A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings
The reconstruction of individual haplotypes can facilitate the interpretation of disease risks; however, high costs and technical challenges still hinder their assessment in clinical settings. Second-generation sequencing is the gold standard for variant discovery but, due to the production of short...
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MDPI AG
2020-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/21/23/9177 |
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| author | Simone Maestri Maria Giovanna Maturo Emanuela Cosentino Luca Marcolungo Barbara Iadarola Elisabetta Fortunati Marzia Rossato Massimo Delledonne |
| author_facet | Simone Maestri Maria Giovanna Maturo Emanuela Cosentino Luca Marcolungo Barbara Iadarola Elisabetta Fortunati Marzia Rossato Massimo Delledonne |
| author_sort | Simone Maestri |
| collection | DOAJ |
| description | The reconstruction of individual haplotypes can facilitate the interpretation of disease risks; however, high costs and technical challenges still hinder their assessment in clinical settings. Second-generation sequencing is the gold standard for variant discovery but, due to the production of short reads covering small genomic regions, allows only indirect haplotyping based on statistical methods. In contrast, third-generation methods such as the nanopore sequencing platform developed by Oxford Nanopore Technologies (ONT) generate long reads that can be used for direct haplotyping, with fewer drawbacks. However, robust standards for variant phasing in ONT-based target resequencing efforts are not yet available. In this study, we presented a streamlined proof-of-concept workflow for variant calling and phasing based on ONT data in a clinically relevant 12-kb region of the <i>APOE</i> locus, a hotspot for variants and haplotypes associated with aging-related diseases and longevity. Starting with sequencing data from simple amplicons of the target locus, we demonstrated that ONT data allow for reliable single-nucleotide variant (SNV) calling and phasing from as little as 60 reads, although the recognition of indels is less efficient. Even so, we identified the best combination of ONT read sets (600) and software (BWA/Minimap2 and HapCUT2) that enables full haplotype reconstruction when both SNVs and indels have been identified previously using a highly-accurate sequencing platform. In conclusion, we established a rapid and inexpensive workflow for variant phasing based on ONT long reads. This allowed for the analysis of multiple samples in parallel and can easily be implemented in routine clinical practice, including diagnostic testing. |
| first_indexed | 2024-03-10T14:24:15Z |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T14:24:15Z |
| publishDate | 2020-12-01 |
| publisher | MDPI AG |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-624d9da92bd24abbb69b9123fcaf52bb2023-11-20T23:09:45ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-12-012123917710.3390/ijms21239177A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical SettingsSimone Maestri0Maria Giovanna Maturo1Emanuela Cosentino2Luca Marcolungo3Barbara Iadarola4Elisabetta Fortunati5Marzia Rossato6Massimo Delledonne7Department of Biotechnology, University of Verona, 37134 Verona, ItalyDepartment of Biotechnology, University of Verona, 37134 Verona, ItalyDepartment of Biotechnology, University of Verona, 37134 Verona, ItalyDepartment of Biotechnology, University of Verona, 37134 Verona, ItalyDepartment of Biotechnology, University of Verona, 37134 Verona, ItalyDepartment of Biotechnology, University of Verona, 37134 Verona, ItalyDepartment of Biotechnology, University of Verona, 37134 Verona, ItalyDepartment of Biotechnology, University of Verona, 37134 Verona, ItalyThe reconstruction of individual haplotypes can facilitate the interpretation of disease risks; however, high costs and technical challenges still hinder their assessment in clinical settings. Second-generation sequencing is the gold standard for variant discovery but, due to the production of short reads covering small genomic regions, allows only indirect haplotyping based on statistical methods. In contrast, third-generation methods such as the nanopore sequencing platform developed by Oxford Nanopore Technologies (ONT) generate long reads that can be used for direct haplotyping, with fewer drawbacks. However, robust standards for variant phasing in ONT-based target resequencing efforts are not yet available. In this study, we presented a streamlined proof-of-concept workflow for variant calling and phasing based on ONT data in a clinically relevant 12-kb region of the <i>APOE</i> locus, a hotspot for variants and haplotypes associated with aging-related diseases and longevity. Starting with sequencing data from simple amplicons of the target locus, we demonstrated that ONT data allow for reliable single-nucleotide variant (SNV) calling and phasing from as little as 60 reads, although the recognition of indels is less efficient. Even so, we identified the best combination of ONT read sets (600) and software (BWA/Minimap2 and HapCUT2) that enables full haplotype reconstruction when both SNVs and indels have been identified previously using a highly-accurate sequencing platform. In conclusion, we established a rapid and inexpensive workflow for variant phasing based on ONT long reads. This allowed for the analysis of multiple samples in parallel and can easily be implemented in routine clinical practice, including diagnostic testing.https://www.mdpi.com/1422-0067/21/23/9177nanopore sequencingvariant callinghaplotype phasingdiagnostic testing |
| spellingShingle | Simone Maestri Maria Giovanna Maturo Emanuela Cosentino Luca Marcolungo Barbara Iadarola Elisabetta Fortunati Marzia Rossato Massimo Delledonne A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings International Journal of Molecular Sciences nanopore sequencing variant calling haplotype phasing diagnostic testing |
| title | A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings |
| title_full | A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings |
| title_fullStr | A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings |
| title_full_unstemmed | A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings |
| title_short | A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings |
| title_sort | long read sequencing approach for direct haplotype phasing in clinical settings |
| topic | nanopore sequencing variant calling haplotype phasing diagnostic testing |
| url | https://www.mdpi.com/1422-0067/21/23/9177 |
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