公式中的 -111 %为待测孕妇血浆样品的 X-UR%值， Y-UR%为待测孕妇血 浆样品的 Y-UR%值。 Furthermore, according to an embodiment of the present invention, when it is determined that at least one of the twins is a male fetus, the method further includes: first, estimating the fetal DNA concentration based on the obtained N x and N Y respectively, so as to obtain the first fetus respectively L DNA M%chrX, and second fetal DNA concentration %chrY. According to an embodiment of the present invention, fetal DNA concentration estimation can be performed by the following steps: selecting normal male and normal female plasma sample sets as controls, respectively calculating 丫-111% and -UR% of each plasma sample; respectively calculating each male plasma Sample Y-UR 0 /. The arithmetic mean of X-UR%, denoted as Y M and X M , was calculated for each female plasma sample as Y-UR°/^n X-UR 0 /. The arithmetic mean value, denoted as Y F and X F ; then, the first fetal DNA M%chrX estimated using the X chromosome is calculated according to the formula (X-UR%-X F )/(X M -X F ), according to The formula (Y-UR%-Y F )/(Y M -Y F ) calculates the second fetal DNA concentration 3⁄4%chrY estimated using the Y chromosome, where -111% of the formula is the X of the plasma sample of the pregnant woman to be tested The -UR% value, Y-UR% is the Y-UR% value of the plasma sample of the pregnant woman to be tested.
然后， 确定所述％<±1¾和％(；1«^的显著差异性 p-value。 进而， 基于 p-value可以确定双 胞胎中两胎儿的性别。 具体地， 根据本发明的实施例， 如果 p-value>0.1 , 可以确定双胞胎 中的胎儿均为男胎， 如果 p-value<0.1 , 且。 /。chrX 大于％chrY, 则可以确定该双胞胎中存在 一个男胎和一个女胎。 这是因为， p-vahie>0.1时， ％chrX和％。1^丫差异较小， 说明两个胎 儿贡献的落于 X、 Y染色体上的 DNA浓度几乎相等， 只有当两个胎儿均为男胎时， 两个胎 儿贡献的落于 X、 Y染色体上的 DNA浓度才会相等（因为此时两个胎儿提供了两条 X染色 体和两条 Y染色体）； 如果两个胎儿中是一个男胎一个女胎， 则两个胎儿贡献的落于 X、 Y 染色体上的 DNA浓度会是 X染色体上的 DNA浓度高于 Y染色体上的 DNA浓度（因为此 时两个胎儿提供了三条 X染色体和一条 Y染色体）。 此外， 如果胎儿存在染色体变异（例如 非整倍体）， 还可以参考利用变异染色体估算的胎儿 DNA浓度推断双胞胎胎儿的性别， 具 体地， 如果利用变异染色体估计得到的胎儿 DNA浓度约为利用 Υ染色体估计得到的胎儿 DNA浓度的 1/2, 则推断两胎儿为两个男胎。 需要说明的是， 在本文中参考利用变异染色体 估算的胎儿 DNA浓度推断双胞胎胎儿性别时，是以默认假设只有一个胎儿出现异常为前提 的 （因为两个胎儿都异常的几率很小）， 进而， 利用异常染色体估算得到的胎儿浓度只是其 中一个胎儿提供的 DNA浓度。如果两个胎儿均为男胎，那么利用 Υ染色体估算得到的胎儿 浓度则是两个胎儿提供的 DNA浓度，理论上是上述用变异染色体估算得到的胎儿浓度的两 倍。从而， 当利用变异染色体估计得到的胎儿 DNA浓度约为利用 Υ染色体估计得到的胎儿 DNA浓度的 1/2时， 可以推断两胎儿均为男胎。 Then, the significant difference p-values of the %<±13⁄4 and %(;1«^ are determined. Further, the sex of the two fetuses in the twins can be determined based on the p-value. Specifically, according to an embodiment of the present invention, P-value>0.1, it can be determined that the fetus in the twins is a male fetus. If p-value<0.1, and /chrX is greater than %chrY, it can be determined that there is one male fetus and one female fetus in the twin. Because, when p-vahie>0.1, the difference between %chrX and %11 is small, indicating that the concentration of DNA on the X and Y chromosomes contributed by the two fetuses is almost equal, only when both fetuses are male. The concentration of DNA on the X and Y chromosomes contributed by the two fetuses will be equal (because the two fetuses provide two X chromosomes and two Y chromosomes); if the two fetuses are a male fetus and a female The concentration of DNA on the X and Y chromosomes contributed by the two fetuses is that the DNA concentration on the X chromosome is higher than the DNA concentration on the Y chromosome (because of this When the two fetuses provide three X chromosomes and one Y chromosome). In addition, if there is a chromosomal variation in the fetus (for example, aneuploidy), the sex of the twin fetus can also be inferred by using the fetal DNA concentration estimated by the variant chromosome. Specifically, if the estimated fetal DNA concentration using the variant chromosome is about using the Υ chromosome Estimating the 1/2 of the fetal DNA concentration obtained, it is concluded that the two fetuses are two male fetuses. It should be noted that, in this paper, the gender of the fetus is estimated by using the estimated fetal DNA concentration of the mutant chromosome, which is premised on the assumption that only one fetus is abnormal (because the probability of abnormality of both fetuses is small), and further, The fetal concentration estimated using abnormal chromosomes is only the DNA concentration provided by one of the fetuses. If both fetuses are male, then the fetal concentration estimated using the sputum chromosome is the DNA concentration provided by the two fetuses, theoretically twice the fetal concentration estimated using the mutated chromosome. Thus, when the fetal DNA concentration estimated using the mutant chromosome is about 1/2 of the fetal DNA concentration estimated using the sputum chromosome, it can be inferred that both fetuses are male fetuses.
进一步， 当针对多个双胞胎孕妇的血液 DNA样本进行检测时， 可以在构建测序文库的 过程中， 在测序文库中引入标签序列 Index, 由此， 可以通过针对不同的样本采用不同的标 签序列， 实现同时对多个检测样本进行测序 , 并通过标签有效地区分各样本的测序数据， 进而能够分别获得各样本的 M、 Nx、 Ny的值， 从而能够分别进行后续的双胞胎中是否存在 男胎的判断。 Further, when detecting blood DNA samples of a plurality of twin pregnant women, the tag sequence Index can be introduced into the sequencing library during the process of constructing the sequencing library, thereby implementing different tag sequences for different samples. At the same time, multiple test samples are sequenced, and the sequencing data of each sample is effectively distinguished by the label, and then the values of M, N x and N y of each sample can be respectively obtained, so that whether there are male tires in the subsequent twins respectively Judgment.
具体地， 根据本发明的一些实施例， 本发明的确定双胞胎中是否存在男胎的方法还可 以包括： 统计比对到 X和 Y染色体上的唯一比对测序短序列数（Nx、 Ny )， 计算该唯一比 对测序短序列数与全基因组唯一比对测序短序列数（M ) 的比值， 分别记 ί故 X-UR%和 Y-UR% , 当丫-1¾%大于第一阈值且 Y-UR% X-UR%大于第二阈值时， 判定双胞胎中存在男 胎。 再结合孕妇血浆中胎儿 DNA浓度， 确定该双胞胎的性别， 具体方法前面已详细描述， 不再赘述。 根据本发明的实施例， 第一阈值为 0.0004 (即大于 0.0004时判定为存在男胎）， 第二阈值为 0.0075 (即大于 0.0075时判定为存在男胎）。 Specifically, according to some embodiments of the present invention, the method of determining whether a male fetus is present in a twin may further comprise: statistically comparing the number of short sequences sequenced to the unique alignment on the X and Y chromosomes (N x , N y Calculating the ratio of the number of short sequences sequenced by the unique alignment to the short sequence number (M) of the whole genome unique alignment sequence, respectively, X-UR% and Y-UR%, when 丫-13⁄4% is greater than the first threshold When Y-UR% X-UR% is greater than the second threshold, it is determined that there is a male fetus in the twins. The gender of the twins is determined by combining the fetal DNA concentration in the plasma of the pregnant woman. The specific method has been described in detail above and will not be described again. According to an embodiment of the invention, the first threshold is 0.0004 (i.e., greater than 0.0004 is determined to be the presence of a male fetus) and the second threshold is 0.0075 (i.e., greater than 0.0075 is determined to be the presence of a male fetus).
