CN109207600A - The method and system of affiliation between identification biological sample - Google Patents
The method and system of affiliation between identification biological sample Download PDFInfo
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- CN109207600A CN109207600A CN201710548169.4A CN201710548169A CN109207600A CN 109207600 A CN109207600 A CN 109207600A CN 201710548169 A CN201710548169 A CN 201710548169A CN 109207600 A CN109207600 A CN 109207600A
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Abstract
The invention proposes a kind of methods of affiliation between identification biological sample.This method comprises: (1) constructs sequencing library based on the first sample of nucleic acid from the first organism and the second sample of nucleic acid from the second organism；(2) the obtained sequencing library in step (1) is sequenced；(3) it is based on the sequencing result of step (2), determines first sample of nucleic acid and second sample of nucleic acid in the base type of predetermined SNP site；(4) based on obtained in step (3) as a result, determining the affiliation of first organism and second organism.Using the method for affiliation between identification biological sample according to an embodiment of the present invention, parentage exclusion probability effectively realizes the paternity test of small pregnant week fetus (such as 6~8 weeks) up to 99.99%.
Description
Technical field
The present invention relates to field of biotechnology, in particular it relates to the method for identifying affiliation between biological sample
And system, more particularly it relates to identify the method for affiliation between biological sample, system, for sequencing library into
The purposes of the kit of row screening enrichment and the reagent of the predetermined SNP site of detection in Preparation equipment.
Background technique
Traditional antenatal paternity test method be directly by obtain fetus villus, amniotic fluid, bleeding of the umbilicus or fetal tissue come into
Row paternity test, this invading property technology have certain risk to pregnant woman and fetus, have 1% a possibility that lead to intrauterine infection, sheep
Film rupture, miscarriage, fetal anomaly or death etc..
Contain fetus dissociative DNA in educational circles's discovery maternal bloods in 1997, can be detected out fetal blood since pregnant 5 weeks
It starches dissociative DNA (cfDNA), and increase as pregnant week increases.The fetus cfDNA meeting in blood plasma in 2 hours after pregnant woman's production
It eliminates rapidly.This provides fundamental basis for noninvasive antenatal coherent detection.
Existing paternity test technology has:
Generation STR detection technique: STR (short tandem repeat) is polymorphic locus in human genome, it is by 2-6
Base-pair constitutes core sequence, arranges in tandem sequence repeats, and STR bit point length is generally between 100-300bp, STR between individual
Difference in length constitutes polymorphism, and mendel's law is followed in gene delivery, is widely used to medical jurisprudence individual identification
And paternity test.Market is generally the detection kit of 18 STR bit points or so.
Based on NGS (next generation's sequencing) detection technique: similar products mainly have Illumina FGx, are based on STR and SNP
The Paternity and individual identification in (single nucleotide polymorphism) site, the high throughput sequencing technologies and automatic parting direction of NGS generate
Report.
However, paternity test technology still needs further to develop and improve.
Summary of the invention
The application is to be made based on inventor to the discovery of following facts and problem and understanding:
Usually by 20 sites of capillary electrophoresis detection or so, which is only applicable to fetus for generation STR detection
Amniotic fluid or villus sample, and extract amniotic fluid or fetus villus belongs to invasive sampling method, there is 1% possibility to lead to intrauterine infection
And miscarriage；And it is invasive sampling generally can only 16-24 weeks carry out, thus a generation STR detection can only also solve the pregnancy period this when
Between section paternity test problem；The amplified production segment of traditional STR is longer, and when detecting plasma DNA, there are allele
The problem of loss, therefore traditional STR bit point is not particularly suited for noninvasive antenatal paternity test detection.Detection technique based on NGS, such as
Illumina FGx is not suitable for the detection of this degradation of dna of cfDNA, does not have yet and detects fetus gene from female blood blood plasma
The function of type.
The present invention is directed to solve at least some of the technical problems in related technologies.For this purpose, the present invention mentions
Maternal blood need to be extracted by having gone out a kind of, carry out parting to the fetus polymorphic site in dissociative DNA, and then obtain accurately
The method of affiliation between the identification biological sample of mother and sons' genotype.
