Three-generation sequencing platform-based HLA genotyping method

Abstract

The invention discloses a three-generation sequencing platform-based HLA genotyping method. The method comprises the following steps of (1) performing PCR amplification on HLA genes needed to be genotyped; (2) after a PCR product is detected to be qualified, performing three-generation sequencing to obtain original data; (3) performing long sequence comparison on the original data and a referencegene sequence; (4) after comparison, correcting sequencing errors; (5) performing phase splitting to obtain a haplotype sequence; and (6) performing genotyping judgment. Compared with an existing HLAgenotyping method, the HLA genotyping method has the advantages of few requirements on computing resources, quick genotyping and high resolution, and has important values for application and basic research work of clinical transplantation tissue type matching, population genetics, anthropology, evolutiology and the like.

Description

technical field [0001] The invention belongs to the field of bioinformatics, and in particular relates to an HLA genotyping method based on a three-generation sequencing platform. Background technique [0002] Human leukocyte antigen system is another name for human major histocompatibility complex (Major histocompatibility complex, MHC). It is located on the short arm of human chromosome 6 and consists of a series of closely linked loci. The HLA gene is the most polymorphic in the human genome and one of the most complex genetic systems in humans so far. The protein encoded by the HLA gene has the functions of identifying self and non-self, regulating immune response and so on. The correct and high-precision HLA type plays a decisive role in the success of bone marrow transplantation and organ transplantation. HLA class I (HLA-A, HLA-B, HLA-C) and HLA class II (HLA-DRB1, HLA-DPB1, HLA-DQB1) play a major role in matching. In addition, the type of HLA gene is also closely...

AI Technical Summary

Problems solved by technology

However, PCR-SBT still has the following technical defects: (1) the cost is high and the time is relatively slow; (2) the PCR-SBT typing method is mainly aimed at the detection of exons 2, 3, and 4, which are relatively concentrated in polymorphic sites. Exons 1, 5, 6, and 7 are not sequenced. Due to the high genetic polymorphism of HLA, there are certain polymorphisms in exons 1, 5, 6, and 7. Therefore, the existing method to determine the polymorphism of exons 2-4 may cause some alleles to be unable to be assigned, resulting in ambiguity, which seriously affects clinical work; (3) the full-length sequence of the gene cannot be obtained, and the (4) There are certain random and inferred errors; (5) It is not sensitive to the variation discrimination of variable splicing sites; (6) The typing result can only reach "4 digits". resolution"

Next-generation sequencing is easy to cause misalignment, it is difficult to span repetitive sequences, and the GC bias caused by PCR often leads to wrong coverage of GC-rich regions, affecting the accuracy of variant detection

The technical defects of LAA phase separation are: (1) In the case of high depth, hundreds of thousands of Reads are clustered, which consumes a lot of memory, a lot of calculation, and takes a long time (the time spent is determined by the amplification 2-3k amplicons will take 3 hours, and the memory consumption is determined by the amplicon size and sequencing depth); (2) clustering can only use the original Reads, HLA is the human genome For the genetic system with the highest polymorphism, it is very important to obtain the real SNV / Indel by typing the region. The real SNV / InDel of LAA and the SNV / InDel introduced by the third-generation sequencing are not distinguished, that is, they are clustered, which affects the accuracy of the results In the case of no corrected error rate, the clustered haplotype and the real haplotype have a certain degree of discrepancy; (3) Insensitive to a single SNV / InDel, clustering and phase separation according to a fixed algorithm, the operation not flexible enough

[0015] 1) After blast comparison, the results of incomplete comparison are counted, and the method is not rigorous;

[0016] 2) Low typing accuracy;

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