Noninvasive prenatal haplotype construction method based on long-fragment DNA capture and third-generation sequencing

Abstract

A non-invasive prenatal haplotype construction method based on long-fragment DNA capture and three-generation sequencing, including: constructing a second-generation library using genomic DNA from the peripheral blood of pregnant women and / or their husbands; capturing target genes and flanking regions; constructing Three-generation sequencing library, and three-generation sequencing to obtain sequencing reads; extend from the mutation point of the target gene on the sequencing reads to both ends to find heterozygous SNP sites; the overlapping regions of different sequencing reads contain the same one or When there are multiple SNP sites, the haplotype can be successfully distinguished to both ends until there is a region without sequencing read length coverage or the SNP sites detected by the sequencing read length are all homozygous. The method of the present invention realizes the construction of parental individual haplotypes, and solves the disadvantage that the current non-invasive prenatal detection that relies on parent-child family haplotype analysis is not suitable for family detection that cannot obtain proband samples.

Description

Technical field [0001] The present invention relates to the field of sequencing techniques, and more particularly to a non-invasive production pre-production method based on long fragment DNA capture and three generation sequencing. Background technique [0002] The World Health Organization's 2015 birth defect report shows that there are about 3 genes related to 32,000 birth defects per 100 newborns, and 2,700 new students died of birth defects each year. Studies have shown that most of the birth defects are related to genetic factors, and single genetic defects are one of the important factors. At present, there is no rootic measures for most single genetic defects, and can only replace the treatment, and survive the birth defect Most of them are lifetime disability or intellectual obstacles, which cannot be cured, thereby causing heavy economic and psychological burdens to society and families. Pre-prenatal testing for high-risk pregnant women is an effective means to prevent ...

AI Technical Summary

Problems solved by technology

STR linkage analysis has the problem of fewer STR linkage marker sites. In specific cases, there may be no available STR sites, which requires a lot of pre-experimentation, and most of the STRs are far away from the defect site, so the possibility of misdiagnosis caused by recombination cannot be ruled out.

Based on haplotype analysis, the method of genome capture sequencing or SNP typing of the parental proband's family is often used to first obtain the haplotype associated with the defect site. The multiplex PCR operation is complicated, and the cost of family capture sequencing is high, and it is difficult to popularize. It is necessary to obtain the samples of the parent-child family at the same time, but in practical applications, it is usually encountered that the samples of the offspring of the couple to be tested are not available. For example, Lu Sijia disclosed "a method for SNP-haplotype analysis using multiplex PCR technology" (Publication number: CN105385755A)

Images