Allele-specific quantification of human leukocyte antigen transcript isoforms by nanopore sequencing.

Introduction: While tens of thousands of HLA alleles have been identified by DNA sequencing, the contribution of alternative splicing to HLA diversity is not well characterized. In this study, we sought to determine if long-read sequencing could be used to accurately quantify allele-specific HLA transcripts in primary human lymphocytes. Methods: cDNA libraries were prepared from peripheral blood lymphocytes from 12 donors and sequenced by nanopore long-read sequencing. HLA reads were aligned to donor-specific reference sequences based on the known type of each donor. Allele-specific exon utilization was calculated as the proportion of reads aligning to each allele containing known exons, and transcript isoforms were quantified based on patterns of exon utilization within individual reads. Results: Splice variants were rare among class I HLA genes (median exon retention rate 99%-100%), except for several HLA-C alleles with exon 5 spliced out of up to 15% of reads. Splice variants were also rare among class II HLA genes (median exon retention rate 98%-100%), except for HLA-DQB1. Consistent with previous work, exon 5 of HLA-DQB1 was spliced out in alleles with a mutated splice acceptor site at rs28688207. Surprisingly, a 28% loss of exon 5 was also observed in HLA-DQB1 alleles with an intact splice acceptor site at rs28688207. Discussion: We describe a simple bioinformatic workflow to quantify allele-specific expression of HLA transcript isoforms. Further studies are warranted to characterize the repertoire of HLA transcripts expressed in different cell types and tissues across diverse populations.

Unique Molecular Identifier-Based High-Resolution HLA Typing and Transcript Quantitation Using Long-Read Sequencing.

HLA typing provides essential results for stem cell and solid organ transplants, as well as providing diagnostic benefits for various rheumatology, gastroenterology, neurology, and infectious diseases. It is becoming increasingly clear that understanding the expression of patient HLA transcripts can provide additional benefits for many of these same patient groups. Our study cohort was evaluated using a long-read RNA sequencing methodology to provide rapid HLA genotyping results and normalized HLA transcript expression. Our assay used NGSEngine to determine the HLA genotyping result and normalized mRNA transcript expression using Athlon2. The assay demonstrated an excellent concordance rate of 99.7%. Similar to previous studies, for the class I loci, patients demonstrated significantly lower expression of HLA-C than HLA-A and -B (Mann-Whitney U, p value = 0.0065 and p value = 0.0154, respectively). In general, the expression of class II transcripts was lower than that of class I transcripts. This study demonstrates a rapid high-resolution HLA typing assay using RNA-Seq that can provide accurate HLA genotyping and HLA allele-specific transcript expression in 7-8 h, a timeline short enough to perform the assay for deceased donors.

Performance of a multiplexed amplicon-based next-generation sequencing assay for HLA typing.

BACKGROUND: Next-generation sequencing (NGS) has enabled efficient high-resolution typing of human leukocyte antigen (HLA) genes with minimal ambiguity. Most commercially available assays amplify individual or subgroup of HLA genes by long-range PCR followed by library preparation and sequencing. The AllType assay simplifies the workflow by amplifying 11 transplant-relevant HLA genes in one PCR reaction. Here, we report the performance of this unique workflow evaluated using 218 genetically diverse samples. METHODS: Five whole genes (HLA-A/B/C/DQA1/DPA1) and six near-whole genes (HLA-DRB1/DRB345/DQB1/DPB1; excluding exon 1 and part of intron 1) were amplified in a multiplexed, long-range PCR. Manual library preparation was performed per manufacturer's protocol, followed by template preparation and chip loading on the Ion Chef, and sequencing on the Ion S5 sequencer. Pre-specified rules for quality control and repeat testing were followed; technologists were blinded to the reference results. The concordance between AllType and reference results was determined at 2-field resolution. We also describe the ranges of input DNA and library concentrations, read number per sample and per locus, and key health metrics in relation to typing results. RESULTS: The concordance rates were 98.6%, 99.8% and 99.9% at the sample (n = 218), genotype (n = 1688), and allele (n = 3376) levels, respectively. Three genotypes were discordant, all of which shared the same G group typing results with the reference. Most ambiguous genotypes (116 out of 144, 80.6%) were due to the lack of exon 1 and intron 1 coverage for HLA-DRB1/DRB345/DQB1/DPB1 genes. A broad range of input DNA concentrations and library concentrations were tolerated. Per sample read numbers were adequate for accurate genotyping. Per locus read numbers showed some inter-lot variations, and a trend toward improved inter-locus balance was observed with later lots of reagents. CONCLUSION: The AllType assay on the Ion Chef/Ion S5 platform offers a robust and efficient workflow for clinical HLA typing at the 2-field resolution. The multiplex PCR strategy simplifies the laboratory procedure without compromising the typing accuracy.

