[The Polymorphism Analysis of HLA Class II Alleles Based on Next-Generation Sequencing and Prevention Strategy for Allele Dropout].

OBJECTIVE

To investigate the accuracy of next-generation sequencing technology (NGS) in detecting the polymorphisms of and alleles in randomly-selected unrelated healthy individuals from Shenzhen Han population, investigate the potential reason for allele dropout in routine NGS, and establish an internal quality control system.

METHODS

NGS-based HLA class II genotyping was performed on 1 012 samples using the MiSeqDx platform. The suspected missed alleles indicated by the quality control software and homozygotes were confirmed by PCR-SSOP or PCR-SBT methods.

RESULTS

A total of 139 alleles were detected, including (45), (7), (5), (7), (17), (21), (10) and (27). *09:01(17.09%),15:01(10.72%); *02:02(25.99%),03:01(10.18%); *01:03(36.46%); *01:01(15.42%); *01:02(20.01%),03:02(17.19%); *03:01(19.47%),03:03(17.98%), 05:02(11.66%), 06:01(10.67%); *02:02(54.45%), 01:03(31.18%) and *05:01(39.13%), 02:01(16.90%) alleles were the most common alleles in Shenzhen Han population (frequencies >10%). There was no statistical difference between the gene frequencies of and loci in our study. The HLA Common and Well-Documented Alleles in China (CWD2.4) (χ=12.68, >0.05). 94 cases of homozygous samples detected by NGS were retested by PCR-SSOP or SBT method, and one case of allele dropout at locus was found. SBT method confirmed that the allele of *04:03 was missed. The laboratory internal quality control system was established. Two cases of new alleles were detected and named by WHO Nomenclature Committee for Factors of the HLA System.

CONCLUSION

The HLA genotyping results based on NGS showed a significantly lower ambiguity rate. The HLA class II alleles exhibit genetic polymorphism in the Han population of unrelated healthy individuals in Shenzhen. The independent method based on NGS in clinical histocompatibility testing has limitations and requires internal quality control strategies to avoid allele-dropout events.