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Article

A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings

Department of Biotechnology, University of Verona, 37134 Verona, Italy
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Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(23), 9177; https://doi.org/10.3390/ijms21239177
Received: 13 October 2020 / Revised: 20 November 2020 / Accepted: 27 November 2020 / Published: 1 December 2020
(This article belongs to the Special Issue Functional Genomics in Health and Disease)
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 APOE 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. View Full-Text
Keywords: nanopore sequencing; variant calling; haplotype phasing; diagnostic testing nanopore sequencing; variant calling; haplotype phasing; diagnostic testing
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MDPI and ACS Style

Maestri, S.; Maturo, M.G.; Cosentino, E.; Marcolungo, L.; Iadarola, B.; Fortunati, E.; Rossato, M.; Delledonne, M. A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings. Int. J. Mol. Sci. 2020, 21, 9177. https://doi.org/10.3390/ijms21239177

AMA Style

Maestri S, Maturo MG, Cosentino E, Marcolungo L, Iadarola B, Fortunati E, Rossato M, Delledonne M. A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings. International Journal of Molecular Sciences. 2020; 21(23):9177. https://doi.org/10.3390/ijms21239177

Chicago/Turabian Style

Maestri, Simone, Maria G. Maturo, Emanuela Cosentino, Luca Marcolungo, Barbara Iadarola, Elisabetta Fortunati, Marzia Rossato, and Massimo Delledonne. 2020. "A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings" International Journal of Molecular Sciences 21, no. 23: 9177. https://doi.org/10.3390/ijms21239177

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