CN109280696B - Method for splitting mixed sample by SNP detection technology - Google Patents

Method for splitting mixed sample by SNP detection technology Download PDF

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CN109280696B
CN109280696B CN201710599784.8A CN201710599784A CN109280696B CN 109280696 B CN109280696 B CN 109280696B CN 201710599784 A CN201710599784 A CN 201710599784A CN 109280696 B CN109280696 B CN 109280696B
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曹彦东
赵雪莹
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SHANGHAI CRIMINAL SCIENCE TECHNOLOGY RESEARCH INSTITUTE
Analyses Beijing Biotechnology Co ltd
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Abstract

The invention discloses a method for splitting a mixed sample by using an SNP detection technology. The method uses a high-throughput gene sequencing platform (NGS) to simultaneously detect a plurality of Single Nucleotide Polymorphism Sites (SNPs) by amplicon sequencing, and can solve the problem that a double-person mixed sample cannot be separated in criminal evidence identification, civil judicial expertise identification and clinical application. The method has the technical key points that: 1) in order to ensure that the amplification efficiency of the amplicons is relatively uniform and stable, 219 amplicons obtained by strict condition design and screening are simultaneously amplified in the same tube. 2) In order to ensure that the degraded samples (160bp DNA fragment length) can be successfully amplified, SNPs are selected as biological markers, and are positioned in the middle of a 140bp amplicon. 3) In order to distinguish the sample into a single-person sample or a double-person mixed sample, the mixing proportion of the double-person mixed sample and the respective SNPs genotype set are further split.

Description

Method for splitting mixed sample by SNP detection technology
Technical Field
The invention belongs to the technical field of biology, and particularly relates to mixed sample splitting in forensic DNA individual identification.
Background
At present, in the field of forensic DNA individual identification and paternity testing, STR is mainly used as a biomarker, and along with the development of a new generation of high-throughput gene detection technology, forensic application using SNP as a biomarker begins to completely reveal the horn.
At present, three difficult and complicated materials exist in the field of forensic medicine: 1. mixing a sample: mixed seminal spots in sexual invasion cases, particularly non-seminal spots, mixed blood spots on geriatric organs and the like, DNA signals of a plurality of samples interfere with each other, and a suspect cannot be determined; 2. degrading the sample: when the DNA is degraded into fragments below 200bp, the experimental library building fails due to the fact that the fragments are too short; 3. trace sample: evidence obtained at the crime scene is usually spider-web horse marks, a fingerprint, used rubber gloves and the like, and the experiment library still fails when the initial amount of DNA is too small and only trace level is obtained.
STR Short Tandem Repeat (STR) is also called microsatellite DNA (micro satellite DNA) and is a DNA polymorphic locus widely existing in the human genome. It is composed of 2-6 base pairs to form the core sequence, and is series-connected and repeated, STR gene site length is generally between 100-300bp, and high polymorphism is formed due to DNA fragment length or DNA sequence difference between individuals, and it follows Mendelian inheritance mode in the gene transmission process, and has been widely used in the fields of forensic individual identification and paternity test, etc.
SNP (single nucleotide polymorphism) refers to DNA sequence polymorphism caused by variation of a single nucleotide at the genome level. It is the most common one of the heritable variations in humans. Accounting for more than 90% of all known polymorphisms. SNPs are widely present in the human genome, averaging 1 in every 500-1000 base pairs, and the total number is estimated to be 300 ten thousand or more.
DNA individual recognition was confirmed by analyzing whether the biomarkers STR could match. The method is used for criminal case DNA evidence identification and relies on an STR database of a public security system, and the database is continuously accumulated and updated at present.
The DNA paternity test is to judge whether parents and children are in paternity or not by analyzing whether the biomarkers STR of the offspring and the parents accord with the genetic characteristics according to the theories of forensics, biology and genetics. The detection process comprises the following steps: DNA extraction: extracting DNA in the sample cell nucleus, and purifying (removing impurities such as protein); PCR amplification: the DNA target fragment (STR sequence) is largely copied on a PCR instrument through Polymerase Chain Reaction (PCR) to achieve enrichment amplification effect; 3. adding a detection marker: DNA double strand is separated, and a specific detection marker (used for marking the length of the detected fragment) is added; 4. detecting by a capillary sequencer: DNA carries electric charge, and under the conditions of the same voltage and electrophoresis time, the different lengths of DNA fragments result in different electrophoresis speeds and different display positions of the detection markers through a capillary electrophoresis sequencer; 5. analyzing data and issuing a report: and judging whether the parents and the children are in the parent-child relationship according to whether the STR detection results of the children, the father and the mother accord with the genetic characteristics. The method needs to sample and compare the three to be tested respectively, wherein children need to be independent individuals to accurately sample.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for splitting a mixed sample by an SNP detection technology, in particular to solve the problem of mixed samples (especially 2-person mixing), a high-throughput sequencing platform is used for establishing a library to analyze an SNP set, and the mixed samples (especially 2-person mixing) can be accurately split.
The technical scheme adopted by the invention is as follows: a method for splitting mixed SNP set data comprises the following specific operation steps:
the method comprises the following steps: suppose the mixed sample comprises a sample and a sample, and the mixing ratio is X0：(100-X0)，X0<50 and X0>＝0；
Step two: taking N SNP loci to detect the mixed sample to obtain a mixed sample SNP set, obtaining 2N genotypes of the a sample and the b sample in total, and obtaining mutation frequency Y of each SNP locus;
step three: for mutation frequency Y of each SNP site, the mixing ratio of a and b is 9, which is specifically as follows:
a.2*Y＝X1*GT0+(100-X1)*GT0
b.2*Y＝X2*GT0+(100-X2)*GT1
c.2*Y＝X3*GT0+(100-X3)*GT2
d.2*Y＝X4*GT1+(100-X4)*GT0
e.2*Y＝X5*GT1+(100-X5)*GT1
f.2*Y＝X6*GT1+(100-X6)*GT2
g.2*Y＝X7*GT2+(100-X7)*GT0
h.2*Y＝X8*GT2+(100-X8)*GT1
i.2*Y＝X9*GT2+(100-X9)*GT2
wherein, GT0Representing no mutation to 0, GT1Representing heterozygous mutation as 1, GT2Represents homozygous mutation of 2;
taking the mode of 9N X as the mixing proportion X of the a samples in the mixed sample0；
Step four: sample a mixing ratio X0Is substituted into 9 formulas corresponding to Y (X)1To X9Are all replaced by X0) To obtain (GT) that minimizes the difference between the two sides of the equationai,GTbi) The genotype of the samples a and b at the site i is determined. The method is repeated to obtain SNP set genotypes of N sites.
