CN117965744A - Kit, primer and method for detecting ploidy and maternal cell pollution of fetal samples based on multiplex PCR (polymerase chain reaction) capture technology - Google Patents
Kit, primer and method for detecting ploidy and maternal cell pollution of fetal samples based on multiplex PCR (polymerase chain reaction) capture technology Download PDFInfo
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Abstract
The invention discloses a kit, a primer and a method for detecting ploidy of a fetal sample and maternal cell pollution based on a multiplex PCR (polymerase chain reaction) capture technology. The invention screens and obtains a group of SNP locus combinations capable of detecting ploidy of a fetal sample and pollution of maternal cells, and designs corresponding primer combinations; a maternal pollution detection technology based on multiplex PCR amplification SNP loci is developed; the method can realize the identification of fetal ploidy, the accurate identification of 5% -95% maternal pollution and the identification of non-maternal and non-father relations in paternity and son relations. The technology has the characteristics of simple operation, low price, short experimental time, good compatibility with CNVseq and the like; the defect that the CNV-Seq cannot detect polyploid and maternal pollution is overcome, an effective quality control technical means is provided for samples for CNV-Seq detection, and the defect of STR in the aspect of detecting polyploid and maternal pollution is overcome.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a kit, a primer and a method for detecting ploidy and maternal cell pollution of a fetal sample based on a multiplex PCR (polymerase chain reaction) capture technology.
Background
The occurrence rate of chromosome abnormality of the infants is high, chromosome karyotype analysis is used as a gold standard for prenatal diagnosis of chromosome diseases, and the defects of complex experimental operation, high operation requirement, low flux and resolution and the like can only be detected when chromosome number abnormality and fragments larger than 10kb are deleted, repeated and inverted. Along with the rapid development of high-throughput sequencing technology, genome copy number variation sequencing (copy number variation sequerncing, CNV-seq) can detect copy number abnormality greater than 100kb and low-proportion chimeras, has the advantages of wide detection range, simple operation, good compatibility and the like, and is widely applied.
In practical clinical applications, prenatal diagnostic advice combines CNV-seq technology with STR testing to exclude maternal contamination. At present, antenatal diagnosis mainly adopts amniocentesis sampling and chorionic puncture sampling. However, due to differences in sampling protocols, timing, and operator skill levels, the sampling process may be accompanied by maternal peripheral blood, decidua tissue, etc., resulting in samples containing Maternal Cell Contamination (MCC). Maternal contamination can directly affect the accuracy of the results of subsequent testing techniques, such as those widely used as first-line prenatal diagnosis, CNVseq cannot identify maternal contamination samples and polyploids, resulting in false positives on fetal reality. For this reason, the CNVseq technology needs to be used in conjunction with STR (Short TANDEM REPEAT) or fluorescent quantitative PCR techniques in prenatal diagnosis [ 1 ]. Currently, prenatal diagnostic samples are recommended for STR or SNP typing techniques to exclude maternal contamination. STR is the most dominant detection means at present due to the price advantage in the aspects of parent source pollution and polyploid detection. However, the STR detection requires a specialized instrument, and the result is currently mainly determined manually according to experience [ 2 ], and the result in the peak diagram format is not convenient for an auditor to audit, which results in time and effort consumption and possibly inconsistent with the real condition of the sample. SNP (Single Nucleotide Polymorphism) refers to the case where there are multiple single base alleles at the same genomic position in a population, these genomic loci with polymorphisms are referred to as SNP loci [ 3 ]. SNP array based on whole genome high throughput sequencing overcomes the defect that aCGH and CNVseq cannot detect polyploid, LOH and the like, but has the characteristics of large sample input amount, high price, long period and the like, and limits the application of the SNP array.
【1】 The low-depth whole genome sequencing technology is applied to prenatal diagnosis by the expert's consensus [ J ]. J. Chinese medical genetics, 2019,36 (4): 4.
【2】PENG Zhu,XU Zhen,TU Zheng,YANG Fan,LI Yongjiu,YAN Anxin,NIE Hao,ZHAO Hemiao,ZHAO Xingchun.Evolving Researches into Mixed STR Profiles for Their Analysis[J].Forensic Science and Technology,2022,47(1):10-17.
【3】https://www.genome.gov/genetics-glossary/Single-Nucleotide-Polymorphisms.
Disclosure of Invention
The object of the first aspect of the present invention is to provide a detection reagent.
The object of the second aspect of the present invention is to provide the use of the above detection reagent.
The object of the third aspect of the present invention is to provide a kit.
The fourth aspect of the present invention is directed to a nucleic acid sequencing library construction method for detecting fetal ploidy and/or maternal cell contamination and/or paternity test.
The object of the fifth aspect of the invention is to provide a sequencing library.
The object of a sixth aspect of the present invention is to provide a method for detecting fetal ploidy and/or maternal cell contamination and/or paternity test.
It is an object of a seventh aspect of the present invention to provide a system for detecting fetal ploidy and/or maternal cell contamination and/or paternity test.