如前所述， 由于理论上， 定位到某条染色体上的测序数据的总数与该染色体的长度以 及染色体在生物样本中的含量成比例， 因而， 利用该方法， 通过确定该双胞胎孕妇的血液 DNA样本中 Y染色体的测序数据含量 Y-UR% , 或者 Y-UR% X-UR。/ 值， 可以有效地确 定双胞胎中是否存在男胎。 进而， 基于孕妇血浆中胎儿 DNA浓度， 能够进一步确定双胞胎 中两个胎儿的性别。 计算机可读介质  As mentioned above, since, in theory, the total number of sequencing data located on a chromosome is proportional to the length of the chromosome and the content of the chromosome in the biological sample, the blood DNA of the pregnant woman is determined by the method. The sequencing data content of the Y chromosome in the sample is Y-UR%, or Y-UR% X-UR. / value, can effectively determine whether there are male fetuses in the twins. Further, based on the fetal DNA concentration in the plasma of the pregnant woman, the sex of the two fetuses in the twins can be further determined. Computer readable medium
在本发明的叉一方面， 本发明提出了一种计算机可读介质。 根据本发明的实施例， 该计 算机可读介质上存储有指令， 所述指令适于被处理器执行以便通过下列步骤确定双胞胎中 胎儿性别： In one aspect of the present invention, the present invention provides a computer readable medium. According to an embodiment of the invention, the computer readable medium stores instructions thereon, the instructions being adapted to be executed by the processor to determine in the twins by the following steps Fetal gender:
首先， 获取双胞胎孕妇的血液 DNA的多个测序数据。  First, obtain multiple sequencing data for blood DNA from twin pregnant women.
接下来， 将所述测序数据与人类的参照基因组序列进行比对， 以便获得由多个唯一比 对测序数据构成的唯一比对测序数据集。 根据本发明的实施例， 所述参照基因组序列为 NCBI数据库中版本 36的人类基因组参考序列。 根据本发明的具体实施例， 采用 SOAP和 BWA的至少之一， 将所述测序数据与所述生物的参照基因组序列进行比对。 根据本发明的 实施例， 所述测序数据的长度为 36 100 bp。  Next, the sequencing data is aligned with a human reference genomic sequence to obtain a unique aligned sequencing data set consisting of multiple unique aligned sequencing data. According to an embodiment of the invention, the reference genomic sequence is a human genome reference sequence of version 36 of the NCBI database. According to a particular embodiment of the invention, the sequencing data is aligned with the reference genomic sequence of the organism using at least one of SOAP and BWA. According to an embodiment of the invention, the sequencing data has a length of 36 100 bp.
接着， 确定所述唯一比对测序数据集中所包含的唯一比对测序数据的数目 M。  Next, the number M of unique aligned sequencing data contained in the unique aligned sequencing data set is determined.
接下来， 确定所述唯一比对测序数据集中来源于 Y 染色体的唯一比对测序数据的数目 N 以及来源于 X染色体的唯一比对测序数据的数目 Nx的至少一种。 Next, it is determined that the unique alignment sequencing data sets at least one of the number N of unique alignment sequencing data derived from the Y chromosome and the number N x of unique alignment sequencing data derived from the X chromosome.
然后， 确定 Y染色体的测序数据含量 Y-UR0/。和 X染色体的测序数据含量 X-UR%的至 少一种， 其中， 按照公式 Y-UR%=Ny/M， 确定 Y染色体的测序数据含量 Y- UR%， 按照公式 X-U %=NX/M, 确定 X染色体的测序数据含量 X-U %。 Then, the sequencing data content of the Y chromosome is determined to be Y-UR 0 /. And X-phase sequence data content of at least one of X-UR%, wherein, according to the formula Y-UR%=N y /M, the Y-UR% sequencing data content Y- UR% is determined according to the formula XU %=N X / M, determine the XU % of the sequencing data content of the X chromosome.
最后， 判断所述双胞胎中胎儿的性别， 其中， 当满足下列条件的至少之一时， 确定所 述双胞胎中的至少一个为男胎， 当下列条件均不满足时，确定所述双胞胎均为女 Y-UR% 大于第一阈值； 和丫-1；1 %«-1¾%大于第二阈值。 根据本发明的实施例， 所述第一阈值为 0.0004, 第二阁值为 0.0075。 由此， 当 Y-UR%大于 0.0004或 Y-UR%/X-UR%大于 0.0075时， 可以确定该双胞胎中存在男胎。 才艮据本发明的一个实施例， 当确定所述双胞胎中的至少一 个为男胎时， 可以进一步包括:基于所述 Nx和 Νγ， 分别进行胎儿 DNA浓度估算， 以便分 别得到第一胎儿 DNA浓度。 /。chrX, 以及第二胎儿 DNA浓度％^1 ； 以及确定所述％chrX 和％。1«^的显著差异性 p-vahie, 其中， 如果 p-value>0.1 , 则确定所述双胞胎中的胎儿均为 男胎， 如果 p-value<0.1 , 且％(；1«¾ 大于％chrY, 则确定所述双胞胎中存在一个男胎和一个 女胎。 Finally, determining the sex of the fetus in the twins, wherein, when at least one of the following conditions is satisfied, determining that at least one of the twins is a male fetus, and determining that the twins are female Y when none of the following conditions are satisfied -UR% is greater than the first threshold; and 丫-1; 1%«-13⁄4% is greater than the second threshold. According to an embodiment of the invention, the first threshold is 0.0004 and the second threshold is 0.0075. Thus, when Y-UR% is greater than 0.0004 or Y-UR%/X-UR% is greater than 0.0075, it can be determined that a male fetus is present in the twin. According to an embodiment of the present invention, when it is determined that at least one of the twins is a male fetus, the method further includes: performing fetal DNA concentration estimation based on the N x and Ν γ , respectively, to obtain the first fetus separately DNA concentration. /. chrX, and second fetal DNA concentration %^1; and determining the %chrX and %. 1 significant difference p-vahie, where, if p-value>0.1, it is determined that the fetus in the twins are male, if p-value<0.1, and %(;1«3⁄4 is greater than %chrY And determining that there is one male fetus and one female fetus in the twins.
如前所述， 由于理论上， 定位到某条染色体上的测序数据的总数与该染色体的长度以 及染色体在生物样本中的含量成比例， 因而， 利用该计算机可读介盾， 通过确定该双胞胎 孕妇的血液 DNA样本中 Y染色体的测序数据含量 Y-UR%, 或者 Y-UR%/X-UR%的值， 可 以有效地确定双胞胎中是否存在男胎。 进而， 基于孕妇血浆中胎儿 DNA浓度， 能够进一步 确定双胞胎中两个胎儿的性别。  As mentioned above, since in theory, the total number of sequencing data located on a chromosome is proportional to the length of the chromosome and the content of the chromosome in the biological sample, the computer-readable shield is used to determine the twin The Y-UR%, or Y-UR%/X-UR% value of the Y chromosome in the blood DNA sample of the pregnant woman can effectively determine whether there is a male fetus in the twin. Further, based on the fetal DNA concentration in the maternal plasma, the sex of the two fetuses in the twins can be further determined.
