P139 False homozygosity observed for one sample’s HLA genotyping on two independent next generation sequencing platforms

Abstract

Next generation sequencing (NGS) was performed on a patient sample to genotype the HLA-DRB1 locus (exons 2 and 3) using both the Ion Torrent PGM and Illumina MiSeq platforms. The sample typed as HLA-DRB1∗07:01:01 homozygous on both platforms using either the Twin or TypeSteam software analysis packages. This sample was initially typed by Sequence Specific Oligonucleotide (SSO) hybridization then sequenced using group specific Protrans primers. The SSO and sequencing each typed the sample as 07:01/09:01. Secor reagents were then used to perform Sanger sequencing-based typing (SBT) of exon 2 only. Weak signals were obtained on the SBT forward strand, but the HLA type corroborated the SSO result of 07:01/09:01. Analysis of other samples with 07:01/09:01 allelic combination were found to have genotyped correctly on both NGS platforms. The Staden trev utility program was used to extract Sanger traces from Holotype NGS data. If there were a sufficient number of DRB1∗09 reads, the Staden trev utility program would have allowed for their detection. Unfortunately, the extracted Sanger traces did not definitively show the presence of DRB1∗09. The forward Sanger trace showed evidence of DRB1∗09 in the first ∼60 bases where there are six ambiguous positions between DRB1∗07:01 and 09:01. All six positions demonstrated ambiguities supporting the presence of two alleles. However, starting from position 70, signals were weaker and in the eight remaining positions with expected ambiguities, no conclusive signs of DRB1∗09 were found. Increasing the signal in the utility program allowed for detection of weak ambiguous peaks at all eight of these potentially ambiguous positions. The reverse trace showed stronger support for the presence of DRB1∗09:01, with the ambiguities post position 70 all in evidence. We plan to further sequence the whole DRB1 gene region. This may help illuminate the source of the observed allelic drop out and to rule out mutations that may have impacted the primer binding region for primers designed by two separate manufacturers.