An eighth aspect of the present invention is directed to an electronic device.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided a detection reagent for detecting 105 SNP sites comprising ：rs6697376、rs12027119、rs10889407、rs10783100、rs2587895、rs3766361、rs3748666、rs1557080、rs1916231、rs1395931、rs10199403、rs13086388、rs9866013、rs9868434、rs2571468、rs279844、rs13134862、rs1835522、rs2903386、rs11732263、rs6811238、rs12651593、rs253484、rs6878425、rs1423326、rs7710068、rs7728908、rs9369380、rs4947212、rs6940134、rs9397342、rs1019029、rs2686817、rs2269991、rs6966616、rs2267708、rs12156144、rs12544054、rs10503926、rs2353202、rs2975207、rs3019303、rs4871182、rs10781004、rs3118846、rs10821461、rs1463729、rs3780962、rs1927453、rs12244967、rs56331924、rs537432、rs1278329、rs10128711、rs11037366、rs2905513、rs625232、rs2255301、rs219923、rs12581739、rs1995691、rs10506759、rs2579132、rs7979001、rs12306075、rs10773760、rs1161464、rs9316585、rs7321320、rs9531423、rs9302039、rs9519708、rs6571849、rs1454361、rs3922176、rs6606789、rs4244560、rs2899496、rs654065、rs4786640、rs7195826、rs11644264、rs899258、rs9927268、rs4485351、rs1390901、rs8070085、rs1027895、rs2292972、rs7506104、rs9960366、rs1395858、rs9950061、rs4891291、rs576261、rs12480506、rs4911248、rs1005533、rs2822397、rs1041409、rs2831057、rs2831711、rs2243508、rs987640、rs2040411.
Preferably, the detection reagent comprises a primer.
Preferably, the sequence of the primer is shown as SEQ ID NO. 1-SEQ ID NO. 210.
In a second aspect of the invention there is provided the use of a detection reagent according to the first aspect of the invention in any of the following; 1. detecting fetal ploidy;
2. Preparing a product for detecting fetal ploidy;
3. detecting maternal cell contamination;
4. Preparing a product for detecting maternal cell pollution;
5. Parent-child relationship identification;
6. and preparing a paternity test product.
Preferably, the ploidy comprises a whole genome uniparent diploid, triploid and/or tetraploid.
In a third aspect of the invention, there is provided a kit comprising the detection reagent according to the first aspect of the invention.
Preferably, the 5' end of each primer in the detection reagent is connected with a universal sequence required for library-building sequencing.
Preferably, the universal sequence is selected from the group consisting of a common primer, a linker, a specific tag.
Wherein the public primer is used for connecting the target fragment with the linker and the specific tag through PCR; the specific labels are used for distinguishing samples, and different samples are labeled by different specific labels when the library is constructed, so that high-throughput sequencing is realized. The general sequence required by the library construction and sequencing is required to be selected according to the requirement of a sequencing platform, for example, a common primer can be connected to the 5' end of a specific primer, a common primer is connected to the 3' end of a sequencing joint, a common primer is connected to the 3' end of a tag joint, and an amplicon library of sequencing joint-common primer-target fragment-common primer-tag joint can be obtained through PCR amplification; for another example, the 5 'end of the specific primer is directly connected with a tag and a linker, an amplicon library with the specific tag and the linker can be directly obtained through PCR amplification, in addition, the 5' end of the specific primer is only connected with the tag, the amplicon library with the specific tag can be obtained through PCR amplification, and sequencing is carried out after the linker connection; those skilled in the art will appreciate that the manner in which the 5' end of a specific primer is ligated to the universal sequence required for library-building sequencing is not limited thereto.
Preferably, the kit further comprises an amplification buffer and a purification reagent.
In a fourth aspect of the invention, there is provided a nucleic acid sequencing library construction method for detecting fetal ploidy and/or maternal cell contamination and/or paternity identification comprising the steps of: multiplex PCR amplification is performed using the detection reagent of the first aspect of the present invention or the kit of the third aspect of the present invention to construct a sequencing library.
Preferably, the reaction system of multiplex PCR amplification comprises: gDNA input amount is more than or equal to 50ng, primer group 15-25 pM, sequencing joint 40-60 pM, label joint 40-60 pM, multiplex PCR amplification reagent 10-50 uL; different samples use different tag linkers.
Preferably, the multiplex PCR amplification procedure is: 2-4 min at 95 ℃;33 to 37cycles (10 to 20s at 95 ℃,1 to 3min at 62 ℃, 25 to 35s at 72 ℃), 30s to 1.5min at 72 ℃, and 4 to 8 ℃.
Preferably, the sequencing platform includes, but is not limited to Proton, illumina, roche 454, life Ion Torrent, SOLID, pacBio sequencing platform.
In a fifth aspect of the invention there is provided a sequencing library constructed by the method of the fourth aspect of the invention.
In a sixth aspect of the invention, there is provided a method of detecting fetal ploidy and/or maternal cell contamination and/or paternity test comprising the steps of:
1. sequencing fetal and parental samples using the kit of the third aspect of the invention;
2. acquiring the alt and ref allele depth ratios of 105 SNP loci according to the first aspect of the invention in the sequencing result;
3. judging the genotype of each SNP locus in the fetus and the parent, and further judging the ploidy of the fetus and the parent sample according to the genotype combination of 105 SNP loci; and/or
4. And integrating the detection results of the SNP loci of the fetus and the parental sample, and clustering the SNP loci.
