CN115433777A - Integrated identification method for CNV, SV and SGD abnormalities and abnormal sources of embryos - Google Patents

Integrated identification method for CNV, SV and SGD abnormalities and abnormal sources of embryos Download PDF

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CN115433777A
CN115433777A CN202211314568.1A CN202211314568A CN115433777A CN 115433777 A CN115433777 A CN 115433777A CN 202211314568 A CN202211314568 A CN 202211314568A CN 115433777 A CN115433777 A CN 115433777A
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mother
source
father
cnv
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安松坡
乔国枝
刘沙沙
费嘉
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Beijing Zhongyi Kangwei Medical Devices Co ltd
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Abstract

The invention provides an integrated identification method for embryo CNV, SV and SGD abnormity and abnormity sources, wherein the chromosome CNV identification comprises the steps of dividing a chromosome into a plurality of windows and obtaining the copy number state of each window; the identification of the source and the occurrence period of the CNV parent comprises the following steps: constructing a father haplotype and a mother haplotype; extracting effective SNP loci of the filial generation samples; judging the source of the effective SNP locus and dividing the chromosome of the filial generation sample into different recombination regions; and judging whether the copy number CN of each recombination region is abnormal or not and judging the source of the abnormality. SV, SGD and source identification included: constructing a father haplotype and a mother haplotype; judging the source of the maternal haplotypes and the paternal haplotypes of the offspring samples; and judging the abnormality and the source of the abnormality according to the genetic linkage relationship. The method can realize the integrated accurate detection of the CNV, SV and SGD abnormalities and abnormal sources of the embryos, and is beneficial to improving the success rate of transplantation and breeding healthy offspring.

Description

Integrated identification method for CNV, SV and SGD abnormalities and abnormal sources of embryos
Technical Field
The invention belongs to the field of gene detection, relates to a genetic detection technology before embryo implantation, and particularly relates to an integrated identification method for CNV, SV and SGD abnormalities and abnormal sources of embryos.
Background
For families with monogenic genetic diseases or chromosome structure abnormalities, the technology of the third-generation tube babies can help the families to block pathogenic variation from being inherited to offspring, and the willingness of healthy fertility is realized. The PGT technology, namely the genetic detection technology before embryo implantation, refers to the biopsy of trophectoderm cells of embryos, detects genetic information of 3-10 biopsy cells, judges the carrying state of pathogenic genes or structural variation by utilizing the principle of linkage disequilibrium, and selects healthy embryos from a plurality of embryos cultured in vitro to be transplanted back to a maternal uterus by assisting with the whole genome chromosome copy number analysis technology PGT-A so as to improve the transplanting success rate and grow healthy offspring.
The existing PGT-A detection technology still has the problems of narrow detection range and high false positive, and the occurrence of false positive in the detection result is mainly caused by the following two aspects:
1. the existing PGT-A technology is only limited to the detection of the chromosome copy number variation of aneuploidy, but the detection rate of the aneuploidy change or heterozygosity loss (such as triploid, uniparental disomy and the like) of the chromosome copy number is not high, the incidence rate of the triploid in early villus is about 8/10000, the incidence rate in early embryo is possibly higher, and the detection rate is one of the important reasons of abortion except for the aneuploidy; a significant increase in the risk of occurrence of Mendelian recessive genetic disease in the pure and regional area (ROH); while the presence of uniparental disomy (UPD) on specific chromosomes can cause genetic imprinting related diseases, several of these conditions also increase the risk of embryo transfer failure or birth defects.
2. More and more researches show that the detection result has a certain proportion of false positives mainly including the false positives of the chimeric result due to personnel operation or system errors in the links of biopsy, whole Genome Amplification (WGA), sequencing, data analysis and the like in PGT-A detection.
Therefore, accurate detection of such mutations as embryonic Copy Number Variation (CNV), chromosomal Structure Variation (SV), and monogenic genetic disease (SGD) is a problem that needs to be solved to improve the success rate of transplantation and to develop healthy offspring.
Disclosure of Invention
The invention aims to design an integrated identification method for CNV, SV and SGD abnormality and abnormal sources of embryos, which can realize simultaneous identification of chromosome copy number abnormality, structural abnormality, pathogenic gene carrying states and variation sources of samples, broaden the range and performance of conventional PGT-A detection, increase the euploidy change or heterozygosity loss variation of chromosome copy numbers such as triploid and ROH and the like, effectively reduce false positive of chimeric detection, simultaneously judge parent sources with abnormal embryo copy number according to family information, and have important guiding significance for continuous treatment of infertile and bad fertility history couples or the adoption of strategies for semen and egg treatment in PGT technical application, and can greatly reduce clinical abortion rate.
The technical scheme for realizing the purpose of the invention is as follows:
in the first aspect, the invention designs an embryo CNV abnormality identification method, which comprises a chromosome CNV identification step, a CNV parent source identification step and a CNV generation period identification step;
wherein, the step of identifying the chromosome CNV comprises the following steps:
s1, dividing a chromosome into a plurality of windows which are connected in sequence;
s2, calculating a probe detection signal value LRR of each window, and acquiring the copy number state of each window, wherein the copy number state comprises a default, a monomer, a dimer, a trimer and a tetrad, and the adjacent two states are in a chimeric state;
the CNV parent source identification and CNV generation period identification steps comprise:
s3, acquiring a progeny sample and the genotype of the father and the mother, and constructing a father haplotype and a mother haplotype according to the genetic relationship;
s4, extracting SNP loci with one genotype being heterozygous and the other genotype being homozygous in the parents in the filial generation samples as effective SNP loci;
s5, judging the source of the effective SNP locus of the filial generation according to the genotypes of the father and the mother, and marking the effective SNP locus of the filial generation as the effective SNP locus
Figure 276541DEST_PATH_IMAGE001
Or
Figure 886514DEST_PATH_IMAGE002
Or
Figure 118912DEST_PATH_IMAGE003
Or
Figure 156138DEST_PATH_IMAGE004
；
S6, according to
Figure 919826DEST_PATH_IMAGE001
、
Figure 333490DEST_PATH_IMAGE002
、
Figure 420395DEST_PATH_IMAGE003
、
Figure 159680DEST_PATH_IMAGE004
Dividing the offspring sample chromosome into different recombination regions;
s7, extracting BAF values of child samples with homozygous parent SNP sites and different genotypes, calculating the parent source ratio MAF of bases provided by mothers in the SNP sites in each recombination region of the child samples, and calculating the average value of the parent source ratios of the mother SNP sites in each recombination region in the step S6;
s8, judging whether the copy number CN of the recombination region and the parent source ratio MAF in the step S6 are abnormal or not according to the MAF value, and judging the abnormal source, wherein the judgment comprises the following steps:
s801, when the copy number CN of the recombination region of the offspring sample is not 2, judging that the copy number is abnormal and judging an abnormal source;
s802, when the copy number CN of the recombination area of the child generation sample is 2, judging whether the MAF of the parent source accounts for the ratio is abnormal or not, and if so, judging the abnormal source.