The method for splitting mixed samples according to SNP set data, which comprises the following steps: the number N of the SNP sites is not less than 80.
The method for splitting mixed samples according to SNP set data as set forth in claim 1, wherein: the SNP locus marker upstream primer nucleotide sequence is shown as SEQ ID NO.1-100, and the SNP locus marker downstream primer nucleotide sequence is shown as SEQ ID NO. 101-200.
A method for splitting a mixed sample based on the SNP detection technology of the SNP set data splitting method of claims 1 to 3, which is characterized in that: the method comprises the following steps:
step 1, extracting mixed sample genome DNA, and enriching target DNA fragments by a multiplex reaction PCR method;
step 2, constructing a DNA fragment library, preparing an NGS sequencing template, and sequencing the DNA fragment through the NGS;
step 3, obtaining the SNP set genotypes of the samples a and b according to the method for splitting the mixed sample of the SNP set data of the claim 1;
step 4, obtaining the SNP set genotype of the known sample a, and then deducing the SNP set genotype of the mixed unknown sample b;
preferably, the mixed sample is a pregnant peripheral blood, criminal case sample or organ transplant patient blood sample.
The method for splitting the mixed sample by the SNP detection technology is applied to criminal case sample identification, when the mixed sample is a criminal case sample, the mixed SNP set is split and analyzed by a mixed SNP set data splitting method, the mixed sample is judged to be mixed samples of 2 persons or more, the known sample comprises a victim SNP set, the suspect SNP set is obtained by comparison, and a basis is provided for criminal cases.
The method for splitting the mixed sample by the SNP detection technology is applied to paternity and child identification through the peripheral blood of a pregnant woman, when the mixed sample is the peripheral blood of the pregnant woman, the known sample comprises maternal genome DNA, the unknown sample comprises fetal genome DNA, and the confirmed fetal SNP set and the father SNP set to be detected are subjected to genetic characteristic comparison, so that the relationship between a fetus and the father to be detected is judged.
The method for splitting the mixed sample by the SNP detection technology is applied to the detection of the blood sample of the organ transplantation patient, when the mixed sample is the blood sample of the organ transplantation patient, the genome DNA in the mixed sample is the recipient genome DNA and the donor genome DNA respectively, the mixed SNP set is split in a mixed proportion by the mixed SNP set data splitting method, and the rejection condition is judged according to the mixed proportion.
The invention has the advantages and positive effects that: the DNA fragment where the biological marker is located needs to be shorter, the detection sensitivity is higher, the initial amount of the DNA is very low, and the result is required to be repeatable; the high-throughput sequencing platform is used for detecting the appointed SNP set, 1-5% of sensitivity can be realized on 160bpDNA fragments and 1ngDNA initial quality samples, and if the DNA quality samples are 2 human mixed samples, the mixing ratio is more than 1: 49, the SNP can be accurately split into two SNP sets.
The scheme has multiple applications, and in criminal case sample detection, if a victim SNP set is known, another SNP set can be determined as a suspect SNP set, so that a key clue is provided for criminal case detection; the method can also be used for noninvasive paternity test, a fetal SNP set can be accurately split only by taking peripheral blood more than six weeks of pregnancy, and then the SNP set of a father is compared to determine whether the father is a biological father of a fetus, factors that children are independent individuals are eliminated, and the paternity relationship can be determined as early as possible, for example, the method can be applied to criminal cases and can quickly determine the suspected person of rape; meanwhile, the method can be applied to the detection of the rejection condition of the transplanted organ.
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FIG. 1 is a flow chart of an embodiment of the present invention;
FIG. 2 is a graph of the mixing ratio versus the frequency distribution of mutations;
FIG. 3 shows SNP site distribution;
FIG. 4 is a SNP site density distribution curve.
Detailed Description
The scheme mainly applies the SNP detection technology to the split of the mixed sample, and has the core that a high-throughput sequencing platform is used for detecting the appointed SNP set, so that the sensitivity of 1-5% can be realized on 160bpDNA fragments and 1ngDNA initial quantity detection materials. If the sample is a 2-person mixed sample, the sample can be accurately split into two SNP sets, and particularly applied to criminal cases.
The method for splitting the mixed sample by the SNP detection technology comprises the following steps:
step 1, extracting genome DNA of a mixed sample, taking peripheral blood free DNA of a pregnant woman or an organ transplantation patient as the mixed sample, and removing blood cells by centrifugation; and other mixed samples, such as mixed seminal plaque/blood plaque, need to crack cells, and a mixed SNP set is constructed through NGS, wherein the nucleotide sequence of an SNP site marker upstream primer is shown as SEQ ID NO.1-80, and the nucleotide sequence of an SNP site marker downstream primer is shown as SEQ ID NO. 81-160.
Step 2: and (3) constructing a library, namely amplifying and enriching the DNA obtained in the step one by using a kit of multiplex reaction PCR (polymerase chain reaction), and then adding a sequencing joint (containing a tag sequence for sample identification) to construct a library.
