Next generation sequencing (NGS) assays are state of the art for HLA genotyping. To sequence on an Illumina sequencer, the DNA of interest must be enriched, fragmented, and bookended with known oligonucleotide sequences, a process known as library construction. Many HLA genotyping assays enrich the target loci by long-range PCR (LR-PCR), prior to fragmentation. This PCR step has been reported to introduce errors in the DNA to be sequenced, including inaccurate replication of repeated sequences, and the in vitro recombination of alleles encoded on separate chromosomes. An alternative library construction method involves fragmentation of genomic DNA, followed by hybrid-capture (HC) enrichment of target HLA loci. This HC-based method involves PCR, but with far fewer cycles. Consequently, the HC method had significantly fewer PCR-induced errors, including more faithful replication of repeated sequences, and the near elimination of recombinant sequences. These improvements likely produce more accurate NGS sequencing data of HLA loci.

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http://dx.doi.org/10.1016/j.humimm.2021.02.010DOI Listing

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