Abstract
Identification of human leukocyte antigen (HLA) alleles from next-generation sequencing (NGS) data is challenging because of the high polymorphism and mosaic nature of HLA genes. Owing to the complex nature of HLA genes and consequent challenges in allele assignment, Oxford Nanopore Technologies’ (ONT) single-molecule sequencing technology has been of great interest due to its fitness for sequencing long reads. In addition to the read length, ONT’s advantages are its portability and possibility for a rapid real-time sequencing, which enables a simultaneous data analysis. Here, we describe a targeted RNA-based method for HLA typing using ONT sequencing and SeqNext-HLA SeqPilot software (JSI Medical Systems GmbH). Twelve classical HLA genes were enriched from cDNA of 50 individuals, barcoded, pooled, and sequenced in 10 MinION R9.4 SpotON flow cell runs producing over 30,000 reads per sample. Using barcoded 2D reads, SeqPilot assigned HLA alleles to two-field typing resolution or higher with the average read depth of 1750x. Sequence analysis resulted in 99–100% accuracy at low-resolution level (one-field) and in 74–100% accuracy at high-resolution level (two-field) with the expected alleles. There are still some limitations with ONT RNA sequencing, such as noisy reads, homopolymer errors, and the lack of robust algorithms, which interfere with confident allele assignment. These issues need to be inspected carefully in the future to improve the allele call rates. Nevertheless, here we show that sequencing of multiplexed cDNA amplicon libraries on ONT MinION can produce accurate high-resolution typing results of 12 classical HLA loci. For HLA research, ONT RNA sequencing is a promising method due to its capability to sequence full-length HLA transcripts. In addition to HLA genotyping, the technique could also be applied for simultaneous expression analysis.
Highlights
The human leukocyte antigen (HLA) complex on the short arm of chromosome 6 (6p21.3) is the most polymorphic region in the human genome with over 26,000 known alleles reported by the IPD IMGT/HLA database (Release 3.41.21)
Oxford Nanopore Technologies (ONT) was the first reported next-generation sequencing (NGS) HLA typing method tested for deceased donor allocation (De Santis et al, 2020)
We developed a method, which applied a template switching oligo (TSO) in the reverse transcription (RT) of RNA molecules into cDNA
Summary
The human leukocyte antigen (HLA) complex on the short arm of chromosome 6 (6p21.3) is the most polymorphic region in the human genome with over 26,000 known alleles reported by the IPD IMGT/HLA database (Release 3.41.21). It is extensively studied, and associated with various infectious and autoimmune diseases (Liu et al, 2016; Karnes et al, 2017; Hirata et al, 2019), and transplantation outcomes (Petersdorf et al, 2015). Longer reads might ease the alignment against HLA alleles and less frequently lead to ambiguous typing results Both of these longread sequencing platforms have already proved themselves as an option in HLA research. In addition to genomic sequencing data, ONT was used to genotype HLA class I loci and to determine their gene-level expression from RNA sequencing (RNA-Seq) reads (Montgomery et al, 2020)
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