DNA qualification workflow for next generation sequencing of histopathological samples.

Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory...

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Autores principales:	Michele Simbolo, Marisa Gottardi, Vincenzo Corbo, Matteo Fassan, Andrea Mafficini, Giorgio Malpeli, Rita T Lawlor, Aldo Scarpa
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4092ff80f7aa4b97aba50372a059d4b2
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spelling	oai:doaj.org-article:4092ff80f7aa4b97aba50372a059d4b22021-11-18T07:42:51ZDNA qualification workflow for next generation sequencing of histopathological samples.1932-620310.1371/journal.pone.0062692https://doaj.org/article/4092ff80f7aa4b97aba50372a059d4b22013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23762227/?tool=EBIhttps://doaj.org/toc/1932-6203Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.Michele SimboloMarisa GottardiVincenzo CorboMatteo FassanAndrea MafficiniGiorgio MalpeliRita T LawlorAldo ScarpaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 6, p e62692 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Michele Simbolo Marisa Gottardi Vincenzo Corbo Matteo Fassan Andrea Mafficini Giorgio Malpeli Rita T Lawlor Aldo Scarpa DNA qualification workflow for next generation sequencing of histopathological samples.
description	Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.
format	article
author	Michele Simbolo Marisa Gottardi Vincenzo Corbo Matteo Fassan Andrea Mafficini Giorgio Malpeli Rita T Lawlor Aldo Scarpa
author_facet	Michele Simbolo Marisa Gottardi Vincenzo Corbo Matteo Fassan Andrea Mafficini Giorgio Malpeli Rita T Lawlor Aldo Scarpa
author_sort	Michele Simbolo
title	DNA qualification workflow for next generation sequencing of histopathological samples.
title_short	DNA qualification workflow for next generation sequencing of histopathological samples.
title_full	DNA qualification workflow for next generation sequencing of histopathological samples.
title_fullStr	DNA qualification workflow for next generation sequencing of histopathological samples.
title_full_unstemmed	DNA qualification workflow for next generation sequencing of histopathological samples.
title_sort	dna qualification workflow for next generation sequencing of histopathological samples.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/4092ff80f7aa4b97aba50372a059d4b2
work_keys_str_mv	AT michelesimbolo dnaqualificationworkflowfornextgenerationsequencingofhistopathologicalsamples AT marisagottardi dnaqualificationworkflowfornextgenerationsequencingofhistopathologicalsamples AT vincenzocorbo dnaqualificationworkflowfornextgenerationsequencingofhistopathologicalsamples AT matteofassan dnaqualificationworkflowfornextgenerationsequencingofhistopathologicalsamples AT andreamafficini dnaqualificationworkflowfornextgenerationsequencingofhistopathologicalsamples AT giorgiomalpeli dnaqualificationworkflowfornextgenerationsequencingofhistopathologicalsamples AT ritatlawlor dnaqualificationworkflowfornextgenerationsequencingofhistopathologicalsamples AT aldoscarpa dnaqualificationworkflowfornextgenerationsequencingofhistopathologicalsamples
_version_	1718423031000334336

DNA qualification workflow for next generation sequencing of histopathological samples.

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