Further, in step S5, the method for determining the source of the effective SNP sites in the offspring includes:
when the genotype of the father is heterozygous and the genotype of the mother is homozygous, the effective SNP locus of the filial generation sample is the father locus, and the effective SNP locus of the filial generation sample is judged from which nucleotide chain of the father the effective SNP locus comes from according to the haplotype of the father and is marked as
Figure 128774DEST_PATH_IMAGE001
Or
Figure 831281DEST_PATH_IMAGE002
；
When the genotype of the mother is heterozygous and the genotype of the father is homozygous, the effective SNP locus of the filial generation sample is the maternal locus, which nucleotide chain the effective SNP locus of the filial generation sample comes from is judged according to the haplotype of the mother and marked as
Figure 38272DEST_PATH_IMAGE003
Or
Figure 682880DEST_PATH_IMAGE004
。
Specifically, the above
Figure 139269DEST_PATH_IMAGE001
、
Figure 97998DEST_PATH_IMAGE002
、
Figure 956232DEST_PATH_IMAGE003
、
Figure 519544DEST_PATH_IMAGE004
The marking method comprises the following conditions:
(1) When the father genotype is AB (H1H 2) and the mother genotype is AA (H1H 2), the BAF value of the filial generation is unchanged, and the effective SNP locus of the filial generation is judged to be from the father H2 and marked as
Figure 728809DEST_PATH_IMAGE001
；
(2) When the father genotype is AB (H1H 2) and the mother genotype is BB (H1H 2), the BAF value of the filial generation is unchanged, and the effective SNP locus of the filial generation is judged to be from the father H1 and marked as
Figure 22387DEST_PATH_IMAGE002
；
(3) When the father genotype is BA (H1H 2) and the mother genotype is AA (H1H 2), the offspring BAF mirror image is processed into 1-BAF, and the effective SNP locus of the offspring is judged to come from the father H1 and marked as
Figure 938390DEST_PATH_IMAGE002
；
(4) When the father genotype BA (H1H 2) and the mother genotype BB (H1H 2), the offspring BAF is mirrored to be 1-BAF, and the effective SNP locus of the offspring is judged to be from the father H2 and marked as
Figure 924801DEST_PATH_IMAGE001
；
(5) When the maternal genotype AB (H1H 2) and the paternal genotype AA (H1H 2) are met, the BAF value of the filial generation is unchanged, and the effective SNP locus of the filial generation is judged to be from the maternal H2 and marked as
Figure 372094DEST_PATH_IMAGE004
；
(6) When the maternal genotype AB (H1H 2) and the paternal genotype BB (H1H 2) are met, the BAF value of the filial generation is unchanged, and the effective SNP locus of the filial generation is judged to be from the maternal H1 and marked as
Figure 672625DEST_PATH_IMAGE003
；
(7) When the mother genotype BA (H1H 2) and the father genotype AA (H1H 2) exist, the offspring BAF is mirrored to be 1-BAF, and the effective SNP locus of the offspring is judged to be from the mother H1 and is marked as
Figure 239873DEST_PATH_IMAGE003
；
(8) When the mother genotype BA (H1H 2) and the father genotype BB (H1H 2) exist, the offspring BAF is mirrored to be 1-BAF, and the effective SNP site of the offspring is judged to be from the mother H2 and is marked as
Figure 662764DEST_PATH_IMAGE004
。
Here, it should be noted that: a and B are represented by homologous genotypes, H1 is represented by a first nucleotide strand, H2 is represented by a second nucleotide strand, and for the genotypes AB (H1H 2) and BA (H1H 2) described above, the genotype AB (H1H 2) is understood to mean that the homologous gene at a site in the first nucleotide strand H1 is A and the homologous gene at the site in the second nucleotide strand H2 is B; genotype BA (H1H 2) is understood to mean that the homologous gene at a site in the first strand H1 is B and the homologous gene at that site in the second strand H2 is A.
Further, in the step S801, the method for determining a copy number abnormality source includes:
s8011, in an offspring sample, when the copy number CN is more than or equal to 1 and is more than 0, if the mother source accounts for MAF =0, judging that the offspring sample is inherited from a DNA chain of a father and lacks the DNA chain of the mother; if the maternal-source ratio MAF =1.0, the progeny sample is inherited from the DNA strand of the mother, and the DNA strand inherited from the father is deleted;
s8012, in the offspring sample, when 2 is more than the copy number CN is more than 1, if 0 is more than the mother source occupation ratio MAF is more than 0.5, judging that the abnormality is from mother; if 1 is larger than the mother source occupation ratio MAF is larger than 0.5, judging that the abnormality is from father;
s8013, in the child sample, when the copy number CN is more than 2, if 0 is more than the mother source occupation ratio MAF is more than 0.5, judging that the abnormality is from the father; if 1 is larger than the mother source occupation ratio MAF is larger than 0.5, the abnormality is judged to be originated from the mother.
