Cargando…
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...
Autores principales: | , , , , , , , , , , , , , , , , , , |
---|---|
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 |
_version_ | 1782185218837315584 |
---|---|
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. |
format | Text |
id | pubmed-2919738 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT woodhenrym usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT belvedereornella usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT conwaycaroline usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT dalycatherine usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT chalkleyrebecca usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT bickerdikemelissa usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT mckinleyclaire usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT eganphil usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT rosslisa usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT haywardbruce usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT morganjoanne usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT davidsonleslie usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT maclennanken usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT ongthiank usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT papagiannopouloskostas usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT cookian usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT adamsdavidj usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT taylorgrahamr usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens AT rabbittspamela usingnextgenerationsequencingforhighresolutionmultiplexanalysisofcopynumbervariationfromnanogramquantitiesofdnafromformalinfixedparaffinembeddedspecimens |