需要说明的是， 前面针对用于确定双胞胎中胎儿性别的方法所进行的描述， 也适用于 该计算机可读介质， 在此不再赘述。 用于确定双胞胎中胎儿性别的*** It should be noted that the foregoing description of the method for determining the sex of a fetus in a twin also applies to the computer readable medium, and details are not described herein again. System for determining the sex of a fetus in a twin
在本发明的另一方面， 本发明提出了一种用于确定双胞胎中胎儿性别的***。根据本 发明的实施例， 参照图 2， 该***包括： 测序装置 A100、 比对装置 A200和分析装置 A300。 根据本发明的实施例， 测序装置 A100用于针对双胞胎孕妇的血液 DNA进行测序， 以便获 得多个测序数据， 比对装置 A200与测序装置 A100相连， 用于将所述测序数据与人类的参 照基因组序列进行比对， 以便获得由多个唯一比对测序数据构成的唯一比对测序数据集， 分析装置 A300与比对装置 A200相连， 并且用于确定双胞胎中胎儿性别。 根据本发明的实 施例 , 所述参照基因組序列为 NCBI数据库中版本 36的人类基因组参考序列。 根据本发明 的具体实施例， 采用 SOAP和 BWA的至少之一，将所述测序数据与所述生物的参照基因组 序列进行比对。  In another aspect of the invention, the invention proposes a system for determining the sex of a fetus in a twin. According to an embodiment of the present invention, referring to Fig. 2, the system includes: a sequencing device A100, a comparison device A200, and an analysis device A300. According to an embodiment of the present invention, the sequencing device A100 is configured to sequence blood DNA of a twin pregnant woman to obtain a plurality of sequencing data, and the comparison device A200 is connected to the sequencing device A100 for using the sequencing data with a human reference genome The sequences are aligned to obtain a unique aligned sequencing data set consisting of a plurality of unique aligned sequencing data, and analytical device A300 is coupled to comparison device A200 and is used to determine fetal gender in the twins. According to an embodiment of the invention, the reference genomic sequence is the human genome reference sequence of version 36 of the NCBI database. According to a particular embodiment of the invention, the sequencing data is aligned with the reference genomic sequence of the organism using at least one of SOAP and BWA.
根据本发明的实施例， 分析装置 A300进一步包括： 第一计算模块 A310 , 用于确定所 述唯一比对测序数据集中所包含的唯一比对测序数据的数！] M; 第二计算模块 A320, 用于 确定所述唯一比对测序数据集中来源于 Y染色体的唯一比对测序数据的数目 Ny, 以及来源 于 X染色体的唯一比对测序数据的数目 Nx的至少一种； 第三计算模块 A330 , 用于确定 Y 染色体的测序数据含量 Y-UR。/t^ X染色体的测序数据含量 X-UR%的至少一种， 其中， 按 照公式 Y-UR%=Ny/M, 确定 Y染色体的测序数据含量 Y-UR% , 按照公式 X-UR%=NXZM, 确定 X染色体的测序数据含量 X-U %; 以及判断模块 A340, 用于用于判断所述双胞胎中 胎儿的性别， 其中， 当满足下列条件的至少之一时， 确定所述双胞胎中的至少一个为男胎， 当下列条件均不满足时， 确定所述双胞胎均为女胎： Y-UR%大于第一阈值； 和 Y-UR%/X-UR°/。大于第二阈值。 根据本发明的实施例， 所述第一阈值为 0.0004 , 第二阈值为 0.0075。 由此， 当 Y-UR%大于 0.0004或 Y-UR%/X-UR%大于 0.0075时， 可以确定该双胞胎 中存在男胎。 According to an embodiment of the present invention, the analyzing device A300 further includes: a first calculating module A310, configured to determine the number of unique aligned sequencing data included in the unique aligned sequencing data set! a second calculation module A320, configured to determine the number N y of unique alignment sequencing data derived from the Y chromosome in the unique alignment sequencing data set, and the number of unique alignment sequencing data derived from the X chromosome N x At least one of; a third calculating module A330, configured to determine a sequencing data content Y-UR of the Y chromosome. /t^ X chromosome sequencing data content of at least one of X-UR%, wherein, according to the formula Y-UR%=N y /M, the Y-UR% of the Y chromosome is determined according to the formula X-UR% =N X ZM, determining the sequencing data content XU % of the X chromosome; and determining module A340 for determining the sex of the fetus in the twins, wherein the at least one of the following conditions is determined At least one is a male fetus, and when the following conditions are not satisfied, it is determined that the twins are female fetuses: Y-UR% is greater than a first threshold; and Y-UR%/X-UR°/. Greater than the second threshold. According to an embodiment of the invention, the first threshold is 0.0004 and the second threshold is 0.0075. Thus, when Y-UR% is greater than 0.0004 or Y-UR%/X-UR% is greater than 0.0075, it can be determined that a male fetus is present in the twin.
根据本发明的实施例， 判断模块 A340可以进一步包括验证单元， 该验证单元适于在确 定该双胞胎中的至少一个为男胎时，执行下列操作：基于所述 Nx和 NY,分别进行胎儿 DNA 浓度估算， 以便分别得到第一胎儿 DNA浓度％chrX, 以及第二胎儿 DNA浓度％chrY; 以 及确定所述％ 1«¾和。 /。chrY的显著差异性 p-value, 其中， 如果 p-value>0.1， 则确定所述双 胞胎中的胎儿均为男胎， 如果 p-value<0.1 , 且％(；1^ 大于％chrY, 则确定所述双胞胎中存 在一个男胎和一个女胎。 According to an embodiment of the present invention, the judging module A340 may further include a verification unit adapted to perform the following operations when determining that at least one of the twins is a male fetus: performing the fetus separately based on the N x and N Y The DNA concentration is estimated to obtain a first fetal DNA concentration %chrX, and a second fetal DNA concentration %chrY, respectively; and to determine the % 1 «3⁄4 sum. /. a significant difference p-value of chrY, wherein, if p-value>0.1, it is determined that the fetus in the twins is a male fetus, if p-value<0.1, and %(;1^ is greater than %chrY, then determining There is one male and one female in the twins.
如前所述， 由于理论上， 定位到某条染色体上的测序数据的总数与该染色体的长度以 及染色体在生物样本中的含量成比例， 因而， 利用该***， 通过确定该双胞胎孕妇的血液 DNA样本中 Y染色体的测序数据含量 Y-UR% , 或者 Y-UR%/X-UR%的值， 可以有效地确 定双胞胎中是否存在男胎。 然后基于孕妇血浆中的胎儿 DNA浓度， 可以有效确定双胞胎中 两胎儿的性别。 As mentioned above, since in theory, the total number of sequencing data located on a chromosome is proportional to the length of the chromosome and the content of the chromosome in the biological sample, the system is used to determine the blood DNA of the pregnant woman of the twins. The Y-UR%, or the value of Y-UR%/X-UR%, of the Y chromosome in the sample can be effectively confirmed. Whether there are male fetuses in twins. The sex of the two fetuses in the twins can then be effectively determined based on the fetal DNA concentration in the maternal plasma.