In step S802, when the copy number CN of the child sample reassembly area is 2, the method for determining whether the copy number is abnormal includes: and calculating and judging the mother source ratio MAF, and when the mother source ratio MAF is abnormal, judging whether the recombination region of the filial generation sample is one of haploid, uniparental diploid and triploid.
Furthermore, in step S802, when the recombination region copy number CN is 2 and the parent MAF is abnormal, the method for determining the abnormal source includes:
when the parent source ratio MAF =0, the child sample is a father source UPD, the abnormality is judged to be from the father, and if no heterozygous sites exist in the recombination region, the recombination region is an ROH region;
when the ratio MAF is more than 0.5 and more than 0, the filial generation sample is in a chimeric state, and the abnormal source is judged to be from the father;
when the parent source occupation ratio MAF =0.5, the copy number CN of the child sample is normal;
when the ratio MAF is more than 0.5 and more than 1.0, the filial generation sample is in a chimeric state, and the abnormal source is judged to be from the mother;
when the parent source ratio MAF =1.0, the child sample is the parent source UPD, the abnormal source is judged to be from the mother, and if no heterozygous site exists in the recombination region, the recombination region is the ROH region;
the whole filial sample indicates that the filial sample is derived from one chain provided by the mother and two chains provided by the father when the mother-source ratio MAF =1/3, or indicates that the filial sample is derived from two chains provided by the mother and one chain provided by the father when the mother-source ratio MAF =2/3, and the filial sample is triploid;
when the mother source ratio MAF =0 and the heterozygous SNP site does not exist, the whole progeny sample indicates that only one strand is provided by the father in the progeny sample, or when the mother source ratio MAF =1 and the heterozygous SNP site does not exist, the whole progeny sample indicates that only one strand is provided by the mother in the progeny sample, and the progeny sample is haploid;
in the whole progeny sample, when the parent ratio MAF =0 and the heterozygous SNP site is present, it means that only two strands are provided from the father in the progeny sample, or when the parent ratio MAF =1 and the heterozygous SNP site is present, it means that only two strands are provided from the mother in the progeny sample, and then the progeny sample is a uniparental diploid (UPD).
Further, the above method further includes a CNV occurrence timing discriminating step, including S9, for judging the CNV occurrence timing of the chimeric state between the monomer and the dimer or the chimeric state between the dimer and the trimer in step S8, based on the SPH and the BPH.
Specifically, the CNV occurrence period determination method includes:
if the abnormal region in the recombination region only contains SPH, judging that the CNV generation period is mitosis;
if the abnormal region in the recombination region contains BPH, and the BPH does not cross the centromere, judging that the CNV generation period is second-time division of subtraction;
if the abnormal region in the recombination region contains BPH and BPH crosses the centromere, the CNV generation time is judged to be the first division of the subtraction number.
The SPH (single partial homolog) referred to above refers to a homologous chromosome of the genetic parent; BPH (bath partial homologs) refers to the two homologous chromosomes of the genetic parent.
Further, in step S2, the method for calculating the copy number of the window includes:
s201, acquiring detection signal values RC of each window;
s202, calculating the window copy number according to the window detection signal value RC by adopting a hidden Markov model.
Further, the method for obtaining the window detection signal value RC in step S201 includes:
s2011, extracting SNP sites of each window;
s2012, obtaining LRR information of each SNP locus in the window;
s2013, mapping and detecting LRR information of the SNP sites into relative detection depth;
s2014, calculating an average value of the relative detection depths of all the SNP sites in the window to serve as a window detection signal value RC;
s2015, correcting and normalizing the window detection signal value RC to obtain a corrected window detection signal value RC.
In a second aspect, the invention designs an embryo SV abnormality identification method, which comprises the following steps:
constructing a father haplotype and a mother haplotype by adopting the method of the step S3 in the first aspect;
dividing each chromosome of the offspring sample into a plurality of windows connected in sequence by adopting the method of the step S1 in the first aspect;
and analyzing each window, judging the source of the mother haplotype and the father haplotype of the filial generation sample, and judging whether the filial generation sample has SV and abnormal sources.
In a third aspect, the invention designs an embryo SGD variation identification method, which comprises the following steps:
constructing a father haplotype and a mother haplotype by adopting the method of the step S3 in the first aspect;
dividing each chromosome of the offspring sample into a plurality of windows which are connected in sequence by adopting the method of the step S1 in the first aspect;
analyzing each window, judging the source of the maternal haplotype and the paternal haplotype of the child sample, and judging whether the SGD of the child sample has known variation and variation source.
Compared with the prior art, the invention has the beneficial effects that: the method designed by the invention can realize simultaneous identification of sample copy number abnormality, structural abnormality, pathogenic gene carrying state and variation source, expands the range and performance of the conventional PGT-A detection, increases the ploidy change or heterozygosity deletion variation of chromosome copy numbers such as triploid, ROH and the like, effectively reduces false positive of chimeric detection, can judge the parent source of embryo copy number abnormality according to family information, has important guiding significance on continuous treatment of infertility and poor fertility history couples or the strategy of sperm supply and egg supply treatment, and can greatly reduce the abortion rate clinically.
Detailed Description
The invention is further described below in conjunction with specific embodiments, and the advantages and features of the invention will become more apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.
Example 1:
the embodiment provides an embryo CNV abnormality identification method, which comprises a chromosome CNV identification step, a CNV parent source identification step and a CNV generation period identification step;
wherein, the step of identifying the chromosome CNV comprises the following steps:
s1, dividing a chromosome into a plurality of windows which are connected in sequence;
in this step, the size of each window divided by the chromosome is the same, and the size of each window can be determined according to the resolution.
