(203) Identification of a Potential Novel HLA-B Allele with a Large Exonic Deletion Using HybriType™ and AllType Rapid™ Sequencing with TypeStream™ Visual

Aim: New, probe-based methodologies in NGS have lower bias and background noise compared to LR-PCR based methods resulting in the ability to resolve previously challenging or unresolvable samples. With long-read sequencing, large-scale novel features that were difficult to resolve with NGS can be directly observed in HLA typing. Here, a potential novel HLA-B*55 allele with a large deletion spanning the end of intron 3 and most of exon 4 was identified using the HybriType and AllType Rapid assays.

Methods: Whole blood DNA was extracted using the Chemagic DNA Blood kit paired with the Chemagic Prime 8 automation system. HybriType and AllType Rapid libraries were prepared following the manufacturer protocol and sequencing files were processed in TypeStream Visual (TSV) version 3.2 using default analysis parameters.

Results: Results using a LR-PCR method suggested the HLA-B locus type of B*44:03:02, B*55:02:05. However, a 90% decrease in coverage for the B*55 allele and high background positions were noted in exon 4. Result was repeated using HybriType, a probe-based hybrid capture enrichment assay, resulting in a typing of B*44:03:02:04, B*55[NEW] with novel variants in exon 4 matching the B*44:03:02 sequence. Importantly, an instant drop in coverage starting at I3-486 and ending at E4-822 was observed by using the TSV read viewer suggesting the presence of a deletion too large to be directly detected using NGS. Result was confirmed using AllType Rapid, a long-read sequencing assay, resulting in a homozygous type of B*55[NEW] with multiple novel variants and high background positions matching the B*44:03:02 sequence. No drop in coverage was observed from the previously mentioned region. However, many reads were observed containing a large deletion sequence in the region. Using the TSV sequence search, the HybriType library was found to contain many reads with the deletion sequence. The TSV IMGT database was updated with the new allele sequence and reanalyzed to confirm the correct call of the novel B*55 allele.

Conclusion: A potential novel HLA-B allele containing a large deletion in exon 4 was identified using HybriType, AllType Rapid, and TSV analysis software.