And step 3: and (3) sequencing by using a high-throughput sequencer, for example, sequencing the library obtained in the step two by using IonTorrentPGM/S5/S5XL and IlluminaMiSeq to obtain data, wherein the uniformity parameter of the amplicon is more than 95%, the average coverage depth is more than 200X, and the number of autosomal SNP sites is more than 80. And obtaining the mutation frequency of each site in the SNP set
Step 4, a method for splitting a mixed sample by SNP set data comprises the following specific operation steps:
1): suppose the mixed sample comprises a sample and a sample, and the mixing ratio is X0：(100-X0)，X0<50 and X0>＝0；
2): taking N SNP loci to detect the mixed sample to obtain a mixed sample SNP set, obtaining 2N genotypes of the a sample and the b sample in total, and obtaining mutation frequency Y of each SNP locus;
3): for mutation frequency Y of each SNP site, the mixing ratio of a and b is 9, which is specifically as follows:
a.2*Y＝X1*GT0+(100-X1)*GT0
b.2*Y＝X2*GT0+(100-X2)*GT1
c.2*Y＝X3*GT0+(100-X3)*GT2
d.2*Y＝X4*GT1+(100-X4)*GT0
e.2*Y＝X5*GT1+(100-X5)*GT1
f.2*Y＝X6*GT1+(100-X6)*GT2
g.2*Y＝X7*GT2+(100-X7)*GT0
h.2*Y＝X8*GT2+(100-X8)*GT1
i.2*Y＝X9*GT2+(100-X9)*GT2
wherein, GT0Representing no mutation to 0, GT1Representing heterozygous mutation as 1, GT2Represents homozygous mutation of 2;
taking the mode of 9N X as the mixing proportion X of the a samples in the mixed sample0；
4): sample a mixing ratio X0Is substituted into 9 formulas corresponding to Y (X)1To X9Are all replaced by X0) To obtain the difference between two sides of equationMinimum value (GT)ai,GTbi) The genotype of the samples a and b at the site i is determined. The method is repeated to obtain SNP set genotypes of N sites.
For a: b is 1: 1 or 1: 2, most of which are 2-3 genotype combinations, are difficult to resolve accurately, so that only 1: 1,1: 2, but cannot accurately resolve the SNP genotype sets of the two individuals. According to the scheme, the sample can be distinguished from a single human sample, a two-human mixed sample and a mixed sample of more than three people through a human blood or body fluid sample, and if the sample is a two-human mixed sample, the SNP genotype set of two people can be split. Based on the technical support, the scheme has remarkable effects in public security criminal evidence identification, clinical pregnant woman peripheral blood paternity identification and clinical organ transplantation immune system monitoring as shown in figure 1.
Example 1: mixed sample proportional splitting
1): suppose the mixed sample comprises a sample and a sample, and the mixing ratio is X0：(100-X0)，X0<50 and X0>＝0；
2): taking N SNP loci to detect the mixed sample to obtain a mixed sample SNP set, obtaining 2N genotypes of the a sample and the b sample in total, and obtaining mutation frequency Y of each SNP locus;
3): for mutation frequency Y of each SNP site, the mixing ratio of a and b is 9, which is specifically as follows:
a.2*Y＝X1*GT0+(100-X1)*GT0
b.2*Y＝X2*GT0+(100-X2)*GT1
c.2*Y＝X3*GT0+(100-X3)*GT2
d.2*Y＝X4*GT1+(100-X4)*GT0
e.2*Y＝X5*GT1+(100-X5)*GT1
f.2*Y＝X6*GT1+(100-X6)*GT2
g.2*Y＝X7*GT2+(100-X7)*GT0
h.2*Y＝X8*GT2+(100-X8)*GT1
i.2*Y＝X9*GT2+(100-X9)*GT2
wherein, GT0Representing no mutation to 0, GT1Representing heterozygous mutation as 1, GT2Represents homozygous mutation of 2;
taking the mode of 9N X as the mixing proportion X of the a samples in the mixed sample0. The results of the detection are shown in FIG. 3, which shows the distribution of a plurality of SNP sites. FIG. 4 is a density distribution curve of the SNP sites according to the distribution of FIG. 3 with the mixing ratio of the sample a as an independent variable, and it can be seen that the peak value is 20%, i.e. the mixing ratio can be determined to be 1: 4.
4): sample a mixing ratio X0Is substituted into 9 formulas corresponding to Y (X)1To X9Are all replaced by X0) To obtain (GT) that minimizes the difference between the two sides of the equationai,GTbi) The genotype of the samples a and b at the site i is determined. The method is repeated to obtain SNP set genotypes of N sites.
Example 2: public security criminal material evidence identification
DNA extraction: gDNA extraction of mixed samples of criminal cases.
1) The mixed sperm spot (or mixed blood spot on a syringe) in the invasive case is taken into a centrifuge tube containing the cell lysate. The centrifuge tube is reversed for 5-6 times and mixed evenly.
2) The cells were lysed by incubation for 10min at room temperature. Centrifuge at 2000Xg for 10 minutes at room temperature.
3) The supernatant was removed and approximately 1.4mL of liquid remained in the centrifuge tube.
4) RNase was added, the protein pellet was centrifuged at 2000Xg for 10 minutes.
5) Isopropanol was added to the supernatant, centrifuged at 2000Xg for 1 minute at room temperature, and the supernatant was discarded.
6) Washing with ethanol and drying to obtain DNA.
2. Library construction and template preparation
1) Configuring DNA targeting amplification reaction system
5X Ion AmpliSeqTM HiFi Mix(red cap)4uL,2X Ion AmpliSeqTM Primer Pool 4uL,DNA(1-10ng)<10uL,Nuclease-free Water 20uL。
2) Target amplification
The amplicon in which the SNP is located is called the target and the target amplification is performed using the following procedure:
Figure BDA0001356849550000061
3) joint and purification
When sequencing different libraries on a new plate, a different tag sequence must be attached to each library, the tag sequence being located at the sequencing adapter. Using IonXpressTMThe BarcodeX and Ion P1 Adapter add-on linker can be used for Ion Torrent platform detection; if the Illumina platform is used for detection, blunt end repair, phosphorylation, addition of an a-shaped adult end and then addition of a sequencing linker of the Illumina platform are required for the PCR product. This example uses the Ion Torrent platform.
