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Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the a...

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Autores principales: Wood, Henry M., Belvedere, Ornella, Conway, Caroline, Daly, Catherine, Chalkley, Rebecca, Bickerdike, Melissa, McKinley, Claire, Egan, Phil, Ross, Lisa, Hayward, Bruce, Morgan, Joanne, Davidson, Leslie, MacLennan, Ken, Ong, Thian K., Papagiannopoulos, Kostas, Cook, Ian, Adams, David J., Taylor, Graham R., Rabbitts, Pamela
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2919738/
https://www.ncbi.nlm.nih.gov/pubmed/20525786
http://dx.doi.org/10.1093/nar/gkq510
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author Wood, Henry M.
Belvedere, Ornella
Conway, Caroline
Daly, Catherine
Chalkley, Rebecca
Bickerdike, Melissa
McKinley, Claire
Egan, Phil
Ross, Lisa
Hayward, Bruce
Morgan, Joanne
Davidson, Leslie
MacLennan, Ken
Ong, Thian K.
Papagiannopoulos, Kostas
Cook, Ian
Adams, David J.
Taylor, Graham R.
Rabbitts, Pamela
author_facet Wood, Henry M.
Belvedere, Ornella
Conway, Caroline
Daly, Catherine
Chalkley, Rebecca
Bickerdike, Melissa
McKinley, Claire
Egan, Phil
Ross, Lisa
Hayward, Bruce
Morgan, Joanne
Davidson, Leslie
MacLennan, Ken
Ong, Thian K.
Papagiannopoulos, Kostas
Cook, Ian
Adams, David J.
Taylor, Graham R.
Rabbitts, Pamela
author_sort Wood, Henry M.
collection PubMed
description The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.
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spelling pubmed-29197382010-08-11 Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens Wood, Henry M. Belvedere, Ornella Conway, Caroline Daly, Catherine Chalkley, Rebecca Bickerdike, Melissa McKinley, Claire Egan, Phil Ross, Lisa Hayward, Bruce Morgan, Joanne Davidson, Leslie MacLennan, Ken Ong, Thian K. Papagiannopoulos, Kostas Cook, Ian Adams, David J. Taylor, Graham R. Rabbitts, Pamela Nucleic Acids Res Methods Online The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material. Oxford University Press 2010-08 2010-06-04 /pmc/articles/PMC2919738/ /pubmed/20525786 http://dx.doi.org/10.1093/nar/gkq510 Text en © The Author(s) 2010. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/2.5 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Wood, Henry M.
Belvedere, Ornella
Conway, Caroline
Daly, Catherine
Chalkley, Rebecca
Bickerdike, Melissa
McKinley, Claire
Egan, Phil
Ross, Lisa
Hayward, Bruce
Morgan, Joanne
Davidson, Leslie
MacLennan, Ken
Ong, Thian K.
Papagiannopoulos, Kostas
Cook, Ian
Adams, David J.
Taylor, Graham R.
Rabbitts, Pamela
Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens
title Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens
title_full Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens
title_fullStr Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens
title_full_unstemmed Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens
title_short Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens
title_sort using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of dna from formalin-fixed paraffin-embedded specimens
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2919738/
https://www.ncbi.nlm.nih.gov/pubmed/20525786
http://dx.doi.org/10.1093/nar/gkq510
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