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Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2

BACKGROUND: Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruS...

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Autores principales: Nguyen-Dumont, Tú, Teo, Zhi L, Pope, Bernard J, Hammet, Fleur, Mahmoodi, Maryam, Tsimiklis, Helen, Sabbaghian, Nelly, Tischkowitz, Marc, Foulkes, William D, Giles, Graham G, Hopper, John L, Southey, Melissa C, Park, Daniel J
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829211/
https://www.ncbi.nlm.nih.gov/pubmed/24206657
http://dx.doi.org/10.1186/1755-8794-6-48
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author Nguyen-Dumont, Tú
Teo, Zhi L
Pope, Bernard J
Hammet, Fleur
Mahmoodi, Maryam
Tsimiklis, Helen
Sabbaghian, Nelly
Tischkowitz, Marc
Foulkes, William D
Giles, Graham G
Hopper, John L
Southey, Melissa C
Park, Daniel J
author_facet Nguyen-Dumont, Tú
Teo, Zhi L
Pope, Bernard J
Hammet, Fleur
Mahmoodi, Maryam
Tsimiklis, Helen
Sabbaghian, Nelly
Tischkowitz, Marc
Foulkes, William D
Giles, Graham G
Hopper, John L
Southey, Melissa C
Park, Daniel J
author_sort Nguyen-Dumont, Tú
collection PubMed
description BACKGROUND: Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. METHODS: Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. RESULTS: We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. CONCLUSIONS: These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings.
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spelling pubmed-38292112013-11-16 Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2 Nguyen-Dumont, Tú Teo, Zhi L Pope, Bernard J Hammet, Fleur Mahmoodi, Maryam Tsimiklis, Helen Sabbaghian, Nelly Tischkowitz, Marc Foulkes, William D Giles, Graham G Hopper, John L Southey, Melissa C Park, Daniel J BMC Med Genomics Research Article BACKGROUND: Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. METHODS: Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. RESULTS: We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. CONCLUSIONS: These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings. BioMed Central 2013-11-08 /pmc/articles/PMC3829211/ /pubmed/24206657 http://dx.doi.org/10.1186/1755-8794-6-48 Text en Copyright © 2013 Nguyen-Dumont et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Nguyen-Dumont, Tú
Teo, Zhi L
Pope, Bernard J
Hammet, Fleur
Mahmoodi, Maryam
Tsimiklis, Helen
Sabbaghian, Nelly
Tischkowitz, Marc
Foulkes, William D
Giles, Graham G
Hopper, John L
Southey, Melissa C
Park, Daniel J
Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2
title Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2
title_full Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2
title_fullStr Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2
title_full_unstemmed Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2
title_short Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2
title_sort hi-plex for high-throughput mutation screening: application to the breast cancer susceptibility gene palb2
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829211/
https://www.ncbi.nlm.nih.gov/pubmed/24206657
http://dx.doi.org/10.1186/1755-8794-6-48
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