需要说明的是， 前面所述的比对装置和分析装置的功能可以由前面所述的计算机可读 介盾来执行。 由此， 在本发明的再一方面， 本发明叉提出了另外一种用于确定双胞胎中 胎儿性别的***。 根据本发明的实施例， 该***可以包括： 测序装置， 所述测序装置用 于针对所述生物样本的基因组 DNA进行测序， 以便获得多个测序数据； 以及前面所述 的计算机可读介质。 如前所述， 由于理论上， 定位到某条染色体上的测序数据的总数与该 染色体在生物样本中的含量成比例，因而，利用该***，通过确定该双胞胎孕妇的血液 DNA 样本中 Y染色体的测序数据含量 Y-UR%, 或者 Y-UR%/X-UR%的值， 可以有效地确定双胞 胎中是否存在男胎。 进而， 基于孕妇血浆中的胎儿 DNA浓度， 可以有效确定双胞胎中两胎 儿的性别。  It should be noted that the functions of the comparison device and the analysis device described above can be performed by the computer-readable shield described above. Thus, in yet another aspect of the invention, the present invention proposes another system for determining the sex of a fetus in a twin. According to an embodiment of the present invention, the system may include: a sequencing device for sequencing genomic DNA of the biological sample to obtain a plurality of sequencing data; and the computer readable medium described above. As mentioned above, since, in theory, the total number of sequencing data located on a chromosome is proportional to the content of the chromosome in the biological sample, the system is used to determine the Y chromosome in the blood DNA sample of the twin pregnant woman. The sequencing data content of Y-UR%, or the value of Y-UR%/X-UR%, can effectively determine whether a male fetus is present in the twins. Further, based on the fetal DNA concentration in maternal plasma, the sex of the two fetuses in the twins can be effectively determined.
需要说明的是， 前面针对用于确定双胞胎中胎儿性别的方法所进行的描述， 也适用于 该用于确定双胞胎中胎儿性别的***， 在此不再赘述。  It should be noted that the foregoing description of the method for determining the sex of a fetus in a twin also applies to the system for determining the sex of a fetus in a twin, and will not be described herein.
此外， 还需要说明的是， 本发明的用于确定双胞胎中胎儿性别的方法、 ***和计算机 可读介质具有如下优点：  Furthermore, it should also be noted that the method, system and computer readable medium of the present invention for determining the sex of a fetus in a twin have the following advantages:
1、 能够有效地针对双胞胎孕妇的血液 DNA, 确定双胞胎中是否存在男胎， 进而可以对 双胎的性别进行推测判断， 对胎儿的卵型鉴定有一定的指导意义。  1. It can effectively target the blood DNA of twin pregnant women, determine whether there are male fetuses in the twins, and then predict the sex of the twins, which has certain guiding significance for the identification of the fetus.
2、 临床可行性： 仅需 5M左右的测序数据， 即可准确确定双胞胎中的胎儿性别， 大大 减少了数据产生的成本和时间。  2, clinical feasibility: only need about 5M of sequencing data, you can accurately determine the sex of the fetus in the twins, greatly reducing the cost and time of data generation.
3、 安全性： 采用无创产前技术提取双胞胎孕妇血浆 DNA进行建库测序分析， 能够有 效减少孕妇胎儿流产的风险。  3. Safety: The use of non-invasive prenatal technology to extract plasma DNA from twin pregnant women for library sequencing analysis can effectively reduce the risk of fetal abortion in pregnant women.
4、 准确性： 相对于常规的 B超检查和血清代谢物检查， 本发明的用于确定双胞胎中胎 儿性别的方法、 ***和计算机可读介质， 准确性显著。 下面将结合实施例对本发明的方案进行解释。 本领域技术人员将会理解， 下面的实施 例仅用于说明本发明， 而不应视为限定本发明的范围。 实施例中未注明具体技术或条件的， 按照本领域内的文献所描述的技术或条件 (例如参考 J.萨姆布鲁克等著， 黄培堂等译的《分 子克隆实验指南》， 第三版， 科学出版社）或者按照产品说明书进行。 实施例中未注明具体 条件者， 按照常规条件或制造商建议的条件进行。 所用试剂或仪器未注明生产厂商者， 均 为可以通过市场获得的常规产品。 以下括号内为各个试剂或试剂盒的厂家货号。 所使用的 测序用的接头和标签序列来源于 Illumiiia公司的 Multiplexing Sample Preparation Oligonutide Kit。 4. Accuracy: The method, system and computer readable medium of the present invention for determining the sex of a fetus in a twin are significantly accurate relative to conventional B-ultrasound and serum metabolite examination. The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following examples are merely illustrative of the invention and should not be construed as limiting the scope of the invention. In the examples, the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. If no specific conditions are specified in the examples, they are carried out according to the general conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained through the market. The following brackets indicate the manufacturer number of each reagent or kit. The linker and tag sequences used for sequencing were derived from Illliiia's Multiplexing Sample Preparation Oligonutide Kit.
实施例 1:  Example 1:
针对 1例双胎 T21阳性样本和 1 例双月台 T18阳性样本， 根据本发明的用于确定生物样 本中染色体数目异常以及双胞胎中是否存在男胎的方法， 按照以下步骤，检测验证其染色 体数目异常和双胞胎性别情况：  According to the method for determining abnormal chromosome number in a biological sample and the presence or absence of a male fetus in a twin according to the present invention, the following steps are performed to verify the chromosome number of the patient. Abnormal and twin gender conditions:
1、 DNA提取及建库：  1. DNA extraction and database construction:
按照 TiangenDP327-02Kit操作流程提取上述 2例血浆样品 （即 1例双胎 T21阳性样本 和 1 例双胎 T18 阳性样本， 以下简称 Sl、 S2 ) 的 DNA, 所提取 DNA按照修改后的 Illumina/Solexa标准建库流程进行建库， 在本身集中于 200bp的 DNA分子两端被加上测序 所用接头， 每个样本被加上不同的标签序列 （index ), 然后与 flowcell表面互补接头杂交， 在一定条件下使核酸分子成簇生长， 然后在 IlluminaHiseq2000上通过单末端测序， 得到长 度为 36bp的 DNA片段序列。  The DNA of the above 2 plasma samples (ie, 1 twin T21 positive sample and 1 twin fetal T18 positive sample, hereinafter referred to as Sl, S2) was extracted according to the Tiangen DP327-02 Kit protocol. The extracted DNA was according to the modified Illumina/Solexa standard. The library construction process is carried out, and the linker used for sequencing is added to both ends of the 200 bp DNA molecule. Each sample is added with a different tag sequence (index), and then hybridized with the flowcell surface complementary link under certain conditions. The nucleic acid molecules were clustered and then sequenced by single-end sequencing on IlluminaHiseq2000 to obtain a DNA fragment sequence of 36 bp in length.
具体地， 将获自上述血浆样品的约 10ng 的 DNA, 按照进行修改后的 Illumina/Solexa 标准流程建库， 具体流程参照现有技术 （可参见 http://www.mumina.com7提供的 Illumina/Solexa标准建库说明书， 通过参照将其全文并入本文）。 然后， 经 2100Bioanalyzer (Agilent)确定 DNA文库大小及***片段为约 200bp, QPCR精确定量后备用。  Specifically, about 10 ng of DNA obtained from the above plasma sample was constructed according to the modified Illumina/Solexa standard procedure, and the specific procedure was referred to the prior art (see Illumina/ available at http://www.mumina.com7). The Solexa standard library specification is incorporated by reference in its entirety. Then, the DNA library size and insert were determined to be about 200 bp by the 2100 Bioanalyzer (Agilent), and the QPCR was accurately quantified and used.
2、 测序及序列比对：  2. Sequencing and sequence alignment:
将上述获得的两个文库进行上机测序， 具体地， 按照 Illumina/Solexa 官方公布的 ClusterStation和 Hiseq2000 ( PEsequencing )说明书进行操作， 使每个样品得到约 0.25G数 据量， 其中各样本根据前述 index标签区进行分开。  The two libraries obtained above were subjected to on-machine sequencing, specifically, according to the Illumina/Solexa officially published ClusterStation and Hiseb2000 (PEsequencing) instructions, each sample was obtained with a data volume of about 0.25 G, wherein each sample was subjected to the aforementioned index label. The districts are separated.