4) QPCR quantification
The Library was quantified using Ion Library TaqMan quantification Kit (Cat. No.4468802) Kit for qPCR method. Libraries for amplification typically yield 100-500 pM. After quantification, the mixture was diluted to-100 pM and then template preparation was carried out.
3. High throughput sequencer sequencing
Sequencing to obtain a large number of DNA fragment base reads (reads), and performing on-machine sequencing by adopting a second-generation sequencer comprising an Ion Torrent platform or an Illumina platform sequencer.
4. Analyzing data and issuing reports
And comparing mass data reads obtained by sequencing to a human genome standard reference sequence hg19(GRCh37), calculating and recording mutation frequency of each allele of each SNP, splitting respective genotypes of the mixed samples through SNP set data, and comparing the obtained two SNP set genotypes with the known sample SNP set genotypes to obtain an unknown sample SNP set genotype, namely the suspect SNP set genotype.
Example 3: noninvasive fetal paternity testing
In the existing forensic physical evidence identification, the identification of parent-child relationship needs to provide respective test materials for children, father and mother, such as blood, hair, saliva, oral cells, bone and the like, which can be used for the identification of parents, wherein the children need to be independent individuals to accurately sample. By adopting the technical scheme, the SNP set of the child can be separated from the peripheral blood of the pregnant woman, the factor that the child is an independent individual is eliminated, and the parent-child relationship can be determined as early as possible. Because the concentration of the fetal free-fetus DNA in the pregnant woman peripheral blood free DNA (cell-free-fetus DNA) is 5-15% in more than six weeks, the pregnant woman peripheral blood can be regarded as a mixed test material of two persons, the fetal SNP is collected by a method of splitting a mixed sample by an SNP detection technology, and the fetal SNP is compared with a father sample to be detected, so that early noninvasive paternity test is realized. The specific implementation steps are as follows:
DNA extraction: cfDNA extraction from pregnant woman peripheral blood
1) Material and equipment requirements
Figure BDA0001356849550000071
Matters of Experimental attention
(1) All steps were carried out at room temperature (20-25 ℃) unless otherwise noted.
(2) Avoid generating bubbles when the mixture is evenly mixed by a liquid transfer device.
(3) MagMAX if the storage temperature is too lowTMCell Free DNA lysine/Binding Solution and MagMAXTMPrecipitation may occur in Cell Free DNA Wash Solution by incubating the two solutions at 37 ℃ for 1 hour.
(4)MagMAXTMCell Free DNA Magnetic Beads were vortexed thoroughly to resuspend the Beads before use.
(5) Under the condition of preparing MagMAXTM Cell Free DNA Lysis/Binding SResolution and MagMAXTMWhen Cell Free DNA Magnetic Beads are mixed, it is recommended to prepare a larger amount of 5 to 10% of the total sample mixture.
(6) Plasma samples were stably stored in Streck tubes for 14 days. Treatment of the sample with proteinase K prior to extraction can result in 50% higher cfDNA yield.
2) Manual extraction step of free DNA
Preparation of free plasma samples
(1) Whole blood was centrifuged at 2000Xg for 10min at 4 ℃.
(2) Transfer supernatant plasma to a new centrifuge tube.
(3) The transferred plasma was centrifuged again at 16,000 Xg for 10min at 4 ℃.
Note that: alternatively, the transferred plasma can be centrifuged at 6,000 Xg for 30min at 4 ℃ to remove residual blood or cellular debris.
(the following procedure applies to a whole blood sample collected using an EDTA blood collection tube, for a whole blood sample collected using a Strek tube, please refer to protocol Publication number MAN0014327 in English)
3) Lysis of plasma samples to bind cfDNA to magnetic beads
Free DNA binding solution (MagMax) was added in the order of the amounts shown in the table belowTMCell Free DNA lysine/Binding Solution), magnetic beads (MagMax)TMCell Free DNA Magnetic Beads) and plasma preparation
And mixing the mixed solution fully and uniformly, wherein the mixing ratio is 1.25: 0.015: 1
(1) An appropriate volume of plasma sample was added.
(2) Mix by inversion 10 times to mix the plasma sample well with Binding Solution/beads mix.
(3) Shaking for 10min to allow free DNA to bind to the magnetic beads.
(4) After brief centrifugation to collect the tube and cap the tube with liquid, place the tube in a magnetic rack (DynaMag)TMMagnet) for 5min or until the liquid becomes clear and the magnetic beads form a pellet.
(5) The supernatant was carefully discarded using a pipette.
(6) The centrifuge tube was placed on the magnetic stand for another 1min, and the remaining supernatant was removed by pipette.
4) Washing with washing solution and 80% ethanol
(1) Take 1mlMagMAXTMCell Free DNA Wash Solution vortex resuspend the magnetic beads.
(2) The suspension of magnetic beads was transferred to a new 1.5ml centrifuge tube, which was temporarily retained.
(3) A new 1.5ml centrifuge tube containing a suspension of magnetic beads was placed on the magnetic rack for 20 s.
(4) The supernatant was collected and used to re-rinse the previously retained old centrifuge tubes.
(5) The resulting suspension of magnetic beads was transferred to a 1.5ml centrifuge tube on a magnetic rack, and the old centrifuge tube was discarded.
(6) The 1.5ml centrifuge tube was placed on the magnetic rack for an additional 2min, or until the liquid cleared and the beads formed a pellet.
(7) The supernatant was aspirated off with a 1ml pipette.