5. Performing paternity test according to the clustering analysis result in the step 4; and/or calculating the pollution ratio of the maternal cells in the fetal sample.
Preferably, the parent comprises a male parent and a female parent.
Preferably, the quality control process is performed on the sequencing data after the step 1.
The specific steps in the step 2 include:
Obtaining the alt and ref allele depth ratios (ref. Ratio, alt. Ratio) of the SNP sites described in the first aspect of the invention, wherein ref represents the genotype of the reference genome for each SNP site; alt represents a risk allele;
the SNP loci according to the first aspect of the invention are both bi-allelic SNP loci, and when analyzed, ref can be regarded as the A allele and alt type as the B allele, each SNP locus only comprises two common alleles A and B (Alleles).
According to the depth ratio of the A allele and the B allele of each SNP locus, the corresponding A ratio (ref. Ratio) and B ratio (alt. Ratio) can be obtained, and the calculation mode and the relation of the A ratio and the B ratio are as follows:
A ratio＝ref.depth/(ref.depth+alt.depth)；
B ratio＝alt.depth/(ref.depth+alt.depth)；
A ratio+B ratio＝1。
wherein ref.depth is the allele depth of the ref type of the SNP locus, alt.depth is the allele depth of the alt type of the SNP locus;
The specific steps in the step 3 include: and respectively judging the genotypes and/or the sample ploidy of the fetal and parent samples according to the alt.ratio values of the SNP loci. When the sample ploidy is a uniparent diploid, triploid and tetraploid, respectively, there is a significant difference in the genotype combination of the sample and the expectations of the corresponding alt. The genotype of each SNP locus in the fetus and the parent sample can be respectively judged according to the SNP alt.ratio value; further, the combination of all SN P sites can be integrated to determine the sample ploidy of the parent and fetal samples (e.g., AAA, AAB, ABB, BBB genotypes are present in polyploid samples).
The specific steps in the step 4 include:
And (3) integrating SNP detection results of the analyzed parent and fetal samples. The alt.ratio value of the parental sample is taken as feature 1 (X-axis) and the alt.ratio value of the fetal sample is taken as feature 2 (Y-axis). Meanwhile, the genotype results of the parents and the fetus are integrated, the genotype of the parents is marked by uppercase letters (abbreviated as parent samples), the genotype of the fetus sample is marked by lowercase letters, if AAab indicates that the parents are AA genotype, and the fetus is ab genotype. The features 1 and 2 form two-dimensional data, and on the basis, SNP loci are clustered by using a kmeans clustering algorithm.
The specific steps in the step 5 include:
performing paternity test according to the result of the step 4; and/or, calculating parent source contamination.
The specific steps of parent-child identification are as follows: when there is no true biological father-son/mother relationship between the parent and fetal samples, there may be a paradox in the genotypic combination of the two samples. The method identifies sample combinations without real biological father-son/mother-son relationship by identifying the SNP loci which are contrary to each other; conversely, if no contradicting SNP sites are present, the parent and fetal samples are considered to have a true biological parent-child/mother-child relationship.
1) When the fetus is a whole genome uniparent diploid and diploid sample, if the AAbb or BBaa is detected in the combination of the parent and fetal genotypes, it is determined that no biological parent-child/mother-child relationship exists.
2) When the fetus is a triploid sample, if the existence of AAbbb or BBBaaa of the combination of the parent and fetal genotypes is detected, it is determined that no biological father-son/mother-son relationship exists.
3) When the fetus is a tetraploid sample, if the presence of AAbbbb or BBBaaaa of the parent and fetal genotype combination is detected, it is determined that a biological parent-child/mother-child relationship is not present.
The process of calculation of the parent source pollution is as follows:
the same genotypes of all SNP loci can be clustered according to the cluster analysis, and the pollution proportion of maternal cells in the fetal sample is calculated after the cluster analysis.
By comparing the combination of SNP loci in the two samples, the pollution ratio of maternal cells in the fetal sample can be calculated.
I. Such as diploid, triploid, unrelated sample (non-mother, non-father), complete sample 2 (sample 1 is actually a misplaced sample 2), etc., with a, B representing the allele from the parent, a, B representing the allele from the fetus, in particular:
when the fetus is a whole genome uniparent diploid, the parental and fetal sample allele combination categories include: AAaa, ABaa, ABbb, BBbb;
When the fetus is diploid, the parental and fetal sample allele combination categories include: AAaa, AAab,
ABaa、ABab、ABbb、BBab、BBbb；
When the fetus is triploid, the parental and fetal sample allele combination categories include: AAaaa, AAaaab,
AAabb、ABaaa、ABaab、ABabb、ABbbb、BBaab、BBabb、BBbbb；
When the fetus is tetraploid, the parental and fetal sample allele combination categories include: AAaaaa, AAaaab, AAaabb, AAabbb, ABaaaa, ABaaab, ABaabb, ABabbb, ABbbbb, BBaaab, BBaabb, BBabbb, BBbbbb preferably, in step 3, the sample contamination ratio X is estimated based on the combined type of genotypes of the fetus and the B-ratio of each genotype under the combination.
