Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes

Show simple item record Johansson, Tiira Koskela, Satu Yohannes, Dawit A. Partanen, Jukka Saavalainen, Paivi 2021-04-26T06:14:01Z 2021-04-26T06:14:01Z 2021-03-04
dc.identifier.citation Johansson , T , Koskela , S , Yohannes , D A , Partanen , J & Saavalainen , P 2021 , ' Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes ' , Frontiers in Genetics , vol. 12 , 635601 .
dc.identifier.other PURE: 162659747
dc.identifier.other PURE UUID: b21e6023-9129-4357-ad1d-82d54e1a8bd5
dc.identifier.other WOS: 000629978100001
dc.description.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. en
dc.format.extent 10
dc.language.iso eng
dc.relation.ispartof Frontiers in Genetics
dc.rights cc_by
dc.rights.uri info:eu-repo/semantics/openAccess
dc.subject human leukocyte antigen
dc.subject HLA genotyping
dc.subject nanopore sequencing
dc.subject RNA sequencing
dc.subject MinION
dc.subject 1184 Genetics, developmental biology, physiology
dc.title Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes en
dc.type Article
dc.contributor.organization Immunomics
dc.contributor.organization Biosciences
dc.contributor.organization TRIMM - Translational Immunology Research Program
dc.contributor.organization Department of Medical and Clinical Genetics
dc.contributor.organization University of Helsinki
dc.contributor.organization Research Programs Unit
dc.description.reviewstatus Peer reviewed
dc.relation.issn 1664-8021
dc.rights.accesslevel openAccess
dc.type.version publishedVersion

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