Full-Length HLA Class I Genotyping with the MinION Nanopore Sequencer

Lang, Kathrin; Surendranath, Vineeth; Quenzel, Philipp; Schöfl, Gerhard; Schmidt, Alexander H.; Lange, Vinzenz

doi:10.1007/978-1-4939-8546-3_10

Cited by 22 publications

(14 citation statements)

References 9 publications

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“…For accurate surveillance and vaccine subpopulation selection, a highly precise sequencing and variant calling method is always required. In particular, the recall and precision rate of the current MinION sequence technology were inferior to those of the Illumina MiSeq platform, and these limitations were similar to findings from other studies on genotyping using the MinION sequencer ( Wang et al, 2015 ; Cornelis et al, 2017 ; Lang et al, 2018 ; Runtuwene et al, 2018 ). Thus, our method is currently unsuitable to replace the Illumina platform.…”

Section: Discussionsupporting

confidence: 82%

Whole Genome Sequencing of Influenza A and B Viruses With the MinION Sequencer in the Clinical Setting: A Pilot Study

et al. 2018

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Introduction: Whole genome sequencing (WGS) of influenza viruses is important for preparing vaccines and coping with newly emerging viruses. However, WGS is difficult to perform using conventional next-generation sequencers in developing countries, where facilities are often inadequate. In this study, we developed a high-throughput WGS method for influenza viruses in clinical specimens with the MinION portable sequencer.Methods: Whole genomes of influenza A and B viruses were amplified by multiplex RT-PCR from 13 clinical specimens collected in Tokyo, Japan. Barcode tags for multiplex MinION sequencing were added with each multiplex RT-PCR amplicon by nested PCR with custom barcoded primers. All barcoded amplicons were mixed and multiplex sequencing using the MinION sequencer with 1D2 sequencing kit. In addition, multiplex RT-PCR amplicons generated from each clinical specimen were sequenced using the Illumina MiSeq platform to validate the performance of MinION sequencer. The accuracy, recall, and precision rates of MinION sequencing were calculated by comparing the results of variant calling in the Illumina MiSeq platform and MinION sequencer.Results: Whole genomes of influenza A and B viruses were successfully amplified by multiplex RT-PCR from 13 clinical samples. We identified 6 samples as influenza type A virus H3N2 subtype and 7 as influenza B virus Yamagata lineage using the Illumina MiSeq platform. The overall accuracy, recall, and precision rates of the MinION sequencer were, respectively 99.95%, 89.41%, and 97.88% from 1D reads and 99.97%, 93.28%, and 99.86% from 1D2 reads.Conclusion: We developed a novel WGS method for influenza A and B viruses. It is necessary to improve read accuracy and analytical tools in order to better utilize the MinION sequencer for real-time monitoring of genetic rearrangements and for evaluation of newly emerging viruses.

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Section: Discussionsupporting

confidence: 82%

Whole Genome Sequencing of Influenza A and B Viruses With the MinION Sequencer in the Clinical Setting: A Pilot Study

et al. 2018

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“…One of the first platforms used for HLA typing was the 454/GS FLX sequencer (Roche), which has been progressively supplanted by Illumina and Ion Torrent technologies . Advanced technologies are also used, such as Pacific Biosciences's single‐molecule real‐time sequencing and Oxford Nanopore single‐cell DNA template strand sequencing …”

Section: Introductionmentioning

confidence: 99%

“…[21][22][23][24] Advanced technologies are also used, such as Pacific Biosciences's single-molecule real-time sequencing [25][26][27][28][29][30] and Oxford Nanopore single-cell DNA template strand sequencing. [31][32][33] We recently implemented an NGS method for HLA typing using the AllType NGS kit from One Lambda and Thermo Fisher Scientific reagents on the Ion S5 XL platform. The objective of this study was to evaluate the performance of this method by systematic analysis of warnings given by the analysis software and extensive comparison of HLA typing results obtained via other methods and/or knowledge of common HLA associations as a consequence of linkage disequilibrium.…”

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confidence: 99%

Evaluation of the AllType kit for HLA typing using the Ion Torrent S5 XL platform

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Ralazamahaleo

Blouin

et al. 2019

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HLA genotyping by next‐generation sequencing is now widely performed. We aimed at evaluating the performance of the One Lambda AllType kit using Thermo Fisher Scientific reagents on the Ion S5 XL platform. Reads were analyzed using the TypeStream Visual software. We performed 15 runs between April and September 2018 to type DNA at the HLA‐A/B/C/DRB1/3/4/5/DQA1/DQB1/DPA1/DPB1 loci from 340 samples and 15 positive controls. We observed only seven (0.1%) critical mistakes among the 6009 alleles typed, corresponding to two allele dropouts, one false heterozygous typing assignment, and four phasing abnormalities. Among the 1793 presumably new alleles detected by the analysis software, 11 displayed exon mismatches, of which nine were confirmed as new alleles and two had been described previously. Intron mismatches were observed among the remaining presumably new alleles, of which 371 were considered as probably new, and 1411 were rejected for at least one sequence feature such as homopolymers (n = 1206), nucleotide doublet repeats (n = 26), low read depth (<200 reads, n = 93), high background (>20%, n = 79), or phasing abnormalities (n = 7). A comparison of the AllType results with those obtained using other methods at the second‐field resolution level showed 99.5% (1497/1504) concordance for the HLA‐A/B/C/DRB1/DQB1/DPB1 loci. Similar agreement was observed between the HLA‐C or HLA‐DRB3/4/5 results and common linkage disequilibrium, with 96.6% (657/680) and 97.2% (530/545) concordance, respectively. Therefore, the AllType kit used with the Ion S5 XL platform displayed satisfactory performance for HLA typing in current clinical practice.

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“…This feature simplifies the library preparation and enhances the capability of phasing distant variants. Oxford Nanopore Technologies may offer another option for implementing the "long-amplicon, long-reads" approach for HLA typing [30][31][32], although better bioinformatics tools are needed to overcome the high error rate of the nanopore sequencing reads. On the other end of the spectrum, Lange and colleagues took the "short-amplicon, short-reads" approach and developed a workflow to sequence selected exons only on the Illumina platform.…”

Section: Plos Onementioning

confidence: 99%

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

et al. 2020

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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 transplantrelevant 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

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Full-Length HLA Class I Genotyping with the MinION Nanopore Sequencer

Cited by 22 publications

References 9 publications

Whole Genome Sequencing of Influenza A and B Viruses With the MinION Sequencer in the Clinical Setting: A Pilot Study

Whole Genome Sequencing of Influenza A and B Viruses With the MinION Sequencer in the Clinical Setting: A Pilot Study

Evaluation of the AllType kit for HLA typing using the Ion Torrent S5 XL platform

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

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