Meanwhile, in the chromosome division, each chromosome of the object is divided, for example, when the object is a human chromosome, each chromosome is divided by 600kb as a window according to the human genome.
S2, calculating a probe detection signal value LRR of each window, and acquiring the copy number state of each window, wherein the copy number state comprises a default, a monomer, a dimer, a trimer and a tetrad, and the adjacent two states are in a chimeric state;
in one embodiment, a method for calculating a copy number of a window includes:
s201, acquiring detection signal values RC of all windows;
s202, calculating the window copy number according to the window detection signal value RC by adopting a hidden Markov model;
in an alternative embodiment, the window detection signal value RC is obtained by:
s2011, extracting SNP sites of each window;
s2012, obtaining LRR information of each SNP locus in the window;
s2013, mapping and detecting LRR information of the SNP sites into relative detection depth;
in particular according to the formula
Figure 581041DEST_PATH_IMAGE005
Mapping the SNP locus to relative detection depth, wherein D is the mapped relative detection depth, e is a natural constant, LRR is an LRR detection value of the SNP locus, and a and b are fitting parameters;
s2014, calculating an average value of relative detection depths of all SNP sites in a window to serve as a window detection signal value RC;
s2015, correcting and normalizing the window detection signal value RC to obtain a corrected window detection signal value RC;
specifically, the window reads data is subjected to mapcapability and GC content correction, the sample data size is normalized to the same level, and the baseline is used for normalization correction to obtain a window detection signal value RCid so as to eliminate system deviation. The baseline adopted in the correction can be an existing public baseline or reconstructed by using samples in the same batch, and the calculation formula of the baseline correction is as follows:
Figure 419684DEST_PATH_IMAGE006
，
Figure 857750DEST_PATH_IMAGE007
wherein, RCid is a detection signal value after the correction of the ith window of the chromosome, and RCi is a detection signal value RC of the ith window of the chromosome;
in an alternative embodiment, the step S202 of calculating the window copy number according to the window detection signal value RC by using Hidden Markov Model (HMM) includes the following steps:
setting chromosome states as 1, 10, 20, 30 and 40 which respectively represent a deletion body, a monomer, a disome, a trisome and a tetrasome, wherein the state between two adjacent states represents a chimeric state, for example, the state between 1 and 10, and the state between 10 and 20 is a deletion chimeric state; 20 and 30, and 30 and 40 are in a recombined chimeric state;
setting the transition probability between adjacent states as 0.999 and the transition probability between different states as 0.025, calculating to obtain a transition matrix required in HMM calculation by counting window detection signal values (rcids) after correction of each window of a known karyotype sample, calculating the state probability of different windows of each chromosome in the sample through an initial matrix, the transition matrix and the RCid values, defining the windows of adjacent and same states as a potential CNV by connection, and finally determining a copy number analysis result by using a threshold method;
specifically, the formula for predicting the window state is:
Figure 451542DEST_PATH_IMAGE008
，
Figure 794799DEST_PATH_IMAGE009
wherein, in the step (A),
Figure 499450DEST_PATH_IMAGE010
wherein, in the process,
Figure 41290DEST_PATH_IMAGE011
represents the number of copies in the t-1 th window
Figure 556716DEST_PATH_IMAGE012
And the signal value is
Figure 184006DEST_PATH_IMAGE013
When the first t window copy numbers are
Figure 364452DEST_PATH_IMAGE014
The probability of (d);
Figure 291956DEST_PATH_IMAGE015
is the copy number state probability of the first t-1 windows, and n is the copy number; i is an observed value, and T is the number of chromosome windows;
Figure 961972DEST_PATH_IMAGE016
the copy number state probability for the first window;
Figure 279821DEST_PATH_IMAGE017
is the initial probability;
Figure 814357DEST_PATH_IMAGE018
the signal value representing the window is
Figure 596368DEST_PATH_IMAGE013
When the number of copies is
Figure 437286DEST_PATH_IMAGE019
The probability of (d);
Figure 242431DEST_PATH_IMAGE020
representing the state of copy number of a window from a previous window
Figure 826996DEST_PATH_IMAGE021
Transition from State to Current Window copy number State
Figure 214246DEST_PATH_IMAGE019
The probability of (c).
The CNV parent source identification and CNV generation period identification steps comprise:
and S3, acquiring the offspring sample and the genotype of the father and the mother, and constructing the haplotype of the father and the haplotype of the mother according to the genetic relationship.
And S4, extracting SNP loci with one genotype being heterozygous and the other genotype being homozygous in the parents in the filial generation samples as effective SNP loci.
