Abstract
BackgroundSeveral recent studies showed that next-generation sequencing (NGS)-based human leukocyte antigen (HLA) typing is a feasible and promising technique for variant calling of highly polymorphic regions. To date, however, no method with sufficient read depth has completely solved the allele phasing issue. In this study, we developed a new method (HLAscan) for HLA genotyping using NGS data.ResultsHLAscan performs alignment of reads to HLA sequences from the international ImMunoGeneTics project/human leukocyte antigen (IMGT/HLA) database. The distribution of aligned reads was used to calculate a score function to determine correctly phased alleles by progressively removing false-positive alleles. Comparative HLA typing tests using public datasets from the 1000 Genomes Project and the International HapMap Project demonstrated that HLAscan could perform HLA typing more accurately than previously reported NGS-based methods such as HLAreporter and PHLAT. In addition, the results of HLA-A, −B, and -DRB1 typing by HLAscan using data generated by NextGen were identical to those obtained using a Sanger sequencing–based method. We also applied HLAscan to a family dataset with various coverage depths generated on the Illumina HiSeq X-TEN platform. HLAscan identified allele types of HLA-A, −B, −C, −DQB1, and -DRB1 with 100% accuracy for sequences at ≥ 90× depth, and the overall accuracy was 96.9%.ConclusionsHLAscan, an alignment-based program that takes read distribution into account to determine true allele types, outperformed previously developed HLA typing tools. Therefore, HLAscan can be reliably applied for determination of HLA type across the whole-genome, exome, and target sequences.
Highlights
Several recent studies showed that next-generation sequencing (NGS)-based human leukocyte antigen (HLA) typing is a feasible and promising technique for variant calling of highly polymorphic regions
Human major histocompatibility complex (MHC) proteins are encoded by the human leukocyte antigen (HLA) locus, which maps to a 3.6 Mbp stretch on human chromosome 6p21.3
The method is relatively high-throughput and inexpensive compared with PCRSSO and polymerase chain reaction (PCR)-sequencing–based typing (SBT), and enables highly accurate HLA typing by producing hundreds of base pairs of long sequence reads at high coverage depth [11,12,13]
Summary
Several recent studies showed that next-generation sequencing (NGS)-based human leukocyte antigen (HLA) typing is a feasible and promising technique for variant calling of highly polymorphic regions. As an alternative, targeted amplicon sequencing ( known as the PCRNGS approach) was recently developed. This technology uses standard PCR to capture regions of interest, and the resultant amplicons are subjected to nextgeneration sequencing (NGS). The method is relatively high-throughput and inexpensive compared with PCRSSO and PCR-SBT, and enables highly accurate HLA typing by producing hundreds of base pairs of long sequence reads at high coverage depth [11,12,13]. Most of the recently generated genome-wide datasets consist of short sequence reads (~101 bp), for reasons related to efficiency and cost, HLA typing from WGS or WES datasets is a feasible and efficient strategy for achieving accurate typing with existing resources [6, 15]
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