Universität zu Köln

Heydt, Carina, Fassunke, Jana, Kuenstlinger, Helen, Ihle, Michaela Angelika, Koenig, Katharina, Heukamp, Lukas Carl, Schildhaus, Hans-Ulrich, Odenthal, Margarete, Buettner, Reinhard and Merkelbach-Bruse, Sabine (2014). Comparison of Pre-Analytical FFPE Sample Preparation Methods and Their Impact on Massively Parallel Sequencing in Routine Diagnostics. PLoS One, 9 (8). SAN FRANCISCO: PUBLIC LIBRARY SCIENCE. ISSN 1932-6203

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Abstract

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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Heydt, Carina UNSPECIFIED UNSPECIFIED UNSPECIFIED Fassunke, Jana UNSPECIFIED UNSPECIFIED UNSPECIFIED Kuenstlinger, Helen UNSPECIFIED UNSPECIFIED UNSPECIFIED Ihle, Michaela Angelika UNSPECIFIED UNSPECIFIED UNSPECIFIED Koenig, Katharina UNSPECIFIED UNSPECIFIED UNSPECIFIED Heukamp, Lukas Carl UNSPECIFIED UNSPECIFIED UNSPECIFIED Schildhaus, Hans-Ulrich UNSPECIFIED UNSPECIFIED UNSPECIFIED Odenthal, Margarete UNSPECIFIED UNSPECIFIED UNSPECIFIED Buettner, Reinhard UNSPECIFIED UNSPECIFIED UNSPECIFIED Merkelbach-Bruse, Sabine UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-432116
DOI:	10.1371/journal.pone.0104566
Journal or Publication Title:	PLoS One
Volume:	9
Number:	8
Date:	2014
Publisher:	PUBLIC LIBRARY SCIENCE
Place of Publication:	SAN FRANCISCO
ISSN:	1932-6203
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language DNA EXTRACTION METHODS; REAL-TIME PCR; NUCLEIC-ACIDS; CLINICAL-SAMPLES; BRAF MUTATIONS; FORMALIN; TISSUE; SPECIMENS; BIOPSIES; AMPLIFICATION Multiple languages Multidisciplinary Sciences Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/43211