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Method for sequencing and detecting fetal genetic variation by using polymorphic site and target site

A genetic variation and locus technology, applied in the field of single nucleotide locus variation, can solve the problems of not being able to detect chromosomal and subchromosomal variation at the same time, different, non-invasive detection methods are not very sensitive and specific, etc. Achieving good development and application prospects

Pending Publication Date: 2022-06-21
高嵩
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Problems solved by technology

However, for microdeletion / microduplication variations at the subchromosomal level, the sensitivity and specificity of noninvasive detection methods are not very high, especially for small fragments of microdeletion / microduplication variations (Advani, Barrett et al.2017, Prenat Diagn 37:1067-1075; Hu, Wang et al.2019, Human Genomics 13:14; Srebniak, Knapen et al.2020, Mol Genet Genomic Med 8:e1062)
Although a variety of non-invasive detection methods for monogenic genetic diseases based on next-generation sequencing have been developed (Lun, Tsui et al.2008, Proc Natl Acad Sci US A 105:19920-19925; Lo, Chan et al.2010, Sci Transl Med 2:61ra91; Lv, Wei et al.2015, Clinical Chemistry 61:172-181; Vermeulen, Geeven et al.2017, Am J Hum Genet 101:326-339; Allen, Young et al.2018, Noninvasive Prenatal Testing( NIPT) 157-177; Yin, Du et al.2018, J HumGenet 63:1129-1137; Cutts, Vavoulis et al.2019, Blood 134:1190-1193; Zhang, Li et al.2019, Nat Med 25:439- 447), but these methods have not been widely used in clinical practice, mainly because these methods use different methods for detecting chromosomal or subchromosomal level variation, so they cannot be used to detect chromosomal and subchromosomal level variation at the same time
At the same time, these methods are very costly for the detection of each single-gene genetic disease, resulting in low cost-effective screening of low-prevalence single-gene genetic diseases with these methods

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  • Method for sequencing and detecting fetal genetic variation by using polymorphic site and target site
  • Method for sequencing and detecting fetal genetic variation by using polymorphic site and target site
  • Method for sequencing and detecting fetal genetic variation by using polymorphic site and target site

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Experimental program
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Effect test

Embodiment 1

[0108] For biological maternal plasma DNA samples, the minimum component is fetal DNA and the maximum component is maternal DNA. Since the fetus inherits the genetic material on the chromosome of the biological mother, each polymorphic site in the plasma DNA of the biological mother may be one of the five genotypes, and the concentration of fetal DNA can be estimated through iteration according to the above steps.

[0109] figure 2 is a flow chart for estimating fetal DNA concentrations in plasma DNA samples from pregnant women legally permitted to receive egg donation as described in Example 13.

[0110] Example 14. Estimation of fetal DNA concentration using simulated sequencing of polymorphic sites in maternal plasma DNA samples

The method and steps for estimating the concentration of fetal DNA in the sample by using the relative ratio method of allele counts are briefly described below by taking the counts of each allele of five hypothetical polymorphic sites in the s...

Embodiment 2

[0118] For site Id003, R2 / (R1+R2)=0.496≥0.01, R3 / (R1+R2+R3)=0.009<0.01, the number of alleles is estimated to be two, because R1 / (R1+R2)=0.504 <0.5+α, the genotype is estimated to be AB|AB, FC=NA, TC=R1+R2=6249.

[0119] For site Id004, R2 / (R1+R2)=0.379≥0.01, R3 / (R1+R2+R3)=0.003<0.01, the number of alleles is estimated to be two, because 0.5+α≤R1 / (R1+ R2)=0.621<0.75, the genotype is estimated to be AB|AA, FC=R1-R2=2257, TC=R1+R2=9361.

[0120] For site Id005, R2 / (R1+R2)=0.430≥0.01, R3 / (R1+R2+R3)=0.126≥0.01, the number of alleles is estimated to be two, because R2 / R1=0.756≥0.5, the gene The type estimate is AB|AC, FC=R1-R2+R3=1990, TC=R1+R2+R3=8046.

[0121] (3) Estimate the concentration of fetal DNA

[0122] 1) Enter the values ​​of FC and TC

[0123] Estimation method Estimated fetal DNA concentration linear regression 0.2441 Robust regression 0.2441 median of ratios 0.2465 average ratio 0.2450 median ratio 0.2473 ratio of m...

Embodiment 3

[0128] (a) For each polymorphic locus, according to each allele count and f 0 , According to the method described in Example 11 and Example 12, the genotype of the locus was estimated, and theoretically the amplification count (FC) derived from fetal DNA and the total count (TC) derived from maternal and fetal DNA.

[0129] For example, for site number Id0006, R1 to R4 are 3322, 936, 36 and 28 respectively, then O 1 =3322,O 2 =936, O 3 =36,O 4 =28. Since R2 / (R1+R2)≥0.01 and R3 / (R1+R2+R3)<0.01, this locus has two detected allele numbers above the noise threshold.

[0130] The goodness of fit test for all possible genotypes at this locus is as follows:

AA|AA: TC=R1+R2=4258, E 1 =(1-α)×TC=4215.42, E 2 =α×TC=42.58,

[0131] AA|AB: TC=R1+R2=4258, E 1 =(1-f 0 / 2)×TC=4045.10, E 2 = f 0 / 2×TC=212.90,

[0132] AB|AA: TC=R1+R2=4258, E 1 =(1+f 0 ) / 2×TC=2341.90, E 2 =(1-f 0 ) / 2×TC=1916.10,

[0133] AB|AB: TC=R1+R2=4258, E 1 =1 / 2×TC=2129.00, E 2 =1 / 2×TC=212...

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Abstract

The invention provides a method for noninvasive detection of fetal genetic variation. The method comprises the following steps: firstly, carrying out targeted sequencing on polymorphic sites on a reference genome and then carrying out allele copy counting on each polymorphic site, and estimating the percentage of fetal genetic materials in a pregnant woman plasma sample; then, allele copy counting is performed on polymorphic sites on a target genome or a target spot to be detected, and whether the target spot to be detected in a sample varies at the chromosome level, the sub-chromosome level or the single genetic site level or not is detected in cooperation with goodness-of-fit test or an allele counting relative distribution diagram. The method is suitable for simultaneously detecting chromosome epiploidy variation, subchromosome level micro-deletion and micro-repetition variation and short sequence level variation in a pregnant woman plasma sample, and has good development and application prospects.

Description

technical field [0001] The invention relates to the field of genetic variation detection, especially aneuploidy variation at the chromosome level, micro-deletion / micro-duplication variation at the sub-chromosomal level or insertion-deletion and single-nucleotide site variation of short sequences. Background technique [0002] In 1997, cell-free DNA of fetal origin was found in maternal plasma (Lo, Corbetta et al. 1997, Lancet 350:485-487). Based on this discovery and massively parallel sequencing, multiple research groups have developed methods based on sequencing analysis of maternal plasma DNA (cfDNA) to detect chromosomal aneuploidy, subchromosomal microdeletions / microduplications, or Short sequence indels and single nucleotide site variations at the single gene level (Advani, Barrett et al. 2017, Prenat Diagn 37:1067-1075; Breveglieri, D'Aversa et al. 2019, Mol Diagn Ther 23:291-299 ; Andari, Bussamra et al. 2020, Ceska Gynekol 85:41-48; Guseh 2020, Hum Genet 139:1141-1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858G16B15/30G16B20/20G16B20/40
CPCC12Q1/6858G16B15/30G16B20/20G16B20/40C12Q2531/113C12Q2535/122C12Q2537/165C12Q2537/16G16B40/10
Inventor 高嵩
Owner 高嵩