In the first aspect of the present invention, the invention proposes a kind of methods of affiliation between identification biological sample.According to
The embodiment of the present invention, which comprises (1) based on the first sample of nucleic acid from the first organism and from second
The second sample of nucleic acid of organism constructs sequencing library；(2) the obtained sequencing library in step (1) is sequenced；
(3) it is based on the sequencing result of step (2), determines first sample of nucleic acid and second sample of nucleic acid in predetermined SNP site
Base type；(4) based on obtained in step (3) as a result, determining first organism and second organism
Affiliation, wherein the predetermined SNP site includes that the predetermined SNP site includes being selected from site shown in table 1 at least
One of.
Table 1:
.Using the method for affiliation between identification biological sample according to an embodiment of the present invention, parentage exclusion probability is reachable
99.99%, and effectively realize the paternity test of small pregnant week fetus (such as 6~8 weeks).
In the second aspect of the present invention, the invention proposes a kind of systems of affiliation between identification biological sample.According to
The embodiment of the present invention, the system comprises: building sequencing library device, the building sequencing library device are used to be based on to come from
The first sample of nucleic acid in the first organism and the second sample of nucleic acid from the second organism construct sequencing library；Sequencing
Device, the sequencing device determine base types of devices, the determining base for obtained sequencing library to be sequenced
Types of devices is used to be based on sequencing result, determines first sample of nucleic acid and second sample of nucleic acid in predetermined SNP site
Base type；And decision maker, the decision maker are used to determine first organism based on determining base type
With the affiliation of second organism, wherein the SNP site include selected from the site shown in the table 1 at least it
One.Using the device of affiliation between identification biological sample according to an embodiment of the present invention, parent between biological sample is effectively realized
The identification of edge relationship, parentage exclusion probability effectively realize parent-offspring's mirror of small pregnant week fetus (such as 6~8 weeks) up to 99.99%
It is fixed.
In the third aspect of the present invention, the invention proposes a kind of for carrying out the reagent of screening enrichment to sequencing library
Box.According to an embodiment of the invention, the kit includes: probe, the probe specificity identifies predetermined SNP site, described
Predetermined SNP site includes selected from selected from least one of site shown in table 1.Using according to an embodiment of the present invention for survey
Preface library carries out the kit of screening enrichment, can be effectively enriched with to the predetermined SNP site in sequencing library, and then pass through survey
Sequence, can efficiently realize the identification of affiliation between biological sample, and parentage exclusion probability is realized up to 99.99%, and effectively
The paternity test of small pregnant week fetus (such as 6~8 weeks).
In the fourth aspect of the present invention, the invention proposes detect use of the reagent of predetermined SNP site in Preparation equipment
On the way, the equipment includes selected from selected from position shown in table 1 for determining affiliation between biological sample, the predetermined SNP site
At least one of point.Using equipment prepared by the predetermined SNP site of detection according to an embodiment of the present invention, life can be efficiently realized
The identification of affiliation between object sample, parentage exclusion probability effectively realize small pregnant week fetus (such as 6~8 up to 99.99%
Week) paternity test.
Detailed description of the invention
Fig. 1 be it is according to an embodiment of the present invention identification biological sample between affiliation system structural schematic diagram；
Fig. 2 be according to yet another embodiment of the invention identification biological sample between affiliation system structural schematic diagram；
Fig. 3 is the gel electrophoresis of doubtful male parent according to an embodiment of the present invention and maternal leucocyte genomic DNA integrality
Detection figure；And
Fig. 4 is the detected through gel electrophoresis figure that the genomic DNA implemented according to the present invention interrupts rear main band distribution.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings.Below with reference to
The embodiment of attached drawing description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.