B ratio and X are as follows:
Gene combination B ratio = B ratio expected value of maternal genotype X + B ratio expected value of fetal genotype
(1-X)。
In a seventh aspect of the invention, there is provided a detection system for fetal ploidy and/or maternal cell contamination and/or paternity test comprising:
sequencing module: acquiring the alt and ref allele depth ratio of 105 SNP alleles in fetal and parental samples;
And a data processing module: determining the genotype and/or maternal cell contamination ratio of the fetus and the parent by analyzing the alt and ref allele sequencing depth ratios of each SNP allele in the fetus and the parent sample;
And a result output module: for outputting the data analysis result.
Preferably, the data processing module comprises:
Determining the genotype of the fetus and the parent and/or the pollution proportion of the maternal cells by analyzing the allele depth ratio of the alt and ref of each SNP locus in the fetus and the parent sample, wherein the alt type is regarded as a B allele and the ref is regarded as an A allele during analysis; preferably, the alt and ref allele depth ratios are calculated by:
A ratio＝ref.depth/(ref.depth+alt.depth)；
B ratio＝alt.depth/(ref.depth+alt.depth)；
A ratio+B ratio＝1；
where ref. Depth is the allele depth of the type ref of the SNP site and alt. Depth is the allele depth of the type alt of the SNP site.
The procedure for determining the ploidy of the fetus and the parents includes: judging the ploidy of the fetus and the parent sample according to the genotypes of 105 SNP loci of the parents;
Clustering analysis is adopted on the classified data to obtain genotype combinations of SNP loci of fetal samples in the first aspect of the invention; further determining the ploidy of the fetus according to the number of categories of 105 SNP locus allele combinations of the first aspect of the invention;
the paternity test process comprises the following steps:
clustering analysis is adopted on the classified data to obtain genotype combinations of SNP loci of fetal samples and parental samples in the first aspect of the invention; judging whether the biological father-son/mother-son relationship is true according to genotypes of the fetal sample and the parent sample. In particular, when there is no true biological father-son/mother relationship between the parent and fetal samples, there may be a paradox situation in the genotypic combination of the two samples. The method identifies sample combinations without real biological father-son/mother-son relationship by identifying the SNP loci which are contrary to each other; conversely, if no contradicting SNP sites are present, the parent and fetal samples are considered to have a true biological parent-child/mother-child relationship.
The process for determining the pollution ratio of the parent cells comprises the following steps:
Clustering the same genotypes of all SNP loci according to the genotype combination categories of the fetus and the parent to obtain B ratios of all genotypes under the gene combination, and presuming a sample pollution proportion X;
B ratio and X are as follows:
Gene combination B ratio = B ratio expected value of maternal genotype X + B ratio expected value of fetal genotype
(1-X)。
The beneficial effects of the invention are as follows:
According to the invention, a group of SNP locus combinations capable of detecting fetal ploidy and maternal pollution are obtained through screening, and corresponding primer combinations are designed based on the SNP combinations, so that a kit for detecting fetal ploidy and maternal pollution is obtained; and a maternal pollution detection technology based on multiplex PCR amplification SNP is developed; the method can realize the identification of fetal ploidy, the accurate identification of 5% -95% maternal pollution and the identification of non-maternal and non-father relations in paternity and son relations. The technology has the characteristics of simple operation, low price, short experimental time, good compatibility with CNVseq and the like; the defect that the CNV-Seq cannot detect polyploid and maternal pollution is overcome, and an effective quality control technical means is provided for samples for CNV-Seq detection; make up for the shortages of STR in detecting polyploid and parent source pollution.
Drawings
FIG. 1 is a technical roadmap of the invention.
FIG. 2 is a graph showing the correlation of the contamination ratio of SNP and STR detection parent sources.
FIG. 3 shows SNP detection results of a diploid sample without maternal contamination.
FIG. 4 shows SNP detection results of a diploid 33.3% maternal contamination sample.
FIG. 5 shows SNP detection results of 100% of a completely maternal pollution sample.
FIG. 6 shows SNP detection results of triploid non-maternal pollution samples.
FIG. 7 shows SNP detection results of triploid 30.6% parent source contamination samples.
FIG. 8 shows SNP detection results of a tetraploid sample without maternal contamination.
FIG. 9 shows SNP detection results of whole genome uniparent diploid (maternal) samples.
FIG. 10 shows SNP detection results of whole genome uniparent diploid (parent) samples.
FIG. 11 shows SNP detection results of diploid independent samples (non-parent/non-parent samples).
FIG. 12 shows the contamination ratio of STR and SNP detection master and slave paired samples.
FIG. 13 shows STR results for a sample with 75% SNP contamination of a very high percentage of parent contamination.