然后， 利用比对软件 SOAP2, 将获得的各样本的测序数据与 NCBI 数据库中版本 36 ( hgl8; NCBIBuild36 )的人类基因组参考序列进行不容错比对， 以便分别获得各样本的由 多个唯一比对测序数据构成的唯一比对测序数据集。  Then, using the comparison software SOAP2, the obtained sequencing data of each sample is compared with the human genome reference sequence of version 36 (hgl8; NCBIBuild36) in the NCBI database, so as to obtain multiple unique comparisons of each sample respectively. The unique alignment of the sequencing data consists of sequencing data sets.
3、 数据分析  3, data analysis
a )基本统计： 统计各染色体上所落的测序短序列数（即唯一比对的 reads数， 记为 ^ , 下标 i和 j分别代表预定染色体编号和样本编号，以作区分，下不复述）和平均 GC含量 (记 为 GCi, 。 为了弭除样品数据量的差异， 定义相对测序序列数（ ) 为测序序列数和各染 =丄 ^J = 1°g2 a) Basic statistics: Count the number of sequencing short sequences on each chromosome (ie, the number of unique pairs of reads, denoted as ^, subscripts i and j represent the predetermined chromosome number and sample number, respectively, for distinction, not repeated And the average GC content (denoted as GC i, in order to eliminate the difference in the amount of sample data, define the relative number of sequencing sequences ( ) for the number of sequencing sequences and each dye = 丄 ^ J = 1 °g2
色体的平均测序序列数（ n ' )之比的 2为底对数值， 即 The ratio of the average number of sequencing sequences ( n ' ) of the color bodies is 2, which is the base pair value, that is,
b )数据校正和标准化： ①对对照集各染色体的 R"和 进行线性拟合， 获得拟合斜率 和截距 b) Data correction and standardization: 1 pair of control sets of each particle's R " and linear fit to obtain the fitted slope and intercept
②对受试样本， 计算绽得理论值 " GC i + b", 2 pairs of specimens, calculate the theoretical value " GC i + b ",
③进行校正和标准化， 其中定义平均标准化值（ z" )如下： 3 Perform calibration and standardization, where the average normalized value ( z " ) is defined as follows:
¾= ,-^--^) 中 = ~l i j 和
Figure imgf000013_0001
3⁄4= , -^--^) in = ~ l ij and
Figure imgf000013_0001
c ) 阈值与过滤-: 为了获得可靠的结果， 对每条染色体平均标准化值进行过滤。 如果平 均标准化值小于 -1.28或者大于 1.28 ( 95% CI )将作为阳性结果输出， 结果见表 1、 图 3和 图 4。 其中， 表 1列举了样品 S 1和 S2的核型结果， 图 3 示了利用本发明的方法获得的样 品 S 1的染色体数字核型结果； 图 4显示了利用本发明的方法获得的样品 S2的染色体数字 核型结果。  c) Threshold and Filtering -: To obtain reliable results, filter the average normalized value of each chromosome. If the average normalized value is less than -1.28 or greater than 1.28 (95% CI), it will be output as a positive result. The results are shown in Table 1, Figure 3 and Figure 4. Wherein, Table 1 lists the karyotype results of the samples S1 and S2, Figure 3 shows the chromosomal digital karyotype results of the sample S1 obtained by the method of the present invention; and Figure 4 shows the sample S2 obtained by the method of the present invention. Chromosome digital karyotype results.
然后， 将前述结果与已知的核型结果进行比较， 比较结果见下表 1。 由表 1中的结果可 以看出， 本实施例获得的检测结果和核型结果是完全一致的。 由此， 表明本发明的确定生 物样本中染色体数目异常的方法可行， 且结果准确。  Then, the above results were compared with known karyotype results, and the results are shown in Table 1 below. It can be seen from the results in Table 1 that the test results obtained in this example are identical to the karyotype results. Thus, it was shown that the method for determining the abnormal number of chromosomes in the biological sample of the present invention is feasible and the result is accurate.
d )胎儿性别推断： 分别确定前述各样本的唯一比对测序数据集中来源于 X染色体的唯 一比对测序数据的数目 Nx;分别统计比对到前述各样本的 X和 Υ染色体上的唯一比对测序 短序列数 Nx、 Ny, 其中， Νχ为来源于 X染色体的唯一比对测序数据的数曰， Ny为来源于 Y 染色体的唯一比对测序数据的数 。 然后， 分别计算上述唯一比对测序短序列数与全基 因組唯一比对测序短序列数（M )的比值，分别记做 X-UR0/。和 Y-UR%，其中 X-UR%=NX/M, Y-UR%=Ny/M。 当 Y-UR%大于 0.0004时判定为存在男胎， Y-UR%/X-UR°/。大于 0.0075时判 定为存在男胎。 d) inferring sex: ratio of unique sequencing data were determined from the concentration of each sample X chromosome unique sequencing data than the number N x; statistics were unique to the ratio of the X chromosome and Υ of each sample For sequencing short sequence numbers N x , N y , where Νχ is the number of unique alignment sequencing data derived from the X chromosome, and N y is the number of unique alignment sequencing data derived from the Y chromosome. Then, the ratio of the above-mentioned unique alignment sequencing short sequence number to the whole genome unique alignment sequencing short sequence number (M) was calculated separately, and respectively recorded as X-UR 0 /. And Y-UR%, where X-UR%=N X /M, Y-UR%=N y /M. When the Y-UR% is greater than 0.0004, it is determined that there is a male fetus, Y-UR%/X-UR°/. When it is greater than 0.0075, it is determined that there is a male fetus.
接着， 通过以下步骤进行胎儿 DNA浓度估算： 分别选取正常男性和正常女性血浆样品 集作为对照，分别计算各血浆样品的 Y-UR%和 X-UR%;分别计算各男性血浆样品的 Y-UR% 和 X-UR%的算术平均值， 记作 YM和 XM, 分别计算各女性血浆样品的 Y-UR%和 X-UR%的 算术平均值， 记作 YF和 XF; 然后， 根据公式 (X-UR%-XF)/(XM-XF)计算出利用 X染色体估 计的第一胎儿 DNA ½%chrX,根据公式 (Y-UR%-YF)/(YM-YF)计算得出利用 Y染色体估计 的第二胎儿 DNA ¾%chrY, 其中该公式中 X-UR°/c^待测孕妇血浆样品的 X-UR%值， Y-U %为待测孕妇血浆样品的 Y-UR%¾。 Next, estimate the fetal DNA concentration by the following steps: Select normal male and normal female plasma sample sets as controls, calculate Y-UR% and X-UR% of each plasma sample separately; calculate Y-UR of each male plasma sample separately The arithmetic mean of % and X-UR%, denoted as Y M and X M , respectively, calculate the arithmetic mean of Y-UR% and X-UR% of each female plasma sample, denoted as Y F and X F ; Calculate the first fetal DNA 1⁄2%chrX estimated using the X chromosome according to the formula (X-UR%-X F )/(X M -X F ) according to the formula (Y-UR%-Y F )/(Y M - Y F ) Calculate the second fetal DNA 3⁄4%chrY estimated using the Y chromosome, where X-UR°/c^ X-UR% of the plasma sample of the pregnant woman to be tested, YU % is the plasma sample of the pregnant woman to be tested Y-UR%3⁄4.