The method of affiliation between identification biological sample
In the first aspect of the present invention, the invention proposes a kind of methods of affiliation between identification biological sample.According to
The embodiment of the present invention, which comprises (1) based on the first sample of nucleic acid from the first organism and from second
The second sample of nucleic acid of organism constructs sequencing library；(2) the obtained sequencing library in step (1) is sequenced；
(3) it is based on the sequencing result of step (2), determines first sample of nucleic acid and second sample of nucleic acid in predetermined SNP site
Base type；(4) based on obtained in step (3) as a result, determining first organism and second organism
Affiliation,
Wherein, the predetermined SNP site includes selected from selected from least one of site shown in table 1.Using according to this hair
The method of affiliation, parentage exclusion probability effectively realize small up to 99.9% between the identification biological sample of bright embodiment
The paternity test of pregnant week fetus (such as 6~8 weeks).
According to an embodiment of the invention, the accumulation parental right probability of first sample of nucleic acid and the second sample of nucleic acid is greater than
10000 be the instruction with affinity between biological sample.
According to an embodiment of the invention, first sample of nucleic acid and the second sample of nucleic acid include being selected from DNA and RNA extremely
It is one of few.According to a particular embodiment of the invention, the DNA is genomic DNA.
According to an embodiment of the invention, first organism is fetus, second organism is doubting for the fetus
Like biology father.
According to a particular embodiment of the invention, first sample of nucleic acid derives from maternal blood.And then it can effectively keep away
Exempt from intrauterine infection and miscarriage.
According to a particular embodiment of the invention, first sample of nucleic acid is maternal plasma dissociative DNA.Outside pregnant woman
All DNA of the blood plasma DNA from fetus, and then avoid and directly extract amniotic fluid or fetus villus and carry out mentioning for foetal DNA
It takes, effectively prevents the risk of intrauterine infection brought by invasive sampling and miscarriage.
Second sample of nucleic acid source is not particularly limited, as long as a effective amount of doubtful biology father can be extracted
Genomic DNA.According to a particular embodiment of the invention, second biological sample is from second organism
Blood, skin, hair, saliva, muscle and sperm at least one.
According to a particular embodiment of the invention, when first sample of nucleic acid be maternal plasma dissociative DNA, it is above-mentioned
The method of affiliation can further include between identification biological sample: a, based on the third nucleic acid from third organism
Sample constructs sequencing library, and the third organism is the pregnant woman, and the third biological sample is white thin from the pregnant woman
Born of the same parents, the third biological sample are pregnant woman's leucocyte genomic DNA；B, for the sequencing library obtained in step (a)
It is sequenced；And c, it is based on the sequencing result of step (b), determines the third sample of nucleic acid in the base of predetermined SNP site
Type；D, based on obtained as a result, excluding pregnant woman's genomic DNA in maternal plasma dissociative DNA in rapid (c).?
Know both include foetal DNA in pregnant woman's maternal blood plasma DNA, also include female parent DNA, by the above method, can incite somebody to action
Maternal DNA background in maternal plasma dissociative DNA excludes, and then clearly separates foetal DNA, the first organism and second
The determination of affiliation is more true and reliable between organism, and parentage exclusion probability further effectively improves.
The selection of the utilized platform of the sequencing is also not particularly limited, as long as high-flux sequence can be realized.Root
According to the embodiment of the present invention, the sequencing is enterprising in 2000/4000 microarray dataset of BGISeq-500 or Illimina Hiseq
Capable.
According to a particular embodiment of the invention, before carrying out the sequencing, screening enrichment is carried out to the sequencing library
Processing, wherein the screening enrichment processing is directed to the probe of the predetermined SNP site using specificity.It is directed to using specificity
Sequence with predetermined SNP site in the probe enrichment sequencing library of predetermined SNP site, and then be sequenced, it can effectively improve
The detector efficiency of predetermined SNP site, parentage exclusion probability further effectively improve.
