Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequencing in routine diagnostics.

Over the last years, massively parallel sequencing has rapidly evolved and has now transitioned into molecular pathology routine laboratories. It is an attractive platform for analysing multiple genes at the same time with very little input material. Therefore, the need for high quality DNA obtained...

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Autores principales:	Carina Heydt, Jana Fassunke, Helen Künstlinger, Michaela Angelika Ihle, Katharina König, Lukas Carl Heukamp, Hans-Ulrich Schildhaus, Margarete Odenthal, Reinhard Büttner, Sabine Merkelbach-Bruse
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/1b0a555199ba457291e8cd96ba50cc96
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spelling	oai:doaj.org-article:1b0a555199ba457291e8cd96ba50cc962021-11-25T06:05:27ZComparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequencing in routine diagnostics.1932-620310.1371/journal.pone.0104566https://doaj.org/article/1b0a555199ba457291e8cd96ba50cc962014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/25105902/?tool=EBIhttps://doaj.org/toc/1932-6203Over the last years, massively parallel sequencing has rapidly evolved and has now transitioned into molecular pathology routine laboratories. It is an attractive platform for analysing multiple genes at the same time with very little input material. Therefore, the need for high quality DNA obtained from automated DNA extraction systems has increased, especially to those laboratories which are dealing with formalin-fixed paraffin-embedded (FFPE) material and high sample throughput. This study evaluated five automated FFPE DNA extraction systems as well as five DNA quantification systems using the three most common techniques, UV spectrophotometry, fluorescent dye-based quantification and quantitative PCR, on 26 FFPE tissue samples. Additionally, the effects on downstream applications were analysed to find the most suitable pre-analytical methods for massively parallel sequencing in routine diagnostics. The results revealed that the Maxwell 16 from Promega (Mannheim, Germany) seems to be the superior system for DNA extraction from FFPE material. The extracts had a 1.3-24.6-fold higher DNA concentration in comparison to the other extraction systems, a higher quality and were most suitable for downstream applications. The comparison of the five quantification methods showed intermethod variations but all methods could be used to estimate the right amount for PCR amplification and for massively parallel sequencing. Interestingly, the best results in massively parallel sequencing were obtained with a DNA input of 15 ng determined by the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). No difference could be detected in mutation analysis based on the results of the quantification methods. These findings emphasise, that it is particularly important to choose the most reliable and constant DNA extraction system, especially when using small biopsies and low elution volumes, and that all common DNA quantification techniques can be used for downstream applications like massively parallel sequencing.Carina HeydtJana FassunkeHelen KünstlingerMichaela Angelika IhleKatharina KönigLukas Carl HeukampHans-Ulrich SchildhausMargarete OdenthalReinhard BüttnerSabine Merkelbach-BrusePublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 8, p e104566 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Carina Heydt Jana Fassunke Helen Künstlinger Michaela Angelika Ihle Katharina König Lukas Carl Heukamp Hans-Ulrich Schildhaus Margarete Odenthal Reinhard Büttner Sabine Merkelbach-Bruse Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequencing in routine diagnostics.
description	Over the last years, massively parallel sequencing has rapidly evolved and has now transitioned into molecular pathology routine laboratories. It is an attractive platform for analysing multiple genes at the same time with very little input material. Therefore, the need for high quality DNA obtained from automated DNA extraction systems has increased, especially to those laboratories which are dealing with formalin-fixed paraffin-embedded (FFPE) material and high sample throughput. This study evaluated five automated FFPE DNA extraction systems as well as five DNA quantification systems using the three most common techniques, UV spectrophotometry, fluorescent dye-based quantification and quantitative PCR, on 26 FFPE tissue samples. Additionally, the effects on downstream applications were analysed to find the most suitable pre-analytical methods for massively parallel sequencing in routine diagnostics. The results revealed that the Maxwell 16 from Promega (Mannheim, Germany) seems to be the superior system for DNA extraction from FFPE material. The extracts had a 1.3-24.6-fold higher DNA concentration in comparison to the other extraction systems, a higher quality and were most suitable for downstream applications. The comparison of the five quantification methods showed intermethod variations but all methods could be used to estimate the right amount for PCR amplification and for massively parallel sequencing. Interestingly, the best results in massively parallel sequencing were obtained with a DNA input of 15 ng determined by the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). No difference could be detected in mutation analysis based on the results of the quantification methods. These findings emphasise, that it is particularly important to choose the most reliable and constant DNA extraction system, especially when using small biopsies and low elution volumes, and that all common DNA quantification techniques can be used for downstream applications like massively parallel sequencing.
format	article
author	Carina Heydt Jana Fassunke Helen Künstlinger Michaela Angelika Ihle Katharina König Lukas Carl Heukamp Hans-Ulrich Schildhaus Margarete Odenthal Reinhard Büttner Sabine Merkelbach-Bruse
author_facet	Carina Heydt Jana Fassunke Helen Künstlinger Michaela Angelika Ihle Katharina König Lukas Carl Heukamp Hans-Ulrich Schildhaus Margarete Odenthal Reinhard Büttner Sabine Merkelbach-Bruse
author_sort	Carina Heydt
title	Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequencing in routine diagnostics.
title_short	Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequencing in routine diagnostics.
title_full	Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequencing in routine diagnostics.
title_fullStr	Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequencing in routine diagnostics.
title_full_unstemmed	Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequencing in routine diagnostics.
title_sort	comparison of pre-analytical ffpe sample preparation methods and their impact on massively parallel sequencing in routine diagnostics.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/1b0a555199ba457291e8cd96ba50cc96
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Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequencing in routine diagnostics.

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