FIG. 14 shows SNP results of a 75% parent contamination sample, in which the STR was unable to determine the very high proportion of parent contamination.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Example 1: screening of 105 high-frequency variant SNP loci
The inventor screens 105 SNP loci of polymorphic loci (MAF > 0.45) of east Asia population ：rs6697376、rs12027119、rs10889407、rs10783100、rs2587895、rs3766361、rs3748666、rs1557080、rs1916231、rs1395931、rs10199403、rs13086388、rs9866013、rs9868434、rs2571468、rs279844、rs13134862、rs1835522、rs2903386、rs11732263、rs6811238、rs12651593、rs253484、rs6878425、rs1423326、rs7710068、rs7728908、rs9369380、rs4947212、rs6940134、rs9397342、rs1019029、rs2686817、rs2269991、rs6966616、rs2267708、rs12156144、rs12544054、rs10503926、rs2353202、rs2975207、rs3019303、rs4871182、rs10781004、rs3118846、rs10821461、rs1463729、rs3780962、rs1927453、rs12244967、rs56331924、rs537432、rs1278329、rs10128711、rs11037366、rs2905513、rs625232、rs2255301、rs219923、rs12581739、rs1995691、rs10506759、rs2579132、rs7979001、rs12306075、rs10773760、rs1161464、rs9316585、rs7321320、rs9531423、rs9302039、rs9519708、rs6571849、rs1454361、rs3922176、rs6606789、rs4244560、rs2899496、rs654065、rs4786640、rs7195826、rs11644264、rs899258、rs9927268、rs4485351、rs1390901、rs8070085、rs1027895、rs2292972、rs7506104、rs9960366、rs1395858、rs9950061、rs4891291、rs576261、rs12480506、rs4911248、rs1005533、rs2822397、rs1041409、rs2831057、rs2831711、rs2243508、rs987640、rs2040411.
Genome version hg19, designing specific primers according to SNP loci, taking genome DNA as a detection object, performing a large number of experimental screening, optimization and verification, and finally, preferably obtaining 105 pairs of primers with high amplification efficiency and good specificity (table 1).
TABLE 1 Table 105 primer information Table
Example 2: kit for detecting fetal ploidy and maternal pollution based on multiplex PCR (polymerase chain reaction) capture technology
This example provides, by using the specific primer set of example 1, a kit for detecting fetal ploidy, paternity, and maternal contamination based on multiplex PCR capture techniques; the kit can detect fetal ploidy, paternity and maternal contamination by sequencing or other non-sequencing methods.
The kit comprises:
(1) 105 high frequency variant SNP site-specific primer sets: adding a common primer to the 5' end of each specific primer shown in SEQ ID NO. 1-SEQ ID NO. 210, wherein the common primer has the sequence: 5'-AAATGGGCGGTAGGCTTG-3' (SEQ ID NO: 211);
(2) Sequencing adaptors: 5 '-CCTCTCTATGGGCAGTCGGTGAT-common primer-3' (SEQ ID NO: 212);
(3) And (3) tag joint: 5 '-CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNGAT-public primer-3' (SEQ ID NO: 213); n in the sequence represents any base in A/G/C/T, NNNNNNNN is used for identifying libraries constructed by different samples;
(4) Multiplex PCR amplification buffer: multiplex PCR buffers are commonly used in the market, for example: multiplex PCR Kit, 2G Fast Multiplex PCR Kit;
(5) Purifying reagent: the magnetic beads are purified as usual in the market, for example: ampure XP magnetic beads.
Example 3 construction and sequencing of a kit for detecting fetal ploidy, paternity and maternal contamination based on multiplex PCR Capture technique
The embodiment provides a method for constructing a library and sequencing a kit for detecting fetal ploidy, paternity and maternal pollution based on a multiplex PCR capture technology on the basis of the embodiment 2, which comprises the following steps:
1. Multiplex PCR amplification
Preparing a 25uL multiplex PCR reaction system comprising: gDNA input amount is more than or equal to 50ng, the specific primer group is 20pM, the sequencing joint is 50pM, the tag joint is 50pM, and the multiplex PCR amplification reagent is 12.5uL; different samples use different tag linkers.
PCR amplification procedure: 3min at 95 ℃; (95 ℃ 15s,60 ℃ 120s,72 ℃ 30 s) 35 cycles; 72℃1min,4 ℃.
2. Purification of amplified products
According to the number of PCR amplified samples, taking the equal volume of each PCR amplified product to obtain 100 mu L of mixed library of different labels, and carrying out fragment screening and purification on the PCR products by using a purifying reagent.
3. Sequencing
The purified mixed library was quantified using Qubit TM DSDNA HS ASSAY KIT and sequenced on a machine according to the quantitative concentration.
Example 4A method for detecting fetal ploidy, non-affinity relationship, maternal cell contamination
In this example, proton platform was used for sequencing, sequencing template preparation and enrichment see the instructions for Ion PI TM Hi QTMOT2 200 Kit (A26434) procedure, microbeads with template molecules were loaded onto the semiconductor chip of Ion Proton TM sequencer for sequencing, see Ion PITM Hi Q TM Sequence 200 Kit procedure instructions for details, and sequencing method is referred to in example 3.
The flow chart is shown in fig. 1, and the specific steps are as follows:
1. genomic DNA extraction was performed on paired sample 1 and sample 2 (sample 1 is a fetus, sample 2 is a normal diploid non-contaminating sample, and sample 2 is a father or mother sample for paternity identification).
2. The extracted DNA was PCR amplified to library using a multiplex PCR mix system based on 105 SNPs in example 3, and the PCR amplified library was sequenced.
3. And (3) performing quality control on sequencing machine-down data to obtain the depth information of 105 SNP alleles of samples with qualified quality control.