The system of affiliation between identification biological sample
In the second aspect of the present invention, the invention proposes a kind of systems of affiliation between identification biological sample.According to
The embodiment of the present invention, with reference to Fig. 1, the system comprises: building sequencing library device 100, the building sequencing library device
100 for based on the first sample of nucleic acid from the first organism and the second sample of nucleic acid from the second organism, structure
Build sequencing library；Sequencing device 200, the sequencing device 200 is for being sequenced obtained sequencing library；Determine base
Types of devices 300, the determining base types of devices 300 are used to be based on sequencing result, determine first sample of nucleic acid and institute
The second sample of nucleic acid is stated in the base type of predetermined SNP site；And decision maker 400, the decision maker 400 is for being based on
Determining base type determines the affiliation of first organism Yu second organism, wherein the SNP site
Including selected from selected from least one of site shown in table 1.Utilize relationship between identification biological sample according to an embodiment of the present invention
The device of relationship effectively realizes the identification of affiliation between biological sample, parentage exclusion probability up to 99.99%, and effectively
Realize the paternity test of small pregnant week fetus (such as 6~8 weeks).
Another embodiment according to the present invention, with reference to Fig. 2, the system further comprises screening enriching apparatus 500, described
The probe that screening enriching apparatus 500 is used for using specificity for the predetermined SNP site screens the sequencing library
Enrichment processing.Sequence in sequencing library with predetermined SNP site is enriched with for the probe of predetermined SNP site using specificity,
And then be sequenced, the detector efficiency of predetermined SNP site can be effectively improved, parentage exclusion probability further effectively improves.
According to a particular embodiment of the invention, the accumulation parental right probability of first sample of nucleic acid and the second sample of nucleic acid is big
It is the instruction between biological sample with affinity in 10000,
According to a particular embodiment of the invention, first sample of nucleic acid and the second sample of nucleic acid include being selected from DNA and RNA
At least one of.According to a particular embodiment of the invention, the DNA is genomic DNA.
According to an embodiment of the invention, first organism is fetus, second organism is doubting for the fetus
Like biology father.
According to an embodiment of the invention, first organism is fetus, second organism is doubting for the fetus
Like biology father.
According to a particular embodiment of the invention, first sample of nucleic acid derives from maternal blood.And then it can effectively keep away
Exempt from intrauterine infection and miscarriage.
According to a particular embodiment of the invention, first sample of nucleic acid is maternal plasma dissociative DNA.Outside pregnant woman
All blood plasma frees include the DNA from fetus, and then avoid and directly extract amniotic fluid or fetus villus and carry out mentioning for foetal DNA
It takes, effectively prevents the risk of intrauterine infection brought by invasive sampling and miscarriage.
Second sample of nucleic acid source is not particularly limited, as long as a effective amount of doubtful biology father can be extracted
Genomic DNA.According to a particular embodiment of the invention, second biological sample is from second organism
Blood, skin, hair, saliva, muscle and sperm at least one.
The selection of the utilized platform of the sequencing is also not particularly limited, as long as high-flux sequence can be realized.Root
According to the embodiment of the present invention, the sequencing is enterprising in 2000/4000 microarray dataset of BGISeq-500 or Illimina Hiseq
Capable.
For carrying out the kit of screening enrichment to sequencing library
In the third aspect of the present invention, the invention proposes a kind of for carrying out the reagent of screening enrichment to sequencing library
Box.According to an embodiment of the invention, the kit includes: probe, the probe specificity identifies predetermined SNP site, described
Predetermined SNP site includes selected from selected from least one of site shown in table 1.Using according to an embodiment of the present invention for survey
Preface library carries out the kit of screening enrichment, can be effectively enriched with to the predetermined SNP site in sequencing library, and then pass through survey
Sequence, can efficiently realize the identification of affiliation between biological sample, and parentage exclusion probability is realized up to 99.99%, and effectively
The paternity test of small pregnant week fetus (6~8 weeks).