S5, judging the source of the effective SNP locus of the filial generation according to the genotypes of the father and the mother, and marking the effective SNP locus of the filial generation as the effective SNP locus
Figure 429326DEST_PATH_IMAGE001
Or
Figure 252926DEST_PATH_IMAGE002
Or
Figure 906761DEST_PATH_IMAGE003
Or
Figure 132206DEST_PATH_IMAGE004
；
In this step, the method for determining the source of the effective SNP site of the offspring is as follows:
when the genotype of the father is heterozygous and the genotype of the mother is homozygous, the effective SNP locus of the filial generation sample is the father locus, which nucleotide chain the effective SNP locus of the filial generation sample comes from is judged according to the haplotype of the father, and the effective SNP locus is marked as the father nucleotide chain
Figure 518188DEST_PATH_IMAGE001
Or
Figure 845395DEST_PATH_IMAGE002
；
When the mother genotype is heterozygous and the father genotype is homozygous, the offspring sample hasThe effective SNP locus is a maternal locus, which nucleotide chain the effective SNP locus of the filial generation sample comes from is judged according to the haplotype of the mother, and the effective SNP locus is marked as the maternal locus
Figure 37342DEST_PATH_IMAGE003
Or
Figure 320556DEST_PATH_IMAGE004
；
Specifically, the above
Figure 939756DEST_PATH_IMAGE001
、
Figure 3527DEST_PATH_IMAGE002
、
Figure 218739DEST_PATH_IMAGE003
、
Figure 622039DEST_PATH_IMAGE004
The marking method comprises the following conditions:
(1) When the father genotype is AB (H1H 2) and the mother genotype is AA (H1H 2), the BAF value of the filial generation is unchanged, and the effective SNP locus of the filial generation is judged to be from the father H2 and marked as
Figure 412140DEST_PATH_IMAGE001
；
(2) When the father genotype is AB (H1H 2) and the mother genotype is BB (H1H 2), the BAF value of the filial generation is unchanged, and the effective SNP locus of the filial generation is judged to be from the father H1 and marked as
Figure 697628DEST_PATH_IMAGE002
；
(3) When the father genotype is BA (H1H 2) and the mother genotype is AA (H1H 2), the offspring BAF mirror image is processed into 1-BAF, and the effective SNP locus of the offspring is judged to come from the father H1 and marked as
Figure 434640DEST_PATH_IMAGE002
；
(4) When the father genotype BA (H1H 2) and the mother genotype BB (H1H 2), the offspring BAF is mirrored to be 1-BAF, and the effective SNP locus of the offspring is judged to be from the father H2 and marked as
Figure 489183DEST_PATH_IMAGE001
；
(5) When the maternal genotype AB (H1H 2) and the paternal genotype AA (H1H 2) are met, the BAF value of the filial generation is unchanged, and the effective SNP locus of the filial generation is judged to be from the maternal H2 and marked as
Figure 932410DEST_PATH_IMAGE004
；
(6) When the maternal genotype AB (H1H 2) and the paternal genotype BB (H1H 2) are met, the BAF value of the filial generation is unchanged, and the effective SNP locus of the filial generation is judged to be from the maternal H1 and marked as
Figure 580560DEST_PATH_IMAGE003
；
(7) When the genotype BA (H1H 2) of the mother and the genotype AA (H1H 2) of the father exist, the BAF mirror image of the offspring is processed into 1-BAF, and the effective SNP locus of the offspring is judged to be from the mother H1 and marked as
Figure 449159DEST_PATH_IMAGE003
；
(8) When the mother genotype BA (H1H 2) and the father genotype BB (H1H 2) exist, the offspring BAF is mirrored to be 1-BAF, and the effective SNP site of the offspring is judged to be from the mother H2 and is marked as
Figure 561471DEST_PATH_IMAGE004
；
Here, it should be noted that: a and B are homologous genotypes, H1 is a first nucleotide and H2 is a second nucleotide, and for the genotypes AB (H1H 2) and BA (H1H 2) mentioned above, the genotype AB (H1H 2) is understood as meaning that the homologous gene at a site in the first nucleotide H1 is A and the homologous gene at the site in the second nucleotide H2 is B; genotype BA (H1H 2) is understood to mean that the homologous gene at a site in the first strand H1 is B and the homologous gene at that site in the second strand H2 is A.
S6, according to
Figure 444108DEST_PATH_IMAGE001
、
Figure 704188DEST_PATH_IMAGE002
、
Figure 579740DEST_PATH_IMAGE003
、
Figure 812138DEST_PATH_IMAGE004
The progeny sample chromosomes are divided into different recombination regions.
S7, extracting BAF values of child samples with homozygous parent SNP sites and different genotypes, calculating the parent source ratio MAF of bases provided by mothers in the SNP sites in each recombination region of the child samples, and calculating the average value of the parent source ratios of the mother SNP sites in each recombination region in the step S6;
in this step, the calculation formula of the parent-source ratio MAF is as follows
Figure 849364DEST_PATH_IMAGE022
Wherein MAF provides the base proportion for the mother,
Figure 613052DEST_PATH_IMAGE023
is the genotype of the mother, and the genotype of the mother,
Figure 26716DEST_PATH_IMAGE024
father genotype, m represents mother, f represents father.
S8, judging whether the copy number CN of the recombination region and the parent source ratio MAF in the step S6 are abnormal or not and judging the abnormal source according to the MAF value, wherein the judging process comprises the following steps:
s801, when the copy number CN of the recombination region of the sub-generation sample is not 2, judging that the copy number is abnormal and judging an abnormal source;
s802, when the copy number CN of the recombination area of the child generation sample is 2, judging whether the MAF of the parent source accounts for the ratio is abnormal or not, and if so, judging the abnormal source.
In an optional embodiment, in the step S801, the method for determining a copy number abnormality source includes:
s8011, in an offspring sample, when 1 is more than or equal to the copy number CN and is more than 0, if the mother source ratio MAF =0, judging that the offspring sample is inherited from a father DNA chain and lacks from a mother DNA chain; if the maternal-source ratio MAF =1.0, the progeny sample is inherited from the DNA strand of the mother, and the DNA strand inherited from the father is deleted;
s8012, in the offspring sample, when 2 is more than the copy number CN is more than 1, if 0 is more than the mother source occupation ratio MAF is more than 0.5, judging that the abnormality is from mother; if 1 is larger than the mother source occupation ratio MAF is larger than 0.5, judging that the abnormality is from father;
s8013, in the child sample, when the copy number CN is more than 2, if 0 is more than the mother source occupation ratio MAF is more than 0.5, judging that the abnormality is from the father; if 1 is larger than the mother source occupation ratio MAF is larger than 0.5, judging that the abnormality is originated from the mother.
