PMID- 33763116
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20210326
IS  - 1664-8021 (Print)
IS  - 1664-8021 (Electronic)
IS  - 1664-8021 (Linking)
VI  - 12
DP  - 2021
TI  - Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes.
PG  - 635601
LID - 10.3389/fgene.2021.635601 [doi]
LID - 635601
AB  - 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.
CI  - Copyright (c) 2021 Johansson, Koskela, Yohannes, Partanen and Saavalainen.
FAU - Johansson, Tiira
AU  - Johansson T
AD  - Translational Immunology Research Program and Department of Medical and Clinical 
      Genetics, University of Helsinki, Helsinki, Finland.
AD  - Finnish Red Cross Blood Service, Helsinki, Finland.
FAU - Koskela, Satu
AU  - Koskela S
AD  - Finnish Red Cross Blood Service, Helsinki, Finland.
FAU - Yohannes, Dawit A
AU  - Yohannes DA
AD  - Translational Immunology Research Program and Department of Medical and Clinical 
      Genetics, University of Helsinki, Helsinki, Finland.
FAU - Partanen, Jukka
AU  - Partanen J
AD  - Finnish Red Cross Blood Service, Helsinki, Finland.
FAU - Saavalainen, Paivi
AU  - Saavalainen P
AD  - Translational Immunology Research Program and Department of Medical and Clinical 
      Genetics, University of Helsinki, Helsinki, Finland.
AD  - Finnish Red Cross Blood Service, Helsinki, Finland.
LA  - eng
PT  - Journal Article
DEP - 20210304
PL  - Switzerland
TA  - Front Genet
JT  - Frontiers in genetics
JID - 101560621
PMC - PMC7982845
OTO - NOTNLM
OT  - HLA genotyping
OT  - MinION
OT  - RNA sequencing
OT  - human leukocyte antigen
OT  - nanopore sequencing
COIS- The authors declare that the research was conducted in the absence of any 
      commercial or financial relationships that could be construed as a potential 
      conflict of interest.
EDAT- 2021/03/26 06:00
MHDA- 2021/03/26 06:01
PMCR- 2021/03/04
CRDT- 2021/03/25 06:27
PHST- 2020/11/30 00:00 [received]
PHST- 2021/02/11 00:00 [accepted]
PHST- 2021/03/25 06:27 [entrez]
PHST- 2021/03/26 06:00 [pubmed]
PHST- 2021/03/26 06:01 [medline]
PHST- 2021/03/04 00:00 [pmc-release]
AID - 10.3389/fgene.2021.635601 [doi]
PST - epublish
SO  - Front Genet. 2021 Mar 4;12:635601. doi: 10.3389/fgene.2021.635601. eCollection 
      2021.