Motivation: The human leukocyte antigen (HLA) gene cluster plays a crucial role in adaptive immunity and is thus relevant in many biomedical applications. While next-generation sequencing data are often available for a patient, deducing the HLA genotype is difficult because of substantial sequence similarity within the cluster and exceptionally high variability of the loci. Established approaches, therefore, rely on specific HLA enrichment and sequencing techniques, coming at an additional cost and extra turnaround time. Result: We present OptiType, a novel HLA genotyping algorithm based on integer linear programming, capable of producing accurate predictions from NGS data not specifically enriched for the HLA cluster. We also present a comprehensive benchmark dataset consisting of RNA, exome and whole-genome sequencing data. OptiType significantly outperformed previously published in silico approaches with an overall accuracy of 97% enabling its use in a broad range of

/pdf/optitype-precision-hla-typing-from-next-generation-2ljlnroqrr.pdf

OptiType: precision HLA typing from next-generation sequencing data.

A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing. We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed “neXtype” for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week. The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration.

/pdf/cost-efficient-high-throughput-hla-typing-by-miseq-amplicon-jochsidnxj.pdf

Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing

In the past decade, the development of next-generation sequencing (NGS) has paved the way for whole-genome analysis in individuals. Research on the human leukocyte antigen (HLA), an extensively studied molecule involved in immunity, has benefitted from NGS technologies. The HLA region, a 3.6-Mb segment of the human genome at 6p21, has been associated with more than 100 different diseases, primarily autoimmune diseases. Recently, the HLA region has received much attention because severe adverse effects of various drugs are associated with particular HLA alleles. Owing to the complex nature of the HLA genes, classical direct sequencing methods cannot comprehensively elucidate the genomic makeup of HLA genes. Thus far, several high-throughput HLA-typing methods using NGS have been developed. In HLA research, NGS facilitates complete HLA sequencing and is expected to improve our understanding of the mechanisms through which HLA genes are modulated, including transcription, regulation of gene expression and epigenetics. Most importantly, NGS may also permit the analysis of HLA-omics. In this review, we summarize the impact of NGS on HLA research, with a focus on the potential for clinical applications.

/pdf/the-impact-of-next-generation-sequencing-technologies-on-hla-4k9jln6mid.pdf

The impact of next-generation sequencing technologies on HLA research.

Allele-level resolution data at primary HLA typing is the ideal for most histocompatibility testing laboratories. Many high-throughput molecular HLA typing approaches are unable to determine the phase of observed DNA sequence polymorphisms, leading to ambiguous results. The use of higher resolution methods is often restricted due to cost and time limitations. Here we report on the feasibility of using Pacific Biosciences’ Single Molecule Real-Time (SMRT) DNA sequencing technology for high-resolution and high-throughput HLA typing. Seven DNA samples were typed for HLA-A, -B and -C. The results showed that SMRT DNA sequencing technology was able to generate sequences that spanned entire HLA Class I genes that allowed for accurate allele calling. Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. This method has the potential to revolutionize the field of HLA typing. The clinical impact of achieving this level of resolution HLA typing data is likely to considerable, particularly in applications such as organ and blood stem cell transplantation where matching donors and recipients for their HLA is of utmost importance.

/pdf/hla-typing-for-the-next-generation-1dq7fjea3f.pdf

HLA Typing for the Next Generation.

The human leukocyte antigen (HLA) region, the 3.8-Mb segment of the human genome at 6p21, has been associated with more than 100 different diseases, mostly autoimmune diseases. Due to the complex nature of HLA genes, there are difficulties in elucidating complete HLA gene sequences especially HLA gene haplotype structures by the conventional sequencing method. We propose a novel, accurate, and cost-effective method for generating phase-defined complete sequencing of HLA genes by using indexed multiplex next generation sequencing. A total of 33 HLA homozygous samples, 11 HLA heterozygous samples, and 3 parents-child families were subjected to phase-defined HLA gene sequencing. We applied long-range PCR to amplify six HLA genes (HLA-A, -C, -B, DRB1, -DQB1, and –DPB1) followed by transposase-based library construction and multiplex sequencing with the MiSeq sequencer. Paired-end reads (2 × 250 bp) derived from the sequencer were aligned to the six HLA gene segments of UCSC hg19 allowing at most 80 bases mismatch. For HLA homozygous samples, the six amplicons of an individual were pooled and simultaneously sequenced and mapped as an individual-tagging method. The paired-end reads were aligned to corresponding genes of UCSC hg19 and unambiguous, continuous sequences were obtained. For HLA heterozygous samples, each amplicon was separately sequenced and mapped as a gene-tagging method. After alignments, we detected informative paired-end reads harboring SNVs on both forward and reverse reads that are used to separate two chromosomes and to generate two phase-defined sequences in an individual. Consequently, we were able to determine the phase-defined HLA gene sequences from promoter to 3′-UTR and assign up to 8-digit HLA allele numbers, regardless of whether the alleles are rare or novel. Parent–child trio-based sequencing validated our sequencing and phasing methods. Our protocol generated phased-defined sequences of the entire HLA genes, resulting in high resolution HLA typing and new allele detection.

https://link.springer.com/content/pdf/10.1186%2F1471-2164-14-355.pdf

Phase-defined complete sequencing of the HLA genes by next-generation sequencing

Current human leukocyte antigen (HLA) DNA typing methods such as the sequence-based typing (SBT) and sequence-specific oligonucleotide (SSO) methods generally yield ambiguous typing results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment. Here we describe the development and application of the super high-resolution single-molecule sequence-based typing (SS-SBT) of HLA loci at the 8-digit level using next generation sequencing (NGS). NGS which can determine an HLA allele sequence derived from a single DNA molecule is expected to solve the phase ambiguity problem. Eight classical HLA loci-specific polymerase chain reaction (PCR) primers were designed to amplify the entire gene sequences from the enhancer-promoter region to the 3' untranslated region. Phase ambiguities of HLA-A, -B, -C, -DRB1 and -DQB1 were completely resolved and unequivocally assigned without ambiguity to single HLA alleles. Therefore, the SS-SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity.

O Takahashi

Papers

Super high resolution for single molecule‐sequence‐based typing of classical HLA loci at the 8‐digit level using next generation sequencers