4. Obtaining alt and ref allele depth ratios (ref. Ratio) of 105 SNP sites, wherein ref represents the allele of the reference genome for each SNP site; alt represents a risk allele. Since these 105 SNP sites are all bi-allelic, ref can be considered as the a allele and alt type as the B allele at the time of analysis, each SNP site only includes the two common alleles a and B (Alleles). Since normal humans are diploid, only three genotypes AA, AB and BB, the expected B allele depth ratio is only 0, 0.5 and 1, wherein the expected value refers to the theoretical value of the combination of genotypes that are expected to meet the genetic rules and that may exist in the mother and fetus. According to the depth of each SNP locus A allele and B allele, a corresponding A ratio (ref. Ratio) and B ratio (alt. Ratio) can be obtained, and the calculation modes and the relation are as follows:
A ratio＝ref.depth/(ref.depth+alt.depth)；
B ratio＝alt.depth/(ref.depth+alt.depth)；
A ratio+B ratio＝1；
Where ref.depth is the allele depth of the ref type of the SNP site, alt.depth is the allele depth of the alt type of the SNP site.
5. Judging the genotype of each SNP locus in the fetus and the parent, and further judging the ploidy of the fetus and the parent sample according to 105 SNP genotype combinations;
And respectively judging the genotypes and the sample ploidy of the fetal and parent samples according to the alt.ratio values of the SNP loci. As shown in the table below, there is a significant difference in the genotype combination of the sample and the expectations of the corresponding alt.ratio values when the sample ploidy is a uniparent diploid, triploid and tetraploid, respectively. As shown in Table 2, the genotype of each SNP site in the fetal and parent samples can be determined according to the SNP alt.ratio value; further, the combination of all SNP loci can be integrated to judge the sample ploidy of the parent and fetal samples (for example, AAA, AAB, ABB, BBB genotypes exist in a polyploid sample).
TABLE 2
6. And integrating the detection results of the SNP loci of the fetus and the parental sample, and clustering the SNP loci.
And (3) integrating SNP detection results of the analyzed parent and fetal samples. The alt.ratio value of the parental sample is taken as feature 1 (X-axis) and the alt.ratio value of the fetal sample is taken as feature 2 (Y-axis). Meanwhile, the genotype results of the parents and the fetus are integrated, the genotype of the parents is marked by uppercase letters (abbreviated as parent samples), the genotype of the fetus sample is marked by lowercase letters, if AAab indicates that the parents are AA genotype, and the fetus is ab genotype. The features 1 and 2 form two-dimensional data, and on the basis, SNP loci are clustered by using a kmeans clustering algorithm.
7. And (3) paternity test, namely judging whether the parent and fetal samples are real biological father-son/mother-son relations according to the result of the step (6).
Judging whether the parent and the fetal sample are real biological father-son/mother-son relations according to the result of the step 6. In particular, when there is no true biological father-son/mother relationship between the parent and fetal samples, there may be a paradox situation in the genotypic combination of the two samples. The method identifies sample combinations without real biological father-son/mother-son relationship by identifying the SNP loci which are contrary to each other; conversely, if no contradicting SNP sites are present, the parent and fetal samples are considered to have a true biological parent-child/mother-child relationship, exemplified as follows:
1) When the fetus is a whole genome uniparent diploid and diploid sample, if the AAbb or BBaa is detected in the combination of the parent and fetal genotypes, it is determined that no biological parent-child/mother-child relationship exists.
2) When the fetus is a triploid sample, if the existence of AAbbb or BBaaa of the combination of the parent and fetal genotypes is detected, it is determined that no biological father-son/mother-son relationship exists.
3) When the fetus is a tetraploid sample, if the presence of AAbbbb or BBaaaa of the parent and fetal genotype combination is detected, it is determined that a biological parent-child/mother-child relationship is not present.
8. And calculating the pollution ratio of maternal cells in the fetal sample according to the genotype combination of the fetus and the parents.
And clustering the same genotypes of all SNP loci by adopting a kmeans clustering algorithm according to the genotype combination categories of the fetus and the parent to obtain the B ratio of each genotype under the gene combination, and presuming the sample pollution proportion X.
B ratio and X are as follows:
Gene combination B ratio = B ratio expected value of maternal genotype X + B ratio expected value of fetal genotype
(1-X)。
Estimating the sample pollution proportion X according to the ploidy category of the sample 1 and the B ratio of each genotype under the allele combination; b ratio and X are as follows:
maternal and fetal allele combination B ratio = B ratio expected of maternal genotype X + Bratio expected of fetal genotype (1-X).
9. And drawing a SNP sample result graph according to the sample detection result, and outputting the detection result.
Example 5 evaluation of the consistency of SNP and STR detection results
The example adopts SNP to detect different types of mother-child paired samples, including diploid pollution-free samples, triploid pollution samples, different proportions of mother source pollution samples, tetraploid pollution-free samples, genome uniparent diploid (mother source), whole genome uniparent diploid (fatsource) and diploid unpaired sample non-mother/non-father samples, and the consistency of detection results of the two methods is evaluated, and the results are shown in Table 3 and figures 2-11.