然后，结合上述估算的胎儿 DNA浓度，判断双胞胎中两胎儿的性别。具体地， %chrX 和％。1«^差异显著性 p-value>0.1时， 可推断两胎儿为两个男胎， 当％chrX和％。111"¥差异显 著 -\^11½<0.1)且％(；111 比。 /。chrY高时， 可推断两胎儿为一个男胎和一个女胎。 此外， 如果 胎儿存在染色体变异（例如非整倍体）， 可以参考变异染色体估计得到的胎儿 DNA浓度推 断双胞胎胎儿的性别，具体地，如果变异染色体估计得到的胎儿 DNA浓度约为 Y染色体估 计得到的胎儿 DNA浓度的 1/2, 则推断两胎儿为两个男胎。 由表 2可知， 通过 Y-UR%和 Y-UR%/X-UR%的值， 发明人推断， 样品 S1的双胎中至少有一胎为男胎， 进而， 依据前面 所述的推断方法， 因为％chrX和％(；11^估算得到的胎儿 DNA浓度的差异显著性 p-value为 0.408 ,所以可确定双胎为男、女各一胎。同理， S2样品中％chrX和％<± 差异显著性 p-value 为 0.051 , 而且根据 Y染色体估算的胎儿 DNA浓度（％chrY )是 18号变异染色体估算的胎 儿 DNA浓度的 2.55倍， 从而可确定 S2样品为两个男胎。 Then, in combination with the estimated fetal DNA concentration as described above, the sex of the two fetuses in the twins is determined. Specifically, %chrX with%. When 1«^ significant difference p-value>0.1, it can be inferred that the two fetuses are two male fetuses, when %chrX and %. 111"¥ significant difference -\^111⁄2<0.1) and %(;111 ratio. /. When chrY is high, it can be inferred that the two fetuses are a male fetus and a fetus. In addition, if the fetus has chromosomal variation (eg, aneuploid The body of the twin fetus can be inferred by reference to the estimated fetal DNA concentration of the variant chromosome. Specifically, if the estimated fetal DNA concentration of the variant chromosome is about 1/2 of the estimated fetal DNA concentration of the Y chromosome, then the two fetuses are inferred. For the two male tires. As can be seen from Table 2, by the values of Y-UR% and Y-UR%/X-UR%, the inventor concludes that at least one of the twins of the sample S1 is a male tire, and further, according to the front The inference method, because %chrX and %(;11^ estimated fetal DNA concentration difference is significant, the p-value is 0.408, so it can be determined that the twins are male and female. Similarly, in the S2 sample The %chrX and %<± differential significance p-value is 0.051, and the estimated fetal DNA concentration (%chrY) based on the Y chromosome is 2.55 times the estimated fetal DNA concentration of the 18th variant chromosome, thereby determining that the S2 sample is two Male tires.
Figure imgf000014_0001
表 2. 实施例各样本胎儿性别推断结果
Figure imgf000014_0001
Table 2. Fetal gender inference results for each sample in the examples
Figure imgf000014_0002
Figure imgf000014_0002
注： ％chrX表示通过 X染色体上相对唯一比对测序短序列数估算的胎儿 DNA浓度； ％chrY表示通 过 Y染色体上相对唯一比对测序短序列数估算的胎儿 DNA浓度； %clirl8/%chr21表示通过变异的那条 染色体上相对唯一比对测序短序列数估算的胎儿 DNA浓度。 工业实用性  Note: %chrX represents the fetal DNA concentration estimated by the relatively unique alignment of the short sequence number on the X chromosome; %chrY represents the fetal DNA concentration estimated by the relatively unique alignment of the short sequence number on the Y chromosome; %clirl8/%chr21 The fetal DNA concentration estimated by the relatively unique alignment of the sequenced short sequences on the chromosome that is mutated. Industrial applicability
本发明的确定双胞胎中胎儿性别的方法、 ***和计算机可读介质， 可以有效地用于确 定双胞胎孕妇的双胞胎中胎儿的性别。 尽管本发明的具体实施方式已经得到详细的描述， 本领域技术人员将会理解。 根据已 经公开的所有教导， 可以对那些细节进行各种修改和替换， 这些改变均在本发明的保护范 围之内。本发明的全部范围由所附权利要求及其任何等同物给出。 The method, system and computer readable medium of the present invention for determining the sex of a fetus in a twin can be effectively used to determine the sex of a fetus in a twin of a twin pregnant woman. Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions may be made to those details in light of the teachings of the invention, which are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
在本说明书的描述中， 参考术语 "一个实施例"、 "一些实施例"、 "示意性实施例"、 "示 例"、 "具体示例"、 或 "一些示例" 等的描述意指结合该实施例或示例描述的具体特征、 结 构、 材料或者特点包含于本发明的至少一个实施例或示例中。 在本说明书中， 对上述术语 的示意性表述不一定指的是相同的实施例或示例。 而且， 描述的具体特征、 结构、 材料或 者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。  In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples", etc. Particular features, structures, materials or features described in the examples or examples are included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Claims

权利要求书 claims
1、 一种确定双胞胎中胎儿性别的方法， 其特征在于， 包括： 1. A method for determining the gender of fetuses in twins, which is characterized by including:
针对双胞胎孕妇的血液 DNA进行测序， 以便获得多个测序数据； Sequencing the blood DNA of pregnant twins to obtain multiple sequencing data;
将所述测序数据与人类的参照基因组序列进行比对， 以便获得由多个唯一比对测序数 据构成的唯一比对测序数据集； Comparing the sequencing data with a human reference genome sequence to obtain a uniquely aligned sequencing data set composed of multiple uniquely aligned sequencing data;
确定所述唯一比对测序数据集中所包含的唯一比对测序数据的数目 M; Determine the number M of uniquely aligned sequencing data contained in the uniquely aligned sequencing data set;
确定所述唯一比对测序数据集中来源于 Y染色体的唯一比对测序数据的数目 Ny, 以及 来源于 X染色体的唯一比对测序数据的数目 Nx的至少一种； Determine at least one of the number N y of uniquely aligned sequencing data originating from the Y chromosome in the uniquely aligned sequencing data set, and the number N x of uniquely aligned sequencing data originating from the X chromosome;
确定 Y染色体的测序数据含量 Y-UR0/。和 X染色体的测序数据含量 X-UR%的至少一种， 其中， 按照公式 Y-UR%=Ny/M , 确定 Y 染色体的测序数据含量 Y-UR% , 按照公式 X-UR%=NX/M, 确定 X染色体的测序数据含量 X- U %; 以及 Determine the sequencing data content of the Y chromosome Y-UR 0 /. and at least one of the sequencing data content of the X /M, determine the X-chromosome sequencing data content X- U %; and
判断所述双胞胎中胎儿的性别， 其中， 当满足下列条件的至少之一时， 确定所述双胞 胎中的至少一个为男胎， 当下列条件均不满足时， 确定所述双胞胎均为女胎： Determine the gender of the fetuses in the twins. When at least one of the following conditions is met, it is determined that at least one of the twins is a male fetus. When none of the following conditions are met, it is determined that the twins are both female fetuses:
丫-1；1 %大于第一阈值； 和 Ya-1; 1% is greater than the first threshold; and
Y-UR%/X-UR%大于第二闹值。 Y-UR%/X-UR% is greater than the second alarm value.
2、 根据权利要求 1所述的方法， 其特征在于， 所述第一阈值是通过下列步驟确定的： 将至少 30份已知胎儿为女胎的孕妇的血液 DNA 进行平行实验， 以便获得各孕妇的 2. The method according to claim 1, characterized in that the first threshold is determined by the following steps: Conduct parallel experiments on the blood DNA of at least 30 pregnant women whose fetuses are known to be female fetuses, in order to obtain the results of each pregnant woman. of
Y-UR%; Y-UR%;
形成所述各孕妇的 Y-UR%的正态分布曲线； 以及 Form a normal distribution curve of Y-UR% for each pregnant woman; and
确定所述正态分布曲线中心右側区间的 Y-UR%数值作为第一阈值。 Determine the Y-UR% value of the interval on the right side of the center of the normal distribution curve as the first threshold.
3、 根据权利要求 2所述的方法， 其特征在于， 将至少 100份已知胎儿为女胎的孕妇的 血液 DNA进行平行实验。 3. The method according to claim 2, characterized in that a parallel experiment is conducted on the blood DNA of at least 100 pregnant women whose fetuses are known to be female fetuses.
