title: Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequencing in routine diagnostics.
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.
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany.
authors.forename: Carina
authors.initials: C
authors.lastname: Heydt
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Fassunke
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany.
authors.forename: Helen
authors.initials: H
authors.lastname: Künstlinger
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany.
authors.forename: Michaela Angelika
authors.initials: MA
authors.lastname: Ihle
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: König
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany.
authors.forename: Lukas Carl
authors.initials: LC
authors.lastname: Heukamp
authors.affiliation: Institute of Pathology, University Hospital Göttingen, Göttingen, Germany.
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Schildhaus
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany.
authors.forename: Margarete
authors.initials: M
authors.lastname: Odenthal
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Büttner
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Merkelbach-Bruse
mesh_terms: D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004252:DNA Mutational Analysis / Q000379:methods; D005557:Formaldehyde / Q000737:chemistry; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016612:Paraffin Embedding / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D005557:Formaldehyde; D004247:DNA
references.citation: Nat Biotechnol. 2013 Nov;31(11):1023-31
references.pmid: 24142049
references.citation: Genome Biol. 2002 Jun 18;3(7):RESEARCH0034
references.pmid: 12184808
references.citation: Diagn Mol Pathol. 2001 Dec;10(4):265-71
references.pmid: 11763318
references.citation: Mol Cancer. 2009 Oct 08;8:82
references.pmid: 19811662
references.citation: J Mol Diagn. 2007 Sep;9(4):441-51
references.pmid: 17690213
references.citation: BMC Res Notes. 2010 Sep 14;3:239
references.pmid: 20840759
references.citation: Biol Proced Online. 2013 Feb 13;15(1):5
references.pmid: 23406353
references.citation: J Mol Diagn. 2013 Mar;15(2):234-47
references.pmid: 23321017
references.citation: J Mol Diagn. 2005 Feb;7(1):36-9
references.pmid: 15681472
references.citation: Am J Pathol. 2002 Dec;161(6):1961-71
references.pmid: 12466110
references.citation: BMC Cancer. 2014 Jan 10;14:13
references.pmid: 24410877
references.citation: BMC Med Genomics. 2011 Sep 29;4:68
references.pmid: 21958464
references.citation: Lancet Oncol. 2010 Aug;11(8):753-62
references.pmid: 20619739
references.citation: PLoS One. 2013 Jun 06;8(6):e62692
references.pmid: 23762227
references.citation: Virchows Arch. 2010 Sep;457(3):309-17
references.pmid: 20665046
references.citation: Clin Chem Lab Med. 2011 Oct 25;50(2):267-72
references.pmid: 22022984
references.citation: Clin Chem. 2013 Sep;59(9):1376-83
references.pmid: 23649127
references.citation: Pathol Res Pract. 2014 Mar;210(3):142-6
references.pmid: 24355442
references.citation: Genes Chromosomes Cancer. 2013 May;52(5):503-11
references.pmid: 23362162
references.citation: Cancer Discov. 2012 Jan;2(1):23-4
references.pmid: 22585165
references.citation: Methods. 2001 Dec;25(4):409-18
references.pmid: 11846610
references.citation: Clin Chem. 2013 May;59(5):815-23
references.pmid: 23403697
references.citation: Genome Med. 2013 Aug 30;5(8):77
references.pmid: 24001039
references.citation: Exp Mol Pathol. 2012 Feb;92(1):33-43
references.pmid: 21963600
references.citation: PLoS One. 2011;6(12):e28910
references.pmid: 22174923
references.citation: PLoS One. 2007 Jun 20;2(6):e537
references.pmid: 17579711
references.citation: J Mol Diagn. 2013 Sep;15(5):623-33
references.pmid: 23810758
references.citation: Am J Pathol. 1999 Nov;155(5):1467-71
references.pmid: 10550302