TABLE 3 Table 3
Example 6 ploidy of 462 mother and child paired samples were detected and compared using STR and SNP, respectively
In this example, the ploidy of 462 mother-child paired samples were detected and compared with the SNP of this example 1, and the results are shown in table 4, which shows that the SNP-based detection method has 100% consistency, sensitivity and specificity with STR when detecting fetal diploid and triploid.
TABLE 4 Table 4
Example 7 STR comparison of pollution effects of mother sources for SNP detection
Respectively detecting and comparing parent source pollution (MCC) conditions of 401 diploid parent-child paired samples by adopting STR and SNP;
the specific flow of STR detection is as follows:
STR tests were performed on 50 samples using the beijing micro gene technologies company MicroreaderTM, direct ID System kit.
And (3) PCR amplification:
A25 ul PCR reaction system was prepared, comprising 1ul (1-1.5 ng) of template DNA、10ul MicroreaderTm2.5×Buffer D、5ul MicroreaderTM21-D 5×Primer Mix、0.625ul MicroreaderTMPolymerase II、8.375ul nuclease free water.
PCR amplification procedure: 50℃2min,96℃4min, (94℃5s,60℃70 s) 27cycles,60℃30min,4℃hold.
And (3) electrophoresis detection: 8.8ul Hi-Di TM formamide, 0.2. 0.2ul Microreader TM Size Standard Org550 and 1ul PCR product.
Data analysis: opening GENEMAPPER IDX software, importing electrophoresis data, selecting corresponding Analysis parameters such as Panel, analysis Method and Size Standard, and changing the sample type of Ladder to ' ALLELIC LADDER ' in SAMPLE TYPE ' column: analysis of the data begins.
Triploid analysis: the peak areas of different STR sites of the triploid sample meet the genetics rule, for example: 1:1:1, 1:2.
Calculating the pollution ratio of the mother source: and according to different STR heterozygous sites of different samples, calculating the pollution ratio of a mother source and calculating the average value of 3-4 sites, wherein the mother is not inherited to the fetus, and the peak area ratio corresponds to the STR typing.
MCC% was calculated for the peak area of the heterozygous sites relative to the fluorescence units (Relative Fluorescence Units, RFU)
MCC％(STR)＝(C*2)/(C+D+F)；
Note that: c: a site where the mother did not inherit to the fetus; d: the locus where the mother inherits to the fetus; f: the father inherits to the fetal site.
The detection results are shown in fig. 12-14, and the results show that: the detection limit of the parent source pollution of the STR method is 10%, which leads to the fact that <10% of the parent source pollution samples cannot be detected at all in the traditional method, and the method in the embodiment of the invention can accurately detect 5% of the parent source pollution samples; and the STR method can not accurately judge the pollution proportion of samples with the pollution of >70% of mother sources.
The present invention has been described in detail in the above embodiments, but the present invention is not limited to the above examples, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A detection reagent for detecting an SNP site, characterized in that the SNP site comprises ：rs6697376、rs12027119、rs10889407、rs10783100、rs2587895、rs3766361、rs3748666、rs1557080、rs1916231、rs1395931、rs10199403、rs13086388、rs9866013、rs9868434、rs2571468、rs279844、rs13134862、rs1835522、rs2903386、rs11732263、rs6811238、rs12651593、rs253484、rs6878425、rs1423326、rs7710068、rs7728908、rs9369380、rs4947212、rs6940134、rs9397342、rs1019029、rs2686817、rs2269991、rs6966616、rs2267708、rs12156144、rs12544054、rs10503926、rs2353202、rs2975207、rs3019303、rs4871182、rs10781004、rs3118846、rs10821461、rs1463729、rs3780962、rs1927453、rs12244967、rs56331924、rs537432、rs1278329、rs10128711、rs11037366、rs2905513、rs625232、rs2255301、rs219923、rs12581739、rs1995691、rs10506759、rs2579132、rs7979001、rs12306075、rs10773760、rs1161464、rs9316585、rs7321320、rs9531423、rs9302039、rs9519708、rs6571849、rs1454361、rs3922176、rs6606789、rs4244560、rs2899496、rs654065、rs4786640、rs7195826、rs11644264、rs899258、rs9927268、rs4485351、rs1390901、rs8070085、rs1027895、rs2292972、rs7506104、rs9960366、rs1395858、rs9950061、rs4891291、rs576261、rs12480506、rs4911248、rs1005533、rs2822397、rs1041409、rs2831057、rs2831711、rs2243508、rs987640、rs2040411.
2. The detection reagent of claim 1, wherein the detection reagent comprises a primer; preferably, the sequence of the primer is shown as SEQ ID NO. 1-SEQ ID NO. 210.
3. Use of the detection reagent of any one of claims 1 to 2 in any one of the following;
(1) Detecting fetal ploidy;
(2) Preparing a product for detecting fetal ploidy;
(3) Detecting maternal cell contamination;
(4) Preparing a product for detecting maternal cell pollution;
(5) Parent-child relationship identification;
(6) Preparing a paternity test product;
preferably, the ploidy condition comprises a uniparent diploid, triploid and/or tetraploid.
4. A kit comprising the detection reagent of claim 2; preferably, the 5' end of each primer in the kit is connected with a universal sequence required by library building and sequencing;
Preferably, the universal sequence is selected from the group consisting of a common primer, a linker, a specific tag.
