Construction method of blood free DNA (deoxyribonucleic acid) library based on Ion ProtonTM sequencing platform, reagents and application of reagents

Abstract

The invention discloses an Ion Proton-based TM Blood cell-free DNA library construction method, reagents and applications of the sequencing platform, the construction process includes blood cell-free DNA extraction, end repair, fragment selection, adapter ligation, magnetic bead purification, library amplification, library detection, high-throughput sequencing and other steps . The present invention also provides a kind of for constructing based on Ion Proton TM Blood cell-free DNA extraction reagents, end repair reagents, linker ligation reaction reagents, and amplification reagents for the blood cell-free DNA library of the sequencing platform. The invention significantly improves the extraction concentration of fetal free DNA in pregnant women's plasma, and has simple steps and low cost, and improves the success rate of library construction; the optimized library construction system is more convenient for operation and saves costs, and after replacing the magnetic beads, it can be purified and recovered Higher efficiency; library amplification does not require sufficient sample size and secondary detection, and the operation is simpler. The method of the invention is more suitable for non-invasive prenatal diagnosis of fetal chromosomal aneuploidy.

Description

technical field [0001] The invention relates to the field of high-throughput sequencing, specifically an IonProton-based TM The library construction method, reagent and application of the blood cell-free DNA of the sequencing platform. Background technique [0002] According to the 2006 global report on birth defects in the United States, there are at least 7.9 million new cases of birth defects in the world every year, accounting for 6% of the birth population. my country is also a country with a high incidence of birth defects. The current incidence of birth defects is about 5.6%. About 900,000 cases of birth defects. According to reports, 30% of children with severe birth defects in my country can live a normal life after early diagnosis and treatment, about 30% will die after birth, and 40% will become permanently disabled. In addition, it has caused a heavy burden on the economic development of the country and society. According to statistics, the annual treatment cost...

AI Technical Summary

Problems solved by technology

[0014] The total amount of free DNA in plasma is very small, and fetal free DNA in pregnant women only accounts for 3%-6% of pregnant women's plasma. Low, and the extracted free DNA may be partially missing, resulting in a decline in the quality of the library, a decrease in the success rate, and a great impact on the accuracy of the sequencing results; and the obtained small fragment library is mixed and then amplified. Although the amplification deviation between libraries is reduced, it is necessary to collect enough samples before amplification, the operation is more complex and requires secondary detection, and the cost is higher

In addition, the existing library construction method has a large system, which is not conducive to operation, and the waste of reagents is serious, and the cost is high. However, the use of Biocanal Magbeads magnetic beads for purification has low recovery efficiency, and the quality and concentration of the library have declined, resulting in a decline in sequencing quality. , even the sequencing fails

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