In an optional embodiment, in the step S802, when the copy number CN of the child sample reassembly area is 2, the method for determining whether the copy number is abnormal includes: calculating and judging the mother source ratio MAF, and judging whether the offspring sample is one of haploid, uniparental diploid and triploid when the mother source ratio MAF is abnormal;
specifically, when the recombination region copy number CN is 2 and the parent-source ratio MAF is abnormal, the method for determining the abnormal source thereof includes:
when the parent source ratio MAF =0, the child sample is a father source UPD, the abnormality is judged to be originated from the father, and if no heterozygous site exists in the recombination region, the recombination region is an ROH region;
when the parent-source ratio MAF is more than 0.5, the offspring sample is in a chimeric state, and the abnormality is judged to be from the father;
when the parent source occupation ratio MAF =0.5, the copy number CN of the child sample is normal;
when the ratio MAF is more than 0.5 and more than 1.0, the filial generation sample is in a chimeric state, and the abnormal source is judged to be from the mother;
when the parent source ratio MAF =1.0, the child sample is the parent source UPD, the abnormal source is judged to be from the mother, and if no heterozygous sites exist in the recombination region, the recombination region is the ROH region;
the whole filial sample indicates that the filial sample is derived from one chain provided by the mother and two chains provided by the father when the mother-source ratio MAF =1/3, or indicates that the filial sample is derived from two chains provided by the mother and one chain provided by the father when the mother-source ratio MAF =2/3, and the filial sample is triploid;
when the mother source ratio MAF =0 and the heterozygous SNP site does not exist, the whole progeny sample indicates that only one strand is provided by the father in the progeny sample, or when the mother source ratio MAF =1 and the heterozygous SNP site does not exist, the whole progeny sample indicates that only one strand is provided by the mother in the progeny sample, and the progeny sample is haploid;
in the whole progeny sample, when the mother source ratio MAF =0 and the heterozygous SNP site exists, it means that only two strands are provided by the father in the progeny sample, or when the mother source ratio MAF =1 and the heterozygous SNP site exists, it means that only two strands are provided by the mother in the progeny sample, and the progeny sample is the monadic diploid.
In a preferred embodiment, the method for identifying an abnormal embryonic CNV further comprises identifying the CNV occurrence timing when judging the CNV abnormality, that is, S9, judging the CNV occurrence timing in the chimeric state between the monomer and the dimer or the chimeric state between the dimer and the trisomy in step S8 based on SPH and BPH;
specifically, the CNV occurrence period determination method includes:
if the abnormal region in the recombination region only contains SPH, judging that the CNV generation period is mitosis;
if the abnormal region in the recombination region contains BPH, and the BPH does not cross the centromere, judging that the CNV generation period is second-time division of subtraction;
if the abnormal region in the recombination region contains BPH, and the BPH crosses the centromere, the CNV generation period is judged to be the first division of the subtraction.
Example 2:
the embodiment provides an embryo SV abnormality identification method, which comprises the following steps:
the method of step S3 in example 1 was used to construct the paternal and maternal haplotypes.
Dividing each chromosome of the progeny sample into a plurality of windows connected in sequence by adopting the method of the step S1 in the embodiment 1;
and analyzing each window, judging the source of the mother haplotype and the father haplotype of the filial generation sample, and judging whether the filial generation sample has SV abnormality and an abnormal source.
Specifically, firstly, selecting effective sites according to the parental genotype, wherein the selection of the effective sites follows the following rules: the effective sites of the father meet the requirements of 'heterozygous father and homozygous mother'; the effective locus of the mother meets the requirements of father homozygosis and mother heterozygosis.
Secondly, determining the key base in the effective site, wherein the determination method of the key base comprises the following steps: the heterozygous genotype is AB, and when the homozygous genotype is AA, the key basic group is B; when the heterozygous genotype is AB, and the homozygous genotype is BB, the key base is A. According to the rule, key bases in the father effective site and the mother effective site are respectively found out.
Again, the genotype of the progeny sample is labeled, with the label containing the key base being 1, the label not containing the key base being 0, and the label with unknown genotype being-1.
Then, according to Mendelian genetic law, haplotypes of the father and the mother are respectively constructed, and the construction method comprises the following steps: taking the effective sites of mothers in human chromosomes as an example, respectively constructing haplotypes for 23 chromosomes (22 autosomes and chrX), dividing each chromosome into a plurality of continuous windows (bins), analyzing the marks of the filial generation samples on the effective sites of mothers in each window, picking out the filial generation samples which are not simultaneously marked as 1 at the same site on all the effective sites in the windows, and dividing the effective sites of mothers into two haplotypes by the filial generation samples; after all windows are analyzed, splicing according to the positions to obtain a haplotype result of the mother;
the offspring sample judges which haplotype is from the mother according to the locus marked as 1 on the effective locus of the mother; and the father only has one chrX, so the chrX haplotype of the father is directly obtained from the genotype, the haplotype of the father on the autosome is the same as the analysis method of the mother, and the offspring sample judges which haplotype is derived from the father according to the site marked as 1 on the effective site of the father.
When the embryo SV abnormality is identified, under the condition that a progeny sample which is known to be diseased or known to be normal exists in a family, the risk haplotype and the normal haplotype of the progeny sample can be distinguished according to the haplotype near a gene or the haplotype near a translocation chromosome breakpoint.
Specifically, when a child sample is diseased or inherits a derivative chromosome, the haplotype inherited by the sample near a pathogenic gene or a translocation chromosome breakpoint is a risk haplotype, the haplotype inherited by the child sample with the same haplotype is a risk embryo or carrier, and the haplotype inherited by the child sample is a normal haplotype.
When the offspring sample is normal or does not inherit derivative chromosomes, the haplotype inherited by the sample near a pathogenic gene or a translocation chromosome breakpoint is a normal haplotype, the haplotype inheriting the same haplotype is a normal embryo, and the haplotype inheriting the other haplotype is a risk embryo or a carrier.
Example 3:
the embodiment provides an embryo SGD variation identification method, which comprises the following steps:
constructing a father haplotype and a mother haplotype by the method of the step S3 in the embodiment 1;
dividing each chromosome of the progeny sample into a plurality of windows connected in sequence by adopting the method of the step S1 in the embodiment 1;
analyzing each window, judging the source of the maternal haplotype and the paternal haplotype of the child sample, and judging whether the SGD of the child sample has known variation and variation source.