Detect purposes of the reagent of predetermined SNP site in Preparation equipment
In the fourth aspect of the present invention, the invention proposes detect use of the reagent of predetermined SNP site in Preparation equipment
On the way, the equipment includes selected from selected from position shown in table 1 for determining affiliation between biological sample, the predetermined SNP site
At least one of point.According to an embodiment of the invention, the reagent for detecting predetermined SNP site is not particularly limited, including specificity
Identify the probe, primer or antibody etc. of predetermined SNP site.Still another embodiment according to the present invention, the equipment also not by
Especially limitation, it may include carry out the equipment of sequencing identification using high throughput, combined and reflected using probe hybridization or antigen-antibody
Fixed equipment or the equipment identified using PCR.Utilize the reagent institute of the predetermined SNP site of detection according to an embodiment of the present invention
The equipment of preparation, can efficiently realize the identification of affiliation between biological sample, and parentage exclusion probability has up to 99.99%
Effect realizes the paternity test of small pregnant week fetus (such as 6~8 weeks).
To sum up, make a reservation for used by the method and system of affiliation between the application identification biological sample claimed
SNP site be inventor to Chinese Han Population data statistically analyze in human genome project after, according to heterozygosity > 0.5, exclude
The single nucleotide polymorphism molecular labeling that the conditional filtering of rate > 0.4 goes out.And the predetermined SNP site of the application can cover entirely
Genome；It can effectively solve the problems, such as that fetus dissociative DNA content is low in small pregnant week detection or female blood.Fetus dissociative DNA is in mother
Content in close peripheral blood increases as pregnant week increases, and fetus cfDNA content is lower in the general smaller female blood of pregnant week.For tire
The lower cfDNA sample of youngster's ratio carries out the high depth sequencing for a large amount of SNP sites of the application filtered out, can be with parting
The fetus genotype in enough sites is obtained, so that the accumulative parentage exclusion probability or paternity index in these sites are sufficient for patriarchy
Determine.
Probe used by the method and system of affiliation, covering between the application identification biological sample claimed
The < 100bp single-stranded oligonucleotide of target molecule marker site, and captured using the probe of specific recognition SNP, overcome STR multiple
The problems such as closing amplification bring amplification efficiency differentiation and being easy mutation, improves site data volume homogeneity.
Specific embodiments of the present invention are described below in detail, examples of the embodiments are shown in the accompanying drawings.Below by
The specific embodiment being described with reference to the drawings is exemplary, it is intended to is used to explain the present invention, and be should not be understood as to of the invention
Limitation.
Embodiment
Step 1: blood sampling: collecting an example volunteer sample, informed consent form is signed, with STRECK (Cell-Free DNA
Preservative) pipe acquires maternal blood 10mL, and general blood collection tube acquires father's peripheral blood 5mL
Step 2: dividing blood: by 10mL blood of pregnant women at 4 DEG C, 1600g is centrifuged 15min, draws 900uL plasma layer respectively and arrives
1.5mL centrifuge tube；4 DEG C, 16000g is centrifuged 15min, draws 850uL supernatant respectively to new 1.5mL centrifuge tube；4 DEG C, 16000g
It is centrifuged 15min, draws 800uL respectively to new 1.5mL centrifuge tube, the blood plasma after high speed centrifugation twice is used to extract
cfDNA；Intermediate leukocytic cream 300uL+200uLPBS is taken after low-speed centrifugal, for mentioning mother's leucocyte DNA.
Step 3: cfDNA is extracted: 2mL blood plasma is taken, according to QIAGEN kit " QIAamp Circulating
Nucleic Acid Kit " cfDNA is extracted, it is finally dissolved in 50uLNF (removing nucleic acid) water, uses3.0 fluorescent quantitation
Instrument detection.
Extracting genome DNA: mother's leukocytic cream 300uL+200uLPBS retained in step 2, another father's whole blood
500uL extracts genomic DNA with QIAGEN kit " QIAamp DNA Blood Mini Kit ", obtains parent gene group
DNA is usedThe detection of 3.0 fluorescent quantitation instruments.With 2% Ago-Gel, 120V voltage runs glue 35min, detects DNA mass,
Testing result is as shown in Figure 3, it is ensured that genomic DNA is completely undegraded.