(8) The 1.5ml centrifuge tube was retained on the magnetic rack, the magnetic rack was tapped 5 times on the bench top, and then all remaining liquid was pipetted off with a 200ul pipette.
(9) The tube was removed from the magnetic stand, 1ml of freshly prepared 80% ethanol was added, and vortexed for 30 s.
(10) The 1.5ml centrifuge tube was placed on the magnetic rack for an additional 2min, or until the liquid cleared and the beads formed a pellet.
(11) The supernatant was aspirated off with a 1ml pipette.
(12) The 1.5ml centrifuge tubes were retained in a magnetic holder (DynaMag)TM-2Magnet), the beads were air dried for 3-5 min.
(13) The magnetic rack was tapped 5 times on the bench and then all remaining liquid was aspirated off with a 200ul pipette.
5) Elute cfDNA, re-bind to magnetic beads and wash
(1) Add 400ul0.1XTAE to air dried beads and vortex for 5 min.
(2) The 1.5ml centrifuge tube was placed on the magnetic rack for an additional 2min, or until the liquid cleared and the beads formed a pellet.
(3) The supernatant was transferred to a new 1.5ml centrifuge tube.
(4) Add 5ul MagMax to supernatantTMCell Free DNA Magnetic Beads and 500ul MagMaxTMCell Free DNA lysine/Binding Solution and mix well.
(5) Shake for 5min to allow cfDNA to bind to the magnetic beads.
(6) The 1.5ml centrifuge tube was placed on the magnetic rack for an additional 5min, or until the liquid cleared and the beads formed a pellet.
(7) The supernatant was aspirated off with a 1ml pipette.
(8) The centrifuge tube was removed from the magnetic stand and 1ml of MagMax was addedTMCell Free DNA Wash Solution, vortex mix for 30 s.
(9) The 1.5ml centrifuge tube was placed on the magnetic rack for an additional 2min, or until the liquid cleared and the beads formed a pellet.
(10) The supernatant was aspirated off with a 1ml pipette.
(11) The 1.5ml centrifuge tube was retained on the magnetic rack, the magnetic rack was tapped 5 times on the bench top, and then all remaining liquid was pipetted off with a 200ul pipette.
6) Washed twice with 80% ethanol
(1) The tube was removed from the magnetic frame, 1ml of freshly prepared 80% ethanol was added, and vortexed for 30 seconds.
(2) Place 1.5ml centrifuge tube on magnetic rack for 2min or until the liquid becomes clear and the beads form a pellet.
(3) The supernatant was aspirated off with a 1ml pipette.
(4) The 1.5ml centrifuge tube was retained on the magnetic rack, the magnetic rack was tapped 5 times on the bench top, and then all remaining liquid was pipetted off with a 200ul pipette.
(5) Repeating the steps 1-3, and washing with 80% ethanol.
(6) The 1.5ml centrifuge tubes were left on the magnetic rack and the beads were allowed to air dry for 3-5 min.
(7) The 1.5ml centrifuge tube was retained on the magnetic rack, the magnetic rack was tapped 5 times on the bench top, and then all remaining liquid was pipetted off with a 200ul pipette.
7) Elution of cfDNA
(1) Adding 10-15ul MagMAX into a 1.5ml centrifuge tubeTM Cell Free DNA Elution Solution。
(2) Vortex for 5 min.
(3) The 1.5ml centrifuge tube was placed on the magnetic rack for an additional 2min, or until the liquid cleared and the beads formed a pellet.
(4) Extracted cfDNA in supernatant.
(5) The extracted cfDNA can be used directly or transferred to a new centrifuge tube for storage.
(6) Storing at 4 ℃ for 24 h.
(7) Long-term storage at-20 deg.C.
2. Library construction and template preparation
The library construction and template preparation were performed in the same manner as in step 2 of example 2.
3. High throughput sequencer sequencing
Same example 2, step 3 high throughput sequencer sequencing
4. Analyzing data and issuing reports
And comparing mass data reads obtained by sequencing to a human genome standard reference sequence hg19(GRCh37), calculating and recording mutation frequency of each allele of each SNP, splitting respective genotypes of the mixed samples through SNP set data, and comparing the obtained two SNP set genotypes with the known sample SNP set genotypes to obtain an unknown sample SNP set genotype, namely the suspect SNP set genotype.
Wherein the sample with low mixing ratio is a fetal sample (about 10%), and the sample with high mixing ratio is a sample of a pregnant woman and a mother. And detecting the SNP set of the father to be detected, and judging whether the father to be detected is the biological father of the fetus.
Example 4: monitoring of rejection in organ transplant patients
DNA extraction: cfDNA extraction from organ transplant patients
DNA extraction was performed in the same manner as in step 1 of example 3.
2. Library construction and template preparation
Library construction and template preparation were performed as in step 2 of example 2.
3. High throughput sequencer sequencing
Sequencing was performed by the high throughput sequencer as in step 3 of example 3.
4. Analyzing data and issuing reports
And (3) aligning the massive data reads obtained by sequencing to a human genome standard reference sequence hg19(GRCh37), calculating and recording the mutation frequency of each allele of each SNP, and splitting the respective genotypes and the mixing proportion of the mixed samples through SNP set data. And judging the organ transplant rejection according to the change of the mixing ratio of the donor samples. Wherein the low mixing ratio is a sample of an organ donor.