The construction of the paternal haplotype and maternal haplotype, and the determination of the sources of the maternal haplotype and paternal haplotype in the progeny sample are the same as those in example 2, and are not further described herein.
When the embryo SGD is abnormally identified, and when a family does not have a known diseased offspring sample, the risk haplotype and the normal haplotype can be distinguished according to the detection of a mutant embryo, or a translocation chromosome-related copy number abnormal embryo, and the haplotype near a pathogenic gene or the haplotype near a translocation chromosome breakpoint.
Specifically, for the PGT-M family: detecting that the haplotype of the mutated embryo near a pathogenic gene is a risk chromosome, the haplotype of other embryos inherited with the same haplotype is a risk embryo, and the embryos of chromosomes not inherited with the risk chromosome are non-risk embryos;
for the roche translocation family: the short arms of the two chromosomes (chr 13, chr14, chr15, chr21 and chr 22) of the carrier are lost, the long arms are connected, and the two chromosomes are respectively denoted by c and d. If the embryo inherits two haplotypes of the carrier c chromosome, then the genetic carrier d chromosome is the derivative chromosome; if the embryo does not have the haplotype on the c chromosome of the genetic carrier, the d chromosome of the genetic carrier is the normal haplotype; the risk of other embryos can be judged according to the haplotype.
For a balanced translocation family: when the breakpoint of the carrier is c and d, the end with centromere is c and d, the end without centromere is c 'and d', c is connected with d ', d is connected with c'. If the embryo inherits two haplotypes from the carrier on the c-side, the haplotype inherited on the c' -side is a normal haplotype. If the haplotype of the carrier is not inherited on the c side of the embryo, the haplotype inherited on the c' side is the derived haplotype. The judgment method is also suitable for the breakpoint d, and the derivative haplotypes and the normal haplotypes are distinguished by using the haplotypes of the copy number abnormal embryos according to the method, so that the risks of other embryos can be judged through the haplotypes.
For the inverted family, the breakpoints of the carrier are c and d respectively, the two ends are c and d, the inverted part is c 'and d', c is connected with d ', and d is connected with c'. If the embryo inherits two haplotypes from the carrier on the c-side, the haplotype inherited on the c' -side is normal chromosome. If the haplotype of the carrier is not inherited on the c side of the embryo, the haplotype inherited on the c' side is the derivative chromosome. The judgment method is also suitable for the breakpoint d, and the haplotype of the copy number abnormal embryo is used for distinguishing the derivative chromosome from the normal chromosome according to the method, so that the risk of other embryos can be judged through the haplotype.
It is particularly emphasized that the progeny samples (embryos) selected for use in the present invention are obtained by in vitro fertilization, and the cells extracted during in vitro development to cleavage stage or blastocyst stage are not the subject of, or are not the subject of, direct implementation of the living human or animal body, and are not the method for diagnosis and treatment of disease.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (10)

1. An embryo CNV abnormity identification method is characterized by comprising a chromosome CNV identification step, a CNV parent source identification step and a CNV generation period identification step;
wherein, the step of identifying the chromosome CNV comprises the following steps:
s1, dividing a chromosome into a plurality of windows which are connected in sequence;
s2, calculating a probe detection signal value LRR of each window, and acquiring the copy number state of each window, wherein the copy number state comprises a default, a monomer, a dimer, a trimer and a tetrad, and the adjacent two states are in a chimeric state;
the CNV parent source identification and CNV generation period identification steps comprise:
s3, acquiring a progeny sample and the genotype of the father and the mother, and constructing a father haplotype and a mother haplotype according to the genetic relationship;
s4, extracting SNP loci with one genotype being heterozygous and the other genotype being homozygous in the parents in the filial generation samples as effective SNP loci;
s5, judging the source of the effective SNP locus of the filial generation according to the genotypes of the father and the mother, and marking the effective SNP locus of the filial generation as the effective SNP locus
Figure 488890DEST_PATH_IMAGE001
Or
Figure 969681DEST_PATH_IMAGE002
Or
Figure 107402DEST_PATH_IMAGE003
Or
Figure 163082DEST_PATH_IMAGE004
；
S6, according to
Figure 448570DEST_PATH_IMAGE001
、
Figure 920003DEST_PATH_IMAGE002
、
Figure 974546DEST_PATH_IMAGE003
、
Figure 948931DEST_PATH_IMAGE004
Dividing the offspring sample chromosome into different recombination regions;
s7, extracting BAF values of offspring samples with homozygous parent SNP sites and different genotypes, calculating the maternal source ratio MAF of bases provided by the mother in each recombination region of the offspring samples in the SNP sites, and calculating the average value of the maternal source ratios of the parent SNP sites in each recombination region in the step S6;
s8, judging whether the copy number CN of the recombination region and the parent source ratio MAF in the step S6 are abnormal or not according to the MAF value, and judging the abnormal source, wherein the judgment comprises the following steps:
s801, when the copy number CN of the recombination region of the sub-generation sample is not 2, judging that the copy number is abnormal and judging an abnormal source;
s802, when the copy number CN of the recombination region of the child generation sample is 2, judging whether the MAF of the parent source accounts for the abnormal condition, and if so, judging the abnormal source.
