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High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq

Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but...

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Autores principales: Kondrashova, Olga, Love, Clare J., Lunke, Sebastian, Hsu, Arthur L., Waring, Paul M., Taylor, Graham R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646639/
https://www.ncbi.nlm.nih.gov/pubmed/26569395
http://dx.doi.org/10.1371/journal.pone.0143006
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author Kondrashova, Olga
Love, Clare J.
Lunke, Sebastian
Hsu, Arthur L.
Waring, Paul M.
Taylor, Graham R.
author_facet Kondrashova, Olga
Love, Clare J.
Lunke, Sebastian
Hsu, Arthur L.
Waring, Paul M.
Taylor, Graham R.
author_sort Kondrashova, Olga
collection PubMed
description Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.
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spelling pubmed-46466392015-11-25 High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq Kondrashova, Olga Love, Clare J. Lunke, Sebastian Hsu, Arthur L. Waring, Paul M. Taylor, Graham R. PLoS One Research Article Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity. Public Library of Science 2015-11-16 /pmc/articles/PMC4646639/ /pubmed/26569395 http://dx.doi.org/10.1371/journal.pone.0143006 Text en © 2015 Kondrashova et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Kondrashova, Olga
Love, Clare J.
Lunke, Sebastian
Hsu, Arthur L.
Waring, Paul M.
Taylor, Graham R.
High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq
title High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq
title_full High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq
title_fullStr High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq
title_full_unstemmed High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq
title_short High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq
title_sort high-throughput amplicon-based copy number detection of 11 genes in formalin-fixed paraffin-embedded ovarian tumour samples by mlpa-seq
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646639/
https://www.ncbi.nlm.nih.gov/pubmed/26569395
http://dx.doi.org/10.1371/journal.pone.0143006
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