Although the embodiments of the present invention have been described in detail, the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
TABLE I SNP site primer nucleotide sequence
Figure BDA0001356849550000111
Figure BDA0001356849550000121
Figure BDA0001356849550000131
Figure BDA0001356849550000141
Sequence listing
<110> Ansais (Beijing) Biotechnology Ltd
SHANGHAI CRIMINAL SCIENCE TECHNOLOGY Research Institute
<120> method for splitting mixed sample by SNP detection technology
<160> 200
<170> SIPOSequenceListing 1.0
<210> 1
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggcatccttt gtgagctttt taataaaga 29
<210> 2
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ttgggactac aagcaagtga cataaa 26
<210> 3
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
actttcttct tacctagtgg aggttga 27
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
catgcttcgt ccaggaagat aagt 24
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgtacagca ccagaacgga aa 22
<210> 6
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tgaacatgga attcaatcct tcagatca 28
<210> 7
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tgatggccag gtgctgatag ta 22
<210> 8
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tggaattttc tctagctgtt actccaga 28
<210> 9
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gccataagaa ttgcttccct gagat 25
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
acacagcttg ccataaaccc tac 23
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
agagtctcca actgtgacca ct 22
<210> 12
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cctttccatt tcaccgcagt tatg 24
<210> 13
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
caagtacatc ctacagctct tcaaaac 27
<210> 14
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cgaccgatta gatggtgcac at 22
<210> 15
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cgtttgtatt tctctgccag tgaga 25
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gtgtggctgg atggacttag tc 22
<210> 17
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ctctttgggt tggcattctg ttt 23
<210> 18
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
acaagaaaaa gggacagatt acatctgt 28
<210> 19
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
aaataaacaa aatcacaaat gctttcatgg c 31
<210> 20
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
atggcaccat cctcttcctt tac 23
<210> 21
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gcacagtagc attccaccat tg 22
<210> 22
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gtttctggca ggatgcacaa tg 22
<210> 23
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
ctctacaaac aaactgctgt gtttca 26
<210> 24
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gttcttgaaa gtgagattgc tgtaacc 27
<210> 25
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tgtcgtcaag tctatctctt gatgtactt 29
<210> 26
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
tcttaaaggc tgaagcgtca ca 22
<210> 27
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ccactctgtt gcaaagagac taga 24
<210> 28
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ggtcagcaat cctctgctca tt 22
<210> 29
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ttctgagagg attctgcagc aaaa 24
<210> 30
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
gttgctcagg atctgggctt aa 22
<210> 31
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
acccttggca ttgctgaatt ga 22
<210> 33
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ccaggtattc tgcagaaggg atctta 26
<210> 33
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
actatattgt gatgcccaga agttctg 27
<210> 34
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ccttcgggag ctgcatca 18
<210> 35
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gatgtcaaga cctgcctcca at 22
<210> 36
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
cagaaagaag acaagattca aacaagctt 29
<210> 37
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
ataattgttt ctcctttcca ccacagat 28
<210> 38
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
ccagcaaacc ctaccacata gg 22
<210> 39
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
cagctatccg ttttggtcgg at 22
<210> 40
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gtctagagac cagcatgttc tcc 23
<210> 41
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
acgatctctt tcgattaaat cagcca 26
<210> 42
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
gaggtttcgg gatgggaaag ag 22
<210> 43
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
agatgattaa aggaaaagtg aaagctgga 29
<210> 44
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
taggtgaaaa tgcgtggaat agacttt 27
<210> 45
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
ctctgcaaaa caagaatttg ttgagc 26
<210> 46
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
ttttgctgga ttggtctgag gaa 23
<210> 47
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
gtgcccatcc tcaaatacaa agc 23
<210> 48
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
agtgttaccg ctggtgtaat ctc 23
<210> 49
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
tattttcagg gaagaagagc ccagaccta 29
<210> 50
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
tttttatctc tcttgtgtgc ttttaggtg 29
<210> 51
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
cctgccctta actgtctctc tca 23
<210> 52
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
gggaacttct tcatgggtgg t 21
<210> 53
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tgaacaatgt ggcaaaggct ttaac 25
<210> 54
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
aagaggtcca ggatcttcct ca 22
<210> 55
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
gataagtgtc tccaatttaa ctaacgtgga 30
<210> 56
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
ccttacttca gctgcgacca 20
<210> 57
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
tatgggaacc ggacggtcaa ag 22
<210> 58
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
gttggctgtc tcatgctgat g 21
<210> 59
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
tatacaagaa acctttacat gcgaatcaga 30
<210> 60
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
tcaagaaagc tggtggtttc tagtt 25
<210> 61
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
cacacattgt ttgactcaca atacttttgt 30
<210> 62
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
atcaccacca aagaccctag gat 23
<210> 63
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
ggaggaagag aaggctaagg tagaa 25
<210> 64
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
cctggtaagt ttcaaaaccc aaatacg 27
<210> 65
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
ggagctcata tcttattgaa cacca 25
<210> 66
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
tttttccttc ccttcagcca agat 24
<210> 67
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
aaattcaggc tggactttaa atgatcatt 29
<210> 68
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
cactgggtgt gatctttctg aatg 24
<210> 69
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
gcacacatga tcagcacctt ca 22
<210> 70
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
actggtcaga catcgagcta gt 22
<210> 71
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
tggctgtctc cagatgacca 20
<210> 72
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
agccagacct tggccataaa tg 22
<210> 73
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
gccattgtat ataactcttc ttgaaggct 29
<210> 74
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
aagaaataaa gcagtatcac agacaacaga 30
<210> 75
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
tggtctgagt ccttactgtg tctt 24
<210> 76
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
ctgcatttgc ttcccattca gag 23
<210> 77
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