2. The method for identifying CNV abnormality in embryo according to claim 1, wherein the method for determining the source of effective SNP site in offspring in step S5 is:
when the genotype of the father is heterozygous and the genotype of the mother is homozygous, the effective SNP locus of the filial generation sample is the father locus, and the effective SNP locus of the filial generation sample is judged from which nucleotide chain of the father the effective SNP locus comes from according to the haplotype of the father and is marked as
Figure 456136DEST_PATH_IMAGE001
Or
Figure 731259DEST_PATH_IMAGE002
；
When the genotype of the mother is heterozygous and the genotype of the father is homozygous, the effective SNP locus of the filial generation sample is the maternal locus, which nucleotide chain the effective SNP locus of the filial generation sample comes from is judged according to the haplotype of the mother and marked as
Figure 171468DEST_PATH_IMAGE003
Or
Figure 37793DEST_PATH_IMAGE004
。
3. The method for discriminating an embryo CNV abnormality according to claim 1, wherein the method for determining the origin of a copy number abnormality in step S801 includes:
s8011, in an offspring sample, when the copy number CN is more than or equal to 1 and is more than 0, if the mother source accounts for MAF =0, judging that the offspring sample is inherited from a DNA chain of a father and lacks the DNA chain of the mother; if the maternal-source ratio MAF =1.0, the progeny sample is inherited from the DNA strand of the mother, and the DNA strand inherited from the father is deleted;
s8012, in the offspring sample, when 2 is more than the copy number CN is more than 1, if 0 is more than the mother source occupation ratio MAF is more than 0.5, judging that the abnormality is from mother; if 1 is larger than the mother source occupation ratio MAF is larger than 0.5, judging that the abnormality is from father;
s8013, in a child sample, when the copy number CN is more than 2, if 0 is more than the mother source occupation ratio MAF is more than 0.5, judging that the abnormality comes from a father; if 1 is larger than the mother source occupation ratio MAF is larger than 0.5, judging that the abnormality is originated from the mother.
4. The method for discriminating an embryo CNV abnormality according to claim 1, wherein in step S802, when the copy number CN of the daughter sample regrouping region is 2, the method for determining whether there is an abnormality in the copy number is: and calculating and judging the mother source ratio MAF, and when the mother source ratio MAF is abnormal, judging whether the recombination region of the filial generation sample is one of haploid, uniparental diploid and triploid.
5. The method for discriminating abnormality of embryo CNV according to claim 4, wherein in step S802, when the recombination region copy number CN is 2 and the parent-source ratio MAF is abnormal, the method for determining the origin of abnormality comprises:
when the parent source ratio MAF =0, the child sample is a father source UPD, the abnormality is judged to be originated from the father, and if no heterozygous site exists in the recombination region, the recombination region is an ROH region;
when the ratio MAF is more than 0.5 and more than 0, the filial generation sample is in a chimeric state, and the abnormal source is judged to be from the father;
when the parent source ratio MAF =0.5, the child sample copy number CN is normal;
when the ratio MAF is more than 0.5 and is more than 1.0, the filial generation sample is in a chimeric state, and the abnormal source is judged to be from the mother;
when the parent source ratio MAF =1.0, the child sample is the parent source UPD, the abnormal source is judged to be from the mother, and if no heterozygous site exists in the recombination region, the recombination region is the ROH region;
the whole filial sample indicates that the filial sample is derived from one chain provided by the mother and two chains provided by the father when the mother source ratio MAF =1/3, or indicates that the filial sample is derived from two chains provided by the mother and one chain provided by the father when the mother source ratio MAF =2/3, and the filial sample is triploid;
when the mother source ratio MAF =0 and the heterozygous SNP site does not exist, the whole progeny sample indicates that only one strand is provided by the father in the progeny sample, or when the mother source ratio MAF =1 and the heterozygous SNP site does not exist, the whole progeny sample indicates that only one strand is provided by the mother in the progeny sample, and the progeny sample is haploid;
in the whole progeny sample, when the mother source ratio MAF =0 and the heterozygous SNP site exists, it means that only two strands are provided by the father in the progeny sample, or when the mother source ratio MAF =1 and the heterozygous SNP site exists, it means that only two strands are provided by the mother in the progeny sample, and the progeny sample is the monadic diploid.
6. The method of identifying an abnormal CNV according to claim 1, further comprising a CNV occurrence timing identification step of S9, wherein the CNV occurrence timing is determined for the chimeric state between the monomer and the dimer or the chimeric state between the dimer and the trimer in step S8 based on SPH and BPH;
specifically, the CNV occurrence period determination method includes:
if the abnormal region in the recombination region only contains SPH, judging that the CNV generation period is mitosis;
if the abnormal region in the recombination region contains BPH, and the BPH does not cross the centromere, judging that the CNV generation period is second-time division of subtraction;
if the abnormal region in the recombination region contains BPH, and the BPH crosses the centromere, the CNV generation period is judged to be the first division of the subtraction.
7. The method for discriminating embryo CNV abnormality of claim 1, wherein in step S2, the method for calculating the copy number of the window includes:
s201, acquiring detection signal values RC of all windows;
s202, calculating the window copy number according to the window detection signal value RC by adopting a hidden Markov model.
8. The method for discriminating embryo CNV abnormality according to claim 7, wherein the method for acquiring the window detection signal value RC in step S201 is:
s2011, extracting SNP sites of each window;
s2012, obtaining LRR information of each SNP locus in the window;
s2013, mapping and detecting LRR information of the SNP sites into relative detection depth;
s2014, calculating an average value of relative detection depths of all SNP sites in a window to serve as a window detection signal value RC;
s2015, correcting and normalizing the window detection signal value RC to obtain a corrected window detection signal value RC.
9. An embryo SV abnormality identification method is characterized by comprising the following steps:
constructing a paternal haplotype and a maternal haplotype using step S3 described in claim 1;
dividing each chromosome of the progeny sample into a plurality of sequentially connected windows using the step S1 of claim 1;
and analyzing each window, judging the source of the mother haplotype and the father haplotype of the filial generation sample, and judging whether the filial generation sample has SV abnormality and an abnormal source.
10. The method for identifying the SGD variation of the embryo is characterized by comprising the following steps of:
constructing a paternal haplotype and a maternal haplotype using step S3 of claim 1;
dividing each chromosome of the offspring sample into a plurality of windows connected in sequence by using the step S1 of claim 1;
and analyzing each window, judging the sources of the maternal haplotype and the paternal haplotype of the child sample, and judging whether the SGD of the child sample has known variation and variation sources.
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