OR22 High-resolution HLA typing and identification of a novel allele with a splice variant using RNA-SEQ

Aim RNA sequencing (RNA-Seq) is a powerful next-generation sequencing method for discovering, profiling and quantifying RNA transcripts. RNA-Seq can also identify splice junctions and single nucleotide polymorphisms (SNP) in coding exons. In this study, we tested if RNA-Seq reads provide sufficient coverage and depth for high-resolution HLA genotyping. Methods RNA-Seq data was available from 24 individuals from an acutely infected HIV cohort. We used two methods to extract HLA reads from the RNA-Seq data: 1) FR-HIT recruited RNA-Seq reads to the IMGT coding sequence reference database; and 2) the SNP-tolerant aligner GSNAP aligned the reads to a constructed GMAP database consisting of cDNA from HLA genes. The resulting HLA-specific FASTQ reads that were generated were analyzed by commercial HLA typing software. Results We obtained 100% sequence coverage and a median sequence read depth of 331, which was sufficient to genotype 15 HLA loci from each of the donors. HLA genotypes for 8 classical HLA class I and II loci matched genotypes generated from the Sanger sequence-based typing method, as well as targeted next-generation sequencing of genomic DNA. We identified a novel HLA-DQB1 allele in the Thai population with a frequency greater than 5% that was identical to the 05:01:01 allele in all exons except exon 1, where it differed by one SNP at position 81. The DQB1 * 05:01:01 allele, like several other DQB1 alleles, has exon 5 spliced out. This deletion however, cannot be confirmed from the reads that are obtained by sequencing genomic DNA. RNA-Seq confirmed that the novel allele does not express exon 5, as the RNA-Seq data showed a complete absence of exon 5 reads compared to reads from targeted genomic sequences. Conclusions We have successfully genotyped 15 HLA loci and confirmed a splice variant in a novel allele using RNA-Seq. This method highlights the importance of mining RNA-Seq data for high-resolution HLA genotyping. Additional analysis methods may be able to identify allele-specific expression of HLA alleles from RNA-Seq data.