Using Nanopore Whole-Transcriptome Sequencing for Human Leukocyte Antigen Genotyping and Correlating Donor Human Leukocyte Antigen Expression with Flow Cytometric Crossmatch Results.

Transplant centers are increasingly using virtual crossmatching to evaluate recipient and donor compatibility. However, the current state of virtual crossmatching fails to incorporate donor human leukocyte antigen (HLA) expression in the assessment, despite numerous studies that have demonstrated the impact of donor HLA expression on physical crossmatch outcomes. Whole-transcriptome sequencing (RNA-Seq) for HLA enables simultaneous determination of HLA genotyping and relative HLA expression. Ultimately the RNA-Seq needs to be faster to be incorporated into the virtual crossmatching process. However, to demonstrate feasibility, the utility of the MinION sequencer (Oxford Nanopore Technologies, Oxford, UK) was evaluated in combination with RNA-Seq to generate HLA genotypes and to determine HLA class I expression. Although HLA class I expression varied among individuals, the pattern of HLA expression remained relatively consistent (HLA-B > HLA-A = HLA-C). HLA-A and -C had similar expression profiles. The impact of donor HLA expression was evaluated using serum samples containing a single donor-specific antibody (DSA). By making DSA consistent, donor HLA expression variability could be assessed. With consistent DSA mean fluorescence intensity, there was a direct relationship between the donor HLA expression to which the DSA is against and flow cytometric crossmatch median channel shifts.

Suitability of dried DNA for long-range PCR amplification and HLA typing by next-generation sequencing.

Storage and stable shipment of genomic DNA are of great concern to laboratories that may need to perform testing off archived samples. There are some dry-state storage methods that are available that have the potential to provide a way to store samples at room temperature for long periods of time as well as offer a means to ship DNA to other facilities without the same safety concerns that come with shipping liquid samples. The recovered DNA should be of sufficient integrity such that downstream applications can be performed without concern of the sample quality. This work describes sample properties between two methods of DNA storage, dried (room temperature) and traditional (-80 °C). DNA was evaluated for purity, fragment length, and the ability to generate HLA typing using next-generation sequencing.

Buccal swab genomic DNA fragmentation predicts likelihood of successful HLA genotyping by next-generation sequencing.

Many clinical human leukocyte antigen (HLA) laboratories are adopting next-generation sequencing (NGS) technology for HLA genotyping. There have been several reports of the cost-benefit and reduction in turn-around-time provided by NGS. Ninety-six percent of buccal swabs and peripheral blood samples had reportable HLA genotyping by NGS. The HLA loci most likely to fail genotyping from buccal swabs were DQB1, DPB1, and DPA1. Successful buccal swab samples had significantly less genomic DNA fragmentation compared to buccal swab samples that were unsuccessful. Increasing sequencing depth of coverage for heavily fragmented samples rescued HLA genotyping. This information provides laboratories with a quality assurance parameter that reduces the amount of repeat NGS needed to achieve high-resolution HLA genotyping. This information should further reduce laboratory and patient costs for HLA genotyping.