aagcaatttt aatcattagt atggttttta gca 33
<210> 78
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
gggatgcttt acaaagttac tttctataat g 31
<210> 79
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
acagattgag ggcatctgta acag 24
<210> 80
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
ggactgactt taggtgaagg aaact 25
<210> 81
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
gaataggtct attgaattgg ttcagcaac 29
<210> 82
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
aagctcctgt ggctgcatag 20
<210> 83
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
aacagactga cctctcttcc tca 23
<210> 84
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
ctcaatcaag tcactgttca cacttac 27
<210> 85
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
gcaggaactt gaggatccga aa 22
<210> 86
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
gaaatatgaa ggttaccttg ttagcgtct 29
<210> 87
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
gagtgccctc tgtcatttct atca 24
<210> 88
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
ccacatggac aggtagctca 20
<210> 89
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
tttttagtta gccataacag gctggaa 27
<210> 90
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
ggaaactgat ctgctttcaa tgca 24
<210> 91
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
ttttactgga gagaaacctt gtgaatgt 28
<210> 92
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
agggttcccg ttagatgcac ta 22
<210> 93
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
ttgggtgtca taaagtgttc tttctga 27
<210> 94
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
tgggactatc ataccaccca cat 23
<210> 95
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
ttcagaagtg attagaggga ataaaatgca 30
<210> 96
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
gcggtgagag atgtagacga 20
<210> 97
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
atgtacccag gaaagccaaa agaaaaact 29
<210> 98
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
gcagtctctc cttcaatggt aattacc 27
<210> 99
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
cagcagttct gttacttctc ccat 24
<210> 100
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
cttgcacacc ttgctcaaca tc 22
<210> 101
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
agatccggga caaggcattt tt 22
<210> 102
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
aactcttctc ttgcttaaga atctaacctc 30
<210> 103
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
caggttacgg ttacgactgg at 22
<210> 104
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
cataaccagg gttcctgggt tta 23
<210> 105
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
tggcaaacca gagggtattt gg 22
<210> 106
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
aaatcaacac taggcgaagt accaa 25
<210> 107
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
gtaccatcag tgtattccct gaatacag 28
<210> 108
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
acctattccc agctccaggt aa 22
<210> 109
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
gggagatcca ttttgctgac aag 23
<210> 110
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
ctcccaggac actggtcata c 21
<210> 111
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
ttcaggatcc attgcaatct gct 23
<210> 112
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
tttctcctga agtgcattat aaacctgat 29
<210> 113
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
catcatggag atcatgatcc atggt 25
<210> 114
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
gcatgagtca atttcttcaa gcgaa 25
<210> 115
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
gccctcttct gtgactttgc tg 22
<210> 116
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
tagactgagt ccctcactta cctg 24
<210> 117
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
aagttggtta agctgagcta acca 24
<210> 118
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
gctggaaact agaaaagcaa gattcatttc 30
<210> 119
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
atgggtgtcc cttgaaaatt cact 24
<210> 120
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
cttgacatct ttatttctca gactgtagat cag 33
<210> 121
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
gggccttctc tattggaatc atgc 24
<210> 122
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
agatgctggc aagaagcact ta 22
<210> 123
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
gtcattgttt cttatgttca gatgagacct 30
<210> 124
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
gtatgcaggg ctccagtagt tc 22
<210> 125
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
tcagtcaagt ggtcaagtac atgc 24
<210> 126
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
cttacagcgc agagaacgat ct 22
<210> 127
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
agtggtagga tagaaatgtt ctgtctaatc t 31
<210> 128
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
tcccagaatt tcacctgaca tcttc 25
<210> 129
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
ttctggcaca gagtagcgaa tc 22
<210> 130
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
ctttctcggt caatcttttg attcactg 28
<210> 131
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
agaaagaatt tctcaaaaga agccatgaac 30
<210> 132
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
aaacatgtgt gtctctattc tgttgaact 29
<210> 133
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
gggattcata ttgaattcag ggtttacttt 30
<210> 134
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
tcatttgatc cgcaagtcta gca 23
<210> 135
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
gcttaccaga ttaatcagtg cttcaaaatt 30
<210> 136
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
ggttagacat ggccttttgg cata 24
<210> 137
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
ccctcataga gcttaagaat tactgaactc 30
<210> 138
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
aattctgagt gctatgtgtt ggtatcaat 29
<210> 139
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
caggtaggca ttgtagatgt gctt 24
<210> 140
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
atttgggtag aatattccag tctttggg 28
<210> 141
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
ttgacttaca ggaggatgtt gacaag 26
<210> 142
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
cgaaactcgc gaaactgtac actta 25
<210> 143
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
aggaaagtct ggacatacct ttgc 24
<210> 144
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
cctaaaggaa atatctctgc caaagaagtt 30
<210> 145
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
ctctcttttg gccacattga agg 23
<210> 146
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
cccaagtatc ataatttggc tttccttttt 30
<210> 147
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
acatctgagt actgaggcac tcat 24
<210> 148
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
tatgcctcga gctttaaagg ctatataga 29
<210> 149
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
tctcccaaag gactcttccc att 23
<210> 150
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
gcatcagctc caaacatgtg atatct 26
<210> 151
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
tgatagcatc gagttgtacc attgg 25
<210> 152
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
caacttcagg ttaatatgac taaagcagtt ac 32
<210> 153
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
catcttatgt gtagtaaggt gtgaagacac 30
<210> 154
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
gctgttaggt gtactgttgc ttg 23
<210> 155
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
ccatttcaga tacacctttt atacacatgc 30
<210> 156
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
cataggaaga aatgaacatc tgattcttac ca 32
<210> 157
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