Step 4: the library Hiseq is established, the sequencing of upper machine
1. the library cfDNA is established
It repairs end:
Response procedures:
| 20℃ | 30min |
It has reacted and has been purified with 1.8 times of volume Ampure XP beads
End adds dATP after repairing:
| Reacted constituent | Volume |
| Product after reparation | 34uL |
| Nuclease Free Water | 8uL |
| 10×Blue Buffer | 5uL |
| dATP(5mM) | 2uL |
| Klenow 3’-5’exo- | 1uL |
Response procedures:
| 37℃ | 30min |
Connector connection:
| Reacted constituent | Volume |
| DNA sample after adding " A " | 22.5uL |
| 2x Rapid Ligation buffer | 25uL |
| PE Adapter oligo mix(40uM) | 0.5uL |
| T4DNA Ligase(Rapid) | 2uL |
Response procedures:
| 20℃ | 15min |
It has reacted and has been purified with 1.5 times of volume Ampure XP beads.And with Nanodrop detectable concentration.
Pre- amplification:
Response procedures:
It has reacted and has been purified with 1.5 times of volume Ampure XP beads.With3.0 fluorescent quantitation instrument detectable concentrations.
2. the library gDNA is established
Genome interrupts: parent gene group DNA takes 1ug respectively, supplies 80uL with TE buffer, is placed in 96 hole PCR plates
(AXYGEN), instrument by specification ultrasonication segment is interrupted to 250bp or so, with 1.8 times of volume Ampure XP with covaris
Beads purifying.After purification with 2% Ago-Gel, 120V voltage, race glue 35min, detection DNA master tape fragment length, detection knot
Fruit is as shown in figure 4, the results show that master tape size concentrates on 250bp or so after ultrasonication.
It repairs end:
| Reacted constituent | Volume |
| Interrupt rear DNA | 75uL |
| 10×PNK Buffer | 10uL |
| dNTP Mix(10mM) | 4uL |
| T4DNA Polymerase | 5uL |
| T4PNK | 5uL |
| Klenow Fragment | 1uL |
Response procedures:
| 20℃ | 30min |
It has reacted and has been purified with 1.8 times of volume Ampure XP beads
End adds dATP after repairing:
Response procedures:
| 37℃ | 30min |
It has reacted and has been purified with 1.8 times of volume Ampure XP beads
Connector connection:
| Reacted constituent | Volume |
| DNA sample after adding " A " | 20.5uL |
| 2x Rapid Ligation buffer | 25uL |
| PE Adapter oligo mix(40uM) | 1.5uL |
| T4DNA Ligase(Rapid) | 3uL |
Response procedures:
| 20℃ | 15min |
It has reacted and has been purified with 1.5 times of volume Ampure XP beads.And with Nanodrop detectable concentration.
Pre- amplification:
| Reacted constituent | Volume |
| Product after connection | 32uL |
| index primer(10uM) | 4uL |
| 10×Pfx Amplification Buffer | 5uL |
| dNTP Solution Set(10mM) | 2uL |
| MgSO4(50mM) | 1uL |
Response procedures:
It has reacted and has been purified with 1.5 times of volume Ampure XP beads.With3.0 fluorescent quantitation instrument detectable concentrations.
3. hybridization elution
Hybridization elution process is referring to " SureSelectXT Target Enrichment System for Illumina
Paired-End Multiplexed Sequencing Library " process
4. with Agilent2100 and qPCR detection clip size and library concentration.Hiseq4000, BGI Seq500 or its
The sequencing of its platform
5. lower machine data analysis
Bioinformatic analysis is carried out to sequencing result, show that Genotyping is compared, whether identification has affiliation.
The post-sampling 6. child volunteer is born, and sequencing analysis is carried out by above-mentioned steps, with prenatal foetal typing data
Compare, consistency reaches 100%.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (10)
1. a kind of method of affiliation between identification biological sample characterized by comprising
(1) based on the first sample of nucleic acid from the first organism and the second sample of nucleic acid from the second organism, structure
Build sequencing library；
(2) the obtained sequencing library in step (1) is sequenced；
(3) it is based on the sequencing result of step (2), determines first sample of nucleic acid and second sample of nucleic acid in predetermined SNP
The base type in site；
(4) based on obtained in step (3) as a result, determining that the relationship of first organism and second organism is closed
System,
Wherein,
The predetermined SNP site includes selected from least one of 1 witness mark of table.
