Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers: Comparison with Sanger sequencing

Yamamoto, Fumiko; Höglund, Bryan; Fernández-Viña, Marcelo; Tyan, Dolly B.; Rastrou, Melinda; Williams, T. C.; Moonsamy, P. V.; Goodridge, Damian; Anderson, Matthew W.; Erlich, Henry A.; Holcomb, Cherie L.

doi:10.1016/j.humimm.2015.05.002

Cited by 6 publications

(2 citation statements)

References 14 publications

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“…Preliminary work shows promise in obtaining accurate class I allele calling (31). Much of the early work on HLA NGS genotyping was performed on the Roche 454 (26,(32)(33)(34)(35)(36)(37)(38)(39); however, Roche will not be supporting this instrument after 2016. The Thermo Fisher Ion Torrent's most desirable feature is its short run time of 2 h although this run-time savings must first be preceded by a 5-h emulsion PCR step and several other labor-intensive steps to prepare the Ion chip for loading.…”

Section: Discussionmentioning

confidence: 99%

HLA genotyping in the clinical laboratory: comparison of next‐generation sequencing methods

Profaizer

Lázár‐Molnár

et al. 2016

HLA

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Implementation of human leukocyte antigen (HLA) genotyping by next-generation sequencing (NGS) in the clinical lab brings new challenges to the laboratories performing this testing. With the advent of commercially available HLA-NGS typing kits, labs must make numerous decisions concerning capital equipment and address labor considerations. Therefore, careful and unbiased evaluation of available methods is imperative. In this report, we compared our in-house developed HLA NGS typing with two commercially available kits from Illumina and Omixon using 10 International Histocompatibility Working Group (IHWG) and 36 clinical samples. Although all three methods employ long range polymerase chain reaction (PCR) and have been developed on the Illumina MiSeq platform, the methodologies for library preparation show significant variations. There was 100% typing concordance between all three methods at the first field when a HLA type could be assigned. Overall, HLA typing by NGS using in-house or commercially available methods is now feasible in clinical laboratories. However, technical variables such as hands-on time and indexing strategies are sufficiently different among these approaches to impact the workflow of the clinical laboratory.

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Section: Discussionmentioning

confidence: 99%

HLA genotyping in the clinical laboratory: comparison of next‐generation sequencing methods

Profaizer

Lázár‐Molnár

et al. 2016

HLA

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“…Here, we compare a next generation sequencing (NGS) technology with SSOP for accuracy, effort, turnaround time, and level of resolution for genotyping of 11 HLA loci among 289 specimens from five clinical laboratories. While initial NGS systems involved cumbersome 4‐ to 5‐day procedures, the NGS kit used in this study allowed typing to be completed with a 2‐day TAT, with all bench work and sequencer instrument loading accomplished on day 1. Compared with SSOP, this NGS protocol decreased use of consumables, reduced tech‐to‐tech variability, and increased the level of typing resolution at all loci.…”

Section: Introductionmentioning

confidence: 99%

Comparison of sequence‐specific oligonucleotide probe vs next generation sequencing for HLA‐A, B, C, DRB1, DRB3/B4/B5, DQA1, DQB1, DPA1, and DPB1 typing: Toward single‐pass high‐resolution HLA typing in support of solid organ and hematopoietic cell transplant programs

Smith

Pereira

Jaramillo

et al. 2019

HLA

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Many clinical laboratories supporting solid organ transplant programs use multiple HLA genotyping technologies, depending on individual laboratory needs. Sequence‐specific primers and quantitative polymerase chain reaction (qPCR) serve the rapid turnaround necessary for deceased donor workup, while sequence‐specific oligonucleotide probe (SSOP) technology is widely employed for higher volumes. When clinical need mandates high‐resolution data, Sanger sequencing‐based typing (SBT) has been the “gold standard.” However, all those methods commonly yield ambiguous typing results that utilize valuable laboratory resources when resolution is required. In solid organ transplantation, high‐resolution typing may provide critical information for highly sensitized patients with donor‐specific anti‐HLA antibodies (DSA), particularly when DSA involve HLA alleles not discriminated by SSOP typing. Arguments against routine use of SBT include assay complexity, long turnaround times (TAT), and increased costs. Here, we compare a next generation sequencing (NGS) technology with SSOP for accuracy, effort, turnaround time, and level of resolution for genotyping of 11 HLA loci among 289 specimens from five clinical laboratories. Results were concordant except for SSOP misassignments in eight specimens and 21 novel sequences uniquely identified by NGS. With few exceptions, SSOP generated ambiguous results while NGS provided unambiguous three‐field allele assignments. For complete HLA genotyping of up to 24 samples by either SSOP or NGS, bench work was completed on day 1 and typing results were available on day 2. This study provides compelling evidence that, although not viable for STAT typing of deceased donors, a single‐pass NGS HLA typing method has direct application for solid organ transplantation.

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Next-Generation Sequencing of the HLA locus: Methods and impacts on HLA typing, population genetics and disease association studies

2016

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Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers: Comparison with Sanger sequencing

Cited by 6 publications

References 14 publications

HLA genotyping in the clinical laboratory: comparison of next‐generation sequencing methods

HLA genotyping in the clinical laboratory: comparison of next‐generation sequencing methods

Comparison of sequence‐specific oligonucleotide probe vs next generation sequencing for HLA‐A, B, C, DRB1, DRB3/B4/B5, DQA1, DQB1, DPA1, and DPB1 typing: Toward single‐pass high‐resolution HLA typing in support of solid organ and hematopoietic cell transplant programs

Next-Generation Sequencing of the HLA locus: Methods and impacts on HLA typing, population genetics and disease association studies

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