5. A nucleic acid sequencing library construction method for detecting fetal ploidy and/or maternal cell contamination and/or paternity identification, comprising the steps of: a sequencing library is constructed by multiplex PCR amplification using the detection reagent of any one of claims 1 to 2 or the kit of claim 4.
6. A sequencing library constructed by the method of claim 5.
7. A method of detecting fetal ploidy and/or maternal cell contamination and/or paternity test comprising the steps of:
(1) Sequencing fetal and parental samples using the kit of claim 4;
(2) Obtaining the alt and ref allele depth ratios of the SNP sites described in claim 1 in the sequencing result;
(3) Judging the genotype of each SNP locus in the fetus and the parent, and further judging the ploidy of the fetus and the parent sample according to the genotype combination of the SNP; and/or
(4) Integrating the detection results of SNP loci of the fetus and the parental sample, and clustering the SNP loci;
(5) Performing paternity test according to the clustering analysis result; and/or calculating the pollution ratio of the maternal cells in the fetal sample.
8. The method according to claim 7, wherein the specific steps in the step (2) include:
Obtaining the alt and ref allele depth ratios ref.ratio and alt.ratio of the SNP locus of claim 1, wherein ref represents the genotype of the reference genome and ref type is considered as the a allele; alt represents a risk allele and the alt type is considered as a B allele;
the A ratio and B ratio of each SNP site are calculated as follows:
A ratio＝ref.depth/(ref.depth+alt.depth)；
B ratio＝alt.depth/(ref.depth+alt.depth)；
A ratio+B ratio＝1；
wherein ref.depth is the allele depth of the ref type of the SNP locus, alt.depth is the allele depth of the alt type of the SNP locus;
preferably, the specific step of step (3) comprises: judging genotype and/or sample ploidy of the fetus and the parent sample according to alt.ratio values of SNP loci;
Preferably, the specific step of step (4) comprises: integrating and analyzing SNP detection results of parent and fetal samples; taking the alt.ratio value of the parent sample as a characteristic 1, and taking the alt.ratio value of the fetal sample as a characteristic 2; the features 1 and 2 form two-dimensional data, and on the basis, the SNP loci are clustered by using a clustering algorithm.
9. The method of claim 7, wherein the step (5) of paternity testing comprises: when the genotype combination of the parent and the fetal sample is contrary to the SNP locus, the sample combination of the parent and the fetal sample does not exist in the real biological father-son/mother-son relationship; otherwise, if the contradictory SNP locus does not exist, the real biological father-son/mother-son relationship exists between the parent and the fetal sample;
preferably, the specific step of calculating the proportion of maternal cell contamination in step (5) comprises: setting a pollution proportion X;
The B ratio of the gene combination of fetal and parental samples is related to X as:
b ratio of gene combination = B ratio expected of maternal genotype X + B ratio expected of fetal genotype (1-X).
10. A detection system for fetal ploidy and/or maternal cell contamination and/or paternity identification comprising:
Sequencing module: obtaining the alt and ref allele depth ratios of the SNP sites of claim 1 in the sequencing results of fetal and parental samples;
And a data processing module: determining the genotype of the fetus and the parent and/or the pollution ratio of the maternal cells by analyzing the allele depth ratio of alt and ref in the fetus and the parent sample;
And a result output module: for outputting the data analysis result.
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| US20110201507A1 (en) * | 2010-01-19 | 2011-08-18 | Rava Richard P | Sequencing methods and compositions for prenatal diagnoses |
| US20120270212A1 (en) * | 2010-05-18 | 2012-10-25 | Gene Security Network Inc. | Methods for Non-Invasive Prenatal Ploidy Calling |
| CN110846310A (en) * | 2018-08-21 | 2020-02-28 | 深圳华大法医科技有限公司 | Method for carrying out genetic identification on SNP (Single nucleotide polymorphism) site set and embryonic nucleic acid sample and application |
| CN115216539A (en) * | 2022-09-19 | 2022-10-21 | 北京大学第三医院（北京大学第三临床医学院） | Maternal cell pollution detection kit and application thereof |
| CN116497106A (en) * | 2023-06-30 | 2023-07-28 | 北京大学第三医院（北京大学第三临床医学院） | Identification method for maternal pollution in prenatal diagnosis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110201507A1 (en) * | 2010-01-19 | 2011-08-18 | Rava Richard P | Sequencing methods and compositions for prenatal diagnoses |
| US20120270212A1 (en) * | 2010-05-18 | 2012-10-25 | Gene Security Network Inc. | Methods for Non-Invasive Prenatal Ploidy Calling |
| CN110846310A (en) * | 2018-08-21 | 2020-02-28 | 深圳华大法医科技有限公司 | Method for carrying out genetic identification on SNP (Single nucleotide polymorphism) site set and embryonic nucleic acid sample and application |
| CN115216539A (en) * | 2022-09-19 | 2022-10-21 | 北京大学第三医院（北京大学第三临床医学院） | Maternal cell pollution detection kit and application thereof |
| CN116497106A (en) * | 2023-06-30 | 2023-07-28 | 北京大学第三医院（北京大学第三临床医学院） | Identification method for maternal pollution in prenatal diagnosis |
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