ctggcaggct tcttctgatt tc 22
<210> 158
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
caggaagata aaaaggacat cgttcca 27
<210> 159
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
ccctccaaga agttctctca cctaa 25
<210> 160
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
agccactacg ccaaatatat ctgtg 25
<210> 161
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
ttgaatatat ctcctactca gcttcccaa 29
<210> 162
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
gttaagctct accttgctgc tttg 24
<210> 163
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
ctttcttttc atttacccat ttcccttct 29
<210> 164
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
gggtagcagt accttcaaat tttcaaag 28
<210> 165
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
tcttcacact gatgcagttt ctttatagt 29
<210> 166
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
ctcaaccagt caaaatcctg gtatctt 27
<210> 167
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
acaaaattac tataagcagg cttgggat 28
<210> 168
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
caccaaatct ttggtcatga ccttc 25
<210> 169
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
cagcaggagc ttccttcagt tt 22
<210> 170
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
gtgtatatgt tttctttgca gccatttttc 30
<210> 171
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
ctgtcctgct gactgctctt t 21
<210> 172
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
gagttggctc tgtgtatggc tatag 25
<210> 173
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
taaatcggaa gccctggcaa agat 24
<210> 174
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
ctctctagcc ttggataaag agagaatg 28
<210> 175
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
cttgtggcac agtttcccat tc 22
<210> 176
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 176
ctctccatta aaaatgaact tgtctccatc 30
<210> 177
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 177
tggaaaatca aacaaaacca ccagatc 27
<210> 178
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 178
agcagcatag aagatgctca gtg 23
<210> 179
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 179
ccttccaaat gacagtcttt ttccc 25
<210> 180
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 180
agccaaaaac gcctccattg 20
<210> 181
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 181
gccgaagtaa ttttcccaat tttatagtct 30
<210> 182
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 182
acagtgggaa gcagctttgt ag 22
<210> 183
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 183
tctaaattca aggactttaa acctgatggg 30
<210> 184
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 184
cagatgaaaa ccgtcaaatt ctgtgtc 27
<210> 185
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 185
gccctgagtt ccaacatgtc tg 22
<210> 186
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 186
gctgtgagct ctcagtgatg ttataaataa 30
<210> 187
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 187
caattcatgg agcatacctt gttcc 25
<210> 188
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 188
gattcacgtt tctttcagat gaggtttc 28
<210> 189
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 189
tccagtacag taatgccttt gatgtg 26
<210> 190
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 190
tcctagtttt tctaagggat tgttcaagg 29
<210> 191
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 191
tgaggtttga tttgtggctg aaga 24
<210> 192
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 192
ctcagtaatg gtcactttgt ctttgtg 27
<210> 193
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 193
caagctacaa atcctcaaca tctacct 27
<210> 194
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 194
ggttgtgagc tctagttgtg cttt 24
<210> 195
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 195
ttttctcctg gaaggaactg aagtg 25
<210> 196
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 196
caacctgacc aagctggaca t 21
<210> 197
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 197
atcctgcacc ttagccatat tgtc 24
<210> 198
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 198
ccagccaaag aatgctcctg tta 23
<210> 199
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 199
ctttactggc gcttttatca gtgg 24
<210> 200
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 200
tcataaggtc ttttcccagt gtgaatc 27

Claims (4)

1. A method for splitting a mixed sample of SNP aggregate data, the method being a non-disease diagnostic method, characterized by: firstly, determining the mixing ratio of the two samples, and then determining the respective SNP set genotypes of the two samples, wherein the specific operation steps are as follows:
the method comprises the following steps: suppose the mixed sample comprises a sample and a sample, and the mixing ratio is X0:(100-X0)，X0<=50 and X0>=0；
Step two: taking N SNP loci to detect the mixed sample to obtain a mixed sample SNP set, obtaining 2N genotypes of the a sample and the b sample in total, and obtaining mutation frequency Y of each SNP locus;
step three: for mutation frequency Y of each SNP site, the mixing ratio of a and b is 9, which is specifically as follows:
2*Y=X1 *GT0 +(100-X1)*GT0
2*Y=X2 *GT0 +(100-X2)*GT1
2*Y=X3 *GT0 +(100-X3)*GT2
2*Y=X4 *GT1 +(100-X4)*GT0
2*Y=X5 *GT1+(100-X5)*GT1
2*Y=X6 *GT1+(100-X6)*GT2
2*Y=X7 *GT2 +(100-X7)*GT0
2*Y=X8 *GT2 +(100-X8)*GT1
2*Y=X9 *GT2+(100-X9)*GT2
wherein, GT0Representing no mutation to 0, GT1Represents that the heterozygous mutation is 1, and GT2 represents that the homozygous mutation is 2;
taking N X1-X9The mode of (a) is used as the sample mixing ratio X in the mixed sample0;
Step four, mixing the sample a with the proportion X0Carrying back to a formula, and calculating the respective SNP set genotypes of the samples a and b;
the number N of the SNP loci is not less than 80;
the nucleotide sequence of the labeled upstream primer of the SNP locus is shown as SEQ ID NO.1-100,
the nucleotide sequence of the labeled downstream primer of the SNP locus is shown as SEQ ID NO. 101-200.
2. A method for splitting a mixed sample by SNP detection technology, said method being a non-disease diagnostic method, characterized by:
the method comprises the following steps:
step 1, extracting genome DNA of a mixed sample, and enriching target DNA fragments by a multiplex reaction PCR method;
step 2, constructing a DNA fragment library, preparing an NGS sequencing template, and sequencing the DNA fragment through the NGS;
step 3, obtaining the respective SNP set genotypes of the samples a and b according to the method for splitting the mixed sample of the SNP set data of claim 1;
and 4, obtaining the SNP set genotype of the known sample a, and then deducing the SNP set genotype of the mixed unknown sample b.
3. The method for splitting a mixed sample according to the SNP detection technology of claim 2, wherein: the mixed sample is a criminal case sample.
4. The use of the method of splitting a mixed sample according to the SNP detection technique of claim 2 in criminal case sample identification, characterized by: when the mixed sample is a criminal case sample, the mixed SNP set is split and analyzed by a mixed SNP set data splitting method to judge that the mixed sample is a 2-person mixed sample, the known sample comprises a victim SNP set, and the suspect SNP set is obtained by comparison to provide a basis for the criminal case.
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CN110706746B (en) * 2019-11-27 2021-09-17 北京博安智联科技有限公司 DNA mixed typing database comparison algorithm
CN113512595B (en) * 2021-06-11 2023-07-25 深圳雅济科技有限公司 Biomarker, method and application for DNA sample tracking detection

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