4、 根据权利要求 3所述的方法， 其特征在于， 将所述 Y-UR0/。的正态分布曲线 99%置 信区间右端点的 Y-UR%数值作为第一阈值。 4. The method according to claim 3, characterized in that: Y-UR 0 /. The Y-UR% value at the right end point of the 99% confidence interval of the normal distribution curve is used as the first threshold.
5、 根据权利要求 4所述的方法， 其特征在于， 所述第一阈值为 0.0004。 5. The method according to claim 4, characterized in that the first threshold is 0.0004.
6、 根据权利要求 1所述的方法， 其特征在于， 所述笫二阈值是通过下列步骤确定的： 将至少 30份已知胎儿为女胎的孕妇的血液 DNA 进行平行实验， 以便获得各孕妇的 6. The method according to claim 1, characterized in that the second threshold is determined by the following steps: Conducting parallel experiments on the blood DNA of at least 30 pregnant women whose fetuses are known to be female fetuses, in order to obtain the results of each pregnant woman. of
Y-UR%/X-UR%; Y-UR%/X-UR%;
形成所述各孕妇的 Y-UR%/X-UR°/。的正态分布曲线； 以及 The Y-UR%/X-UR°/ of each pregnant woman is formed. the normal distribution curve; and
确定所述正态分布曲线中心右侧区间的 Y-UR%/X-UR%数值作为第二阈值。 The Y-UR%/X-UR% value of the interval on the right side of the center of the normal distribution curve is determined as the second threshold.
7、 根据权利要求 6所述的方法， 其特征在于， 将至少 100份已知胎儿为女胎的孕妇的 血液 DNA进行平行实验。 7. The method according to claim 6, characterized in that parallel experiments are conducted on the blood DNA of at least 100 pregnant women whose fetuses are known to be female fetuses.
8、 根据权利要求 7所述的方法， 其特征在于， 将所述 Y-UR%/X-UR°/ 正态分布曲线 99%置信区间右端点的 Y-UR%/X-UR%数值作为第二阈值。 8. The method according to claim 7, characterized in that, the Y-UR%/X-UR% value of the right end point of the 99% confidence interval of the Y-UR%/X-UR°/ normal distribution curve is used as Second threshold.
9、 根据权利要求 8所述的方法， 其特征在于， 所述第二阈值为 0.0075。 9. The method according to claim 8, characterized in that the second threshold is 0.0075.
10、 根据权利要求 1所述的方法， 其特征在于， 所述双胞胎孕妇的血液 DNA是通过选 自磁珠法和柱层析法的至少一种提取的。 10. The method according to claim 1, characterized in that the blood DNA of the pregnant twins is extracted by at least one method selected from the group consisting of magnetic beads and column chromatography.
11、 根据权利要求 1所述的方法， 其特征在于， 所述测序数据的长度为 36~100 bp。 11. The method according to claim 1, characterized in that the length of the sequencing data is 36~100 bp.
12、根据权利要求 1所述的方法， 其特征在于， 所述参照基因组序列为 NCBI数据库中 版本 36的人类基因组参考序列。 12. The method according to claim 1, characterized in that the reference genome sequence is the human genome reference sequence version 36 in the NCBI database.
13、 根据权利要求 1所述的方法， 其特征在于， 采用 SOAPaUgner/soap2 , 将所述测序 数据与人类的参照基因組序列进行比对。 13. The method according to claim 1, characterized in that SOAPaUgner/soap2 is used to compare the sequencing data with the human reference genome sequence.
14、 根据权利要求 1 所述的方法， 其特征在于， 所述测序是通过选自 Roche/454 GS Junior, Illumina/MiSeq以及 Life Tecnologies/Ion Torrent PGM的至少之一进行的。 14. The method of claim 1, wherein the sequencing is performed by at least one selected from the group consisting of Roche/454 GS Junior, Illumina/MiSeq, and Life Tecnologies/Ion Torrent PGM.
15、 根据权利要求 1 所述的方法， 其特征在于， 当确定所述双胞胎中的至少一个为男 胎时， 进一步包括： 15. The method according to claim 1, characterized in that, when it is determined that at least one of the twins is a male fetus, further comprising:
基于所述 Nx和 ΝΥ, 分别进行胎儿 DNA浓度估算， 以便分别得到第一胎儿 DNA浓度 %chrX, 以及第二胎儿 DNA浓 y¾%chrY; 以及 Based on the N
确定所述％chrX和％±1^的显著差异性 p-value， Determine the significant difference p-value of the %chrX and %±1^,
其中， 如果 p-value>0.1 , 则确定所述双胞胎中的胎儿均为男胎， Among them, if p-value>0.1, it is determined that the fetuses in the twins are all male fetuses,
如果 p-value<0.1 , 且％(；1^¾ 大于％(；1^¥, 则确定所述双胞胎中存在一个男胎和一个女 胎。 If p-value<0.1, and %(1^¾ is greater than %(1^¥), then it is determined that there is a male fetus and a female fetus in the twins.
16、 一种计算机可读介质， 其特征在于， 所述计算机可读介质上存储有指令， 所述指 令适于被处理器执行以便通过下列步骤确定双胞胎中胎儿性别： 16. A computer-readable medium, characterized in that instructions are stored on the computer-readable medium, and the instructions are adapted to be executed by a processor to determine the gender of the fetus in twins through the following steps:
获取双胞胎孕妇的血液 DNA的多个测序数据； Obtain multiple sequencing data of blood DNA of pregnant twins;
将所述测序数据与人类的参照基因組序列进行比对， 以便获得由多个唯一比对测序数 据构成的唯一比对测序数据集； Comparing the sequencing data with a human reference genome sequence to obtain a uniquely aligned sequencing data set composed of multiple uniquely aligned sequencing data;
确定所述唯一比对测序数据集中所包含的唯一比对测序数据的数目 M; Determine the number M of uniquely aligned sequencing data contained in the uniquely aligned sequencing data set;
确定所述唯一比对测序数据集中来源于 Y染色体的唯一比对测序数据的数目 Ny, 以及 来源于 X染色体的唯一比对测序数据的数目 Nx的至少一种； Determine at least one of the number N y of uniquely aligned sequencing data originating from the Y chromosome in the uniquely aligned sequencing data set, and the number N x of uniquely aligned sequencing data originating from the X chromosome;
确定 Y染色体的测序数据含量 Y-UR0/。和 X染色体的测序数据含量 X-UR%的至少一种， 其中， 按照公式 Y-UR%=Ny/M， 确定 Y 染色体的测序数据含量 Y-UR%， 按照公式
Figure imgf000018_0001
确定 X染色体的测序数据含量 X-UR%; 以及 Determine the sequencing data content of the Y chromosome Y-UR 0 /. and at least one of X-chromosome sequencing data content X-UR%, Among them, according to the formula Y-UR%=N y /M, determine the sequencing data content of the Y chromosome Y-UR%, according to the formula
Figure imgf000018_0001
Determine the X-chromosome sequencing data content X-UR%; and
判断所述双胞胎中胎儿的性别， 其中， 当满足下列条件的至少之一时， 确定所述双胞 胎中的至少一个为男胎， 当下列条件均不满足时， 确定所述双胞胎均为女胎： Determine the gender of the fetuses in the twins. When at least one of the following conditions is met, it is determined that at least one of the twins is a male fetus. When none of the following conditions are met, it is determined that the twins are both female fetuses:
Y-UR%大于第一阁值； 和 Y-UR% is greater than the first cabinet value; and
Y-U %/X-UR%大于第二阈值。 Y-U %/X-UR% is greater than the second threshold.
17、 根据权利要求 16所述的计算机可读介质， 其特征在于， 所述笫一阈值为 0.0004。 17. The computer-readable medium according to claim 16, wherein the first threshold is 0.0004.