2. the method according to claim 1, wherein the accumulation of first sample of nucleic acid and the second sample of nucleic acid
It is the instruction with affinity between biological sample that parental right probability, which is greater than 10000,.
3. the method according to claim 1, wherein first sample of nucleic acid and the second sample of nucleic acid include choosing
From at least one of DNA and RNA.
4. the method according to claim 1, wherein first organism is fetus, second organism
For the doubtful biology father of the fetus.
5. according to the method described in claim 4, it is characterized in that, first sample of nucleic acid derive from maternal blood,
Optionally, first sample of nucleic acid is maternal plasma dissociative DNA；
Optionally, second sample of nucleic acid derives from blood, skin, hair, saliva, muscle and the essence of second organism
At least one of liquid.
6. the method according to claim 1, wherein the sequencing is in BGISeq-500 or Illimina
It is carried out in Hiseq2000/4000 microarray dataset,
Optionally, before carrying out the sequencing, screening enrichment processing is carried out to the sequencing library, wherein the screening is rich
Collection processing is directed to the probe of the predetermined SNP site using specificity.
7. according to the method described in claim 5, it is characterized in that, first sample of nucleic acid is free for maternal plasma
DNA, the method further includes:
A constructs sequencing library based on the third sample of nucleic acid from third organism, and the third organism is described pregnant
Woman, the third biological sample derive from pregnant woman's leucocyte, and the third biological sample is pregnant woman's leucocyte genome
DNA；
The sequencing library obtained in step a is sequenced in b；And
C determines the third sample of nucleic acid in the base type of predetermined SNP site based on the sequencing result of step b；
D, based on obtained in step c as a result, excluding pregnant woman's genomic DNA in maternal plasma dissociative DNA.
8. the system of affiliation between a kind of identification biological sample characterized by comprising
Sequencing library device is constructed, the building sequencing library device is used for based on the first nucleic acid sample from the first organism
This and from the second organism the second sample of nucleic acid, construct sequencing library；
Sequencing device, the sequencing device are used to that obtained sequencing library to be sequenced,
It determines that base types of devices, the determining base types of devices are used to be based on sequencing result, determines the first nucleic acid sample
The base type of sheet and second sample of nucleic acid in predetermined SNP site；And
Decision maker, the decision maker are used to determine first organism and described second based on determining base type
The affiliation of organism, wherein the SNP site includes being selected from least one of 1 witness mark of table,
Optionally, it is between biological sample that the accumulation parental right probability of first sample of nucleic acid and the second sample of nucleic acid, which is greater than 10000,
Instruction with affinity,
Optionally, first sample of nucleic acid and the second sample of nucleic acid include being selected from least one of DNA and RNA,
Optionally, first organism is fetus, and second organism is the doubtful biology father of the fetus,
Optionally, first sample of nucleic acid derives from maternal blood,
Optionally, first sample of nucleic acid is maternal plasma dissociative DNA,
Optionally, second biological sample derives from blood, skin, hair, saliva, muscle and the essence of second organism
At least one of liquid,
Optionally, the sequencing is carried out in 2000/4000 microarray dataset of BGISeq-500 or Illimina Hiseq,
Optionally, the system further comprises screening enriching apparatus,
The probe that the screening enriching apparatus is used for using specificity for the predetermined SNP site carries out the sequencing library
Screen enrichment processing.
9. a kind of for carrying out the kit of screening enrichment to sequencing library characterized by comprising
Probe, the probe specificity identify that predetermined SNP site, the predetermined SNP site include selected from site shown in table 1
At least one.
10. detecting purposes of the reagent of predetermined SNP site in Preparation equipment, the equipment is for determining relationship between biological sample
Relationship, the predetermined SNP site, the predetermined SNP site include selected from least one of site shown in table 1.
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Application publication date: 20190115 |