Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes
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’ (O...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2021-03-01
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| Series: | Frontiers in Genetics |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fgene.2021.635601/full |
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| author | Tiira Johansson Tiira Johansson Satu Koskela Dawit A. Yohannes Jukka Partanen Päivi Saavalainen Päivi Saavalainen |
| author_facet | Tiira Johansson Tiira Johansson Satu Koskela Dawit A. Yohannes Jukka Partanen Päivi Saavalainen Päivi Saavalainen |
| author_sort | Tiira Johansson |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-16T17:07:29Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1664-8021 |
| language | English |
| last_indexed | 2024-12-16T17:07:29Z |
| publishDate | 2021-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Genetics |
| spelling | doaj.art-10a3989b593d4ced9e8c949842eb8aa82022-12-21T22:23:31ZengFrontiers Media S.A.Frontiers in Genetics1664-80212021-03-011210.3389/fgene.2021.635601635601Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA GenesTiira Johansson0Tiira Johansson1Satu Koskela2Dawit A. Yohannes3Jukka Partanen4Päivi Saavalainen5Päivi Saavalainen6Translational Immunology Research Program and Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, FinlandFinnish Red Cross Blood Service, Helsinki, FinlandFinnish Red Cross Blood Service, Helsinki, FinlandTranslational Immunology Research Program and Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, FinlandFinnish Red Cross Blood Service, Helsinki, FinlandTranslational Immunology Research Program and Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, FinlandFinnish Red Cross Blood Service, Helsinki, FinlandIdentification 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.https://www.frontiersin.org/articles/10.3389/fgene.2021.635601/fullhuman leukocyte antigenHLA genotypingnanopore sequencingRNA sequencingMinION |
| spellingShingle | Tiira Johansson Tiira Johansson Satu Koskela Dawit A. Yohannes Jukka Partanen Päivi Saavalainen Päivi Saavalainen Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes Frontiers in Genetics human leukocyte antigen HLA genotyping nanopore sequencing RNA sequencing MinION |
| title | Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes |
| title_full | Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes |
| title_fullStr | Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes |
| title_full_unstemmed | Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes |
| title_short | Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes |
| title_sort | targeted rna based oxford nanopore sequencing for typing 12 classical hla genes |
| topic | human leukocyte antigen HLA genotyping nanopore sequencing RNA sequencing MinION |
| url | https://www.frontiersin.org/articles/10.3389/fgene.2021.635601/full |
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