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Genotyping of familial Mediterranean fever gene (MEFV)—Single nucleotide polymorphism—Comparison of Nanopore with conventional Sanger sequencing
BACKGROUND: Through continuous innovation and improvement, Nanopore sequencing has become a powerful technology. Because of its fast processing time, low cost, and ability to generate long reads, this sequencing technique would be particularly suitable for clinical diagnostics. However, its raw data...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8929590/ https://www.ncbi.nlm.nih.gov/pubmed/35298548 http://dx.doi.org/10.1371/journal.pone.0265622 |
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author | Schmidt, Jonas Berghaus, Sandro Blessing, Frithjof Herbeck, Holger Blessing, Josef Schierack, Peter Rödiger, Stefan Roggenbuck, Dirk Wenzel, Folker |
author_facet | Schmidt, Jonas Berghaus, Sandro Blessing, Frithjof Herbeck, Holger Blessing, Josef Schierack, Peter Rödiger, Stefan Roggenbuck, Dirk Wenzel, Folker |
author_sort | Schmidt, Jonas |
collection | PubMed |
description | BACKGROUND: Through continuous innovation and improvement, Nanopore sequencing has become a powerful technology. Because of its fast processing time, low cost, and ability to generate long reads, this sequencing technique would be particularly suitable for clinical diagnostics. However, its raw data accuracy is inferior in contrast to other sequencing technologies. This constraint still results in limited use of Nanopore sequencing in the field of clinical diagnostics and requires further validation and IVD certification. METHODS: We evaluated the performance of latest Nanopore sequencing in combination with a dedicated data-analysis pipeline for single nucleotide polymorphism (SNP) genotyping of the familial Mediterranean fever gene (MEFV) by amplicon sequencing of 47 clinical samples. Mutations in MEFV are associated with Mediterranean fever, a hereditary periodic fever syndrome. Conventional Sanger sequencing, which is commonly applied in clinical genetic diagnostics, was used as a reference method. RESULTS: Nanopore sequencing enabled the sequencing of 10 target regions within MEFV with high read depth (median read depth 7565x) in all samples and identified a total of 435 SNPs in the whole sample collective, of which 29 were unique. Comparison of both sequencing workflows showed a near perfect agreement with no false negative calls. Precision, Recall, and F1-Score of the Nanopore sequencing workflow were > 0.99, respectively. CONCLUSIONS: These results demonstrated the great potential of current Nanopore sequencing for application in clinical diagnostics, at least for SNP genotyping by amplicon sequencing. Other more complex applications, especially structural variant identification, require further in-depth clinical validation. |
format | Online Article Text |
id | pubmed-8929590 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-89295902022-03-18 Genotyping of familial Mediterranean fever gene (MEFV)—Single nucleotide polymorphism—Comparison of Nanopore with conventional Sanger sequencing Schmidt, Jonas Berghaus, Sandro Blessing, Frithjof Herbeck, Holger Blessing, Josef Schierack, Peter Rödiger, Stefan Roggenbuck, Dirk Wenzel, Folker PLoS One Research Article BACKGROUND: Through continuous innovation and improvement, Nanopore sequencing has become a powerful technology. Because of its fast processing time, low cost, and ability to generate long reads, this sequencing technique would be particularly suitable for clinical diagnostics. However, its raw data accuracy is inferior in contrast to other sequencing technologies. This constraint still results in limited use of Nanopore sequencing in the field of clinical diagnostics and requires further validation and IVD certification. METHODS: We evaluated the performance of latest Nanopore sequencing in combination with a dedicated data-analysis pipeline for single nucleotide polymorphism (SNP) genotyping of the familial Mediterranean fever gene (MEFV) by amplicon sequencing of 47 clinical samples. Mutations in MEFV are associated with Mediterranean fever, a hereditary periodic fever syndrome. Conventional Sanger sequencing, which is commonly applied in clinical genetic diagnostics, was used as a reference method. RESULTS: Nanopore sequencing enabled the sequencing of 10 target regions within MEFV with high read depth (median read depth 7565x) in all samples and identified a total of 435 SNPs in the whole sample collective, of which 29 were unique. Comparison of both sequencing workflows showed a near perfect agreement with no false negative calls. Precision, Recall, and F1-Score of the Nanopore sequencing workflow were > 0.99, respectively. CONCLUSIONS: These results demonstrated the great potential of current Nanopore sequencing for application in clinical diagnostics, at least for SNP genotyping by amplicon sequencing. Other more complex applications, especially structural variant identification, require further in-depth clinical validation. Public Library of Science 2022-03-17 /pmc/articles/PMC8929590/ /pubmed/35298548 http://dx.doi.org/10.1371/journal.pone.0265622 Text en © 2022 Schmidt et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Schmidt, Jonas Berghaus, Sandro Blessing, Frithjof Herbeck, Holger Blessing, Josef Schierack, Peter Rödiger, Stefan Roggenbuck, Dirk Wenzel, Folker Genotyping of familial Mediterranean fever gene (MEFV)—Single nucleotide polymorphism—Comparison of Nanopore with conventional Sanger sequencing |
title | Genotyping of familial Mediterranean fever gene (MEFV)—Single nucleotide polymorphism—Comparison of Nanopore with conventional Sanger sequencing |
title_full | Genotyping of familial Mediterranean fever gene (MEFV)—Single nucleotide polymorphism—Comparison of Nanopore with conventional Sanger sequencing |
title_fullStr | Genotyping of familial Mediterranean fever gene (MEFV)—Single nucleotide polymorphism—Comparison of Nanopore with conventional Sanger sequencing |
title_full_unstemmed | Genotyping of familial Mediterranean fever gene (MEFV)—Single nucleotide polymorphism—Comparison of Nanopore with conventional Sanger sequencing |
title_short | Genotyping of familial Mediterranean fever gene (MEFV)—Single nucleotide polymorphism—Comparison of Nanopore with conventional Sanger sequencing |
title_sort | genotyping of familial mediterranean fever gene (mefv)—single nucleotide polymorphism—comparison of nanopore with conventional sanger sequencing |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8929590/ https://www.ncbi.nlm.nih.gov/pubmed/35298548 http://dx.doi.org/10.1371/journal.pone.0265622 |
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