18、 根据权利要求 16所述的计算机可读介质， 其特征在于， 所述第二阈值为 0.0075。18. The computer-readable medium according to claim 16, wherein the second threshold is 0.0075.
19、 根据权利要求 16 所述的计算机可读介质， 其特征在于， 所述测序数据的长度为 36~100 bp。 19. The computer-readable medium according to claim 16, wherein the length of the sequencing data is 36~100 bp.
20、 根据权利要求 16 所述的计算机可读介盾， 其特征在于， 所述参照基因組序列为 NCBI数据库中版本 36的人类基因组参考序列。 20. The computer-readable medium according to claim 16, wherein the reference genome sequence is the human genome reference sequence of version 36 in the NCBI database.
21、 根据权利要求 16所述的计算机可读介 ， 其特征在于， 采用 SOAPaligner/soap2, 将所述测序数据与所述生物的参照基因组序列进行比对。 21. The computer-readable medium according to claim 16, characterized in that SOAPaligner/soap2 is used to compare the sequencing data with the reference genome sequence of the organism.
22、 根据权利要求 16所述的计算机可读介盾， 其特征在于， 当确定所述双胞胎中的至 少一个为男胎时， 进一步包括： 22. The computer-readable medium according to claim 16, wherein when it is determined that at least one of the twins is a male fetus, it further includes:
基于所述 Nx和 ΝΥ, 分别进行胎儿 DNA浓度估算， 以便分别得到第一胎儿 DNA浓度 %chrX, 以及第二胎儿 DNA浓 y¾%chrY; 以及 Based on the N
确定所述％chrX和％±1^的显著差异性 p-value， Determine the significant difference p-value of the %chrX and %±1^,
其中， 如果 p-value>0.1 , 则确定所述双胞胎中的胎儿均为男胎， Among them, if p-value>0.1, it is determined that the fetuses in the twins are all male fetuses,
如果 p-value<0.1 , 且％(；1^¾ 大于％(；1^¥, 则确定所述双胞胎中存在一个男胎和一个女 胎。 If p-value<0.1, and %(1^¾ is greater than %(1^¥), then it is determined that there is a male fetus and a female fetus in the twins.
23、 一种用于确定双胞胎中胎儿性别的***， 其特征在于， 包括： 23. A system for determining the gender of fetuses in twins, characterized by including:
测序装置， 所述测序装置用于针对双胞胎孕妇的血液 DNA进行测序， 以便获得多个测 序数据； A sequencing device, which is used to sequence the blood DNA of pregnant twins in order to obtain multiple sequencing data;
比对装置， 所述比对装置与所述测序装置相连， 用于将所述测序数据与人类的参照基 因组序列进行比对， 以便获得由多个唯一比对测序数据构成的唯一比对测序数据集； A comparison device, the comparison device is connected to the sequencing device, and is used to compare the sequencing data with the human reference genome sequence, so as to obtain unique comparison sequencing data composed of multiple unique comparison sequencing data. set;
分析装置， 所述分析装置与所述比对装置相连， 并且用于确定双胞胎中胎儿性别， 其中， 所述分析装置进一步包括： An analysis device, the analysis device is connected to the comparison device, and is used to determine the gender of the fetuses in twins, wherein the analysis device further includes:
第一计算模块， 所述第一计算模块用于确定所述唯一比对测序数据集中所包含的唯一 比对测序数据的数目 M; 第二计算模块， 所述第二计算模块用于确定所述唯一比对测序数据集中来源于 Y染色 体的唯一比对测序数据的数目 Ny , 以及来源于 X染色体的唯一比对测序数据的数目 Nx的 至少一种； A first calculation module, the first calculation module is used to determine the number M of unique alignment sequencing data contained in the unique alignment sequencing data set; The second calculation module is used to determine the number N y of unique alignment sequencing data originating from the Y chromosome in the unique alignment sequencing data set, and the number of unique alignment sequencing data originating from the X chromosome. At least one of N x ;
第三计算模块，所述第三计算模块用于确定 Υ染色体的测序数据含量 Y-UR%和 X染色 体的测序数据含量 X-UR%的至少一种， 其中， 按照公式 Y-UR%=Ny/M , 确定 Y染色体的 测序数据含量 Y-UR% , 按照公式 X-UR%=NX/M， 确定 X染色体的测序数据含量 X-UR%; 以及 The third calculation module is used to determine at least one of the sequencing data content Y-UR% of the Y chromosome and the sequencing data content X-UR% of the X chromosome, wherein, according to the formula Y-UR%=N y /M, determine the sequencing data content of Y chromosome Y-UR%, according to the formula X-UR%=N X /M, determine the sequencing data content of X chromosome X-UR%; and
判断模块， 所述判断模块用于判断所述双胞胎中胎儿的性别， 其中， 当满足下列条件 的至少之一时， 确定所述双胞胎中的至少一个为男胎， 当下列条件均不满足时， 确定所述 双^胎均为女胎： Determination module, the determination module is used to determine the gender of the fetuses in the twins, wherein when at least one of the following conditions is met, it is determined that at least one of the twins is a male fetus, and when none of the following conditions are met, it is determined The twins are both female:
Y-UR0/。大于第一阈值； 和 Y-UR 0 /. is greater than the first threshold; and
Y-UR% X-UR%大于第二阈值。 Y-UR% X-UR% is greater than the second threshold.
24、 根据权利要求 23所述的***， 其特征在于， 所述第一阈值为 0.0004。 24. The system according to claim 23, wherein the first threshold is 0.0004.
25、 根据权利要求 23所述的***， 其特征在于， 所述笫二阈值为 0.0075。 25. The system according to claim 23, characterized in that the second threshold is 0.0075.
26、 根据权利要求 23所述的***， 其特征在于， 所述判断模块进一步包括验证单元， 所述验证单元适于在确定所述双胞胎中的至少一个为男胎时， 执行下列操作： 26. The system according to claim 23, wherein the judgment module further includes a verification unit, and the verification unit is adapted to perform the following operations when it is determined that at least one of the twins is a male fetus:
基于所述 Nx和 ΝΥ, 分别进行胎儿 DNA浓度估算， 以便分别得到第一胎儿 DNA浓度 %chrX, 以及第二胎儿 DNA浓 y¾%chrY; 以及 Based on the N
确定所述％chrX和％±1^的显著差异性 p-value， Determine the significant difference p-value of the %chrX and %±1^,
其中， 如果 p-value>0.1 , 则确定所述双胞胎中的胎儿均为男胎， Among them, if p-value>0.1, it is determined that the fetuses in the twins are all male fetuses,
如果 p-value<0.1 , 且％(；1^¾ 大于％(；1^¥ , 则确定所述双胞胎中存在一个男胎和一个女 胎。 If p-value<0.1, and %(1^¾ is greater than %(1^¥), then it is determined that there is a male fetus and a female fetus in the twins.
27、 一种用于确定双胞胎中胎儿性别的***， 其特征在于， 包括： 27. A system for determining the gender of fetuses in twins, characterized by including:
测序装置， 所述测序装置用于针对所述双胞胎孕妇的血液 DNA进行测序， 以便获得多 个测序数据； 以及 A sequencing device, the sequencing device is used to sequence the blood DNA of the pregnant twins in order to obtain multiple sequencing data; and
权利要求 16-22任一项所述的计算机可读介质。 The computer-readable medium of any one of claims 16-22.
PCT/CN2012/087582 2012-12-26 2012-12-26 Method, system and computer readable medium for determining fetal sexes of twins WO2014101024A1 (en)

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Publication number Priority date Publication date Assignee Title
US20120219950A1 (en) * 2011-02-28 2012-08-30 Arnold Oliphant Assay systems for detection of aneuploidy and sex determination

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US20120219950A1 (en) * 2011-02-28 2012-08-30 Arnold Oliphant Assay systems for detection of aneuploidy and sex determination

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