Oncotarget

Research Papers:

Single-strand DNA library preparation improves sequencing of formalin-fixed and paraffin-embedded (FFPE) cancer DNA

Mathias Stiller, Antje Sucker, Klaus Griewank, Daniela Aust, Gustavo Bruno Baretton, Dirk Schadendorf and Susanne Horn _

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Oncotarget. 2016; 7:59115-59128. https://doi.org/10.18632/oncotarget.10827

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Abstract

Mathias Stiller1,2,3, Antje Sucker2, Klaus Griewank2, Daniela Aust4, Gustavo Bruno Baretton4, Dirk Schadendorf2, Susanne Horn1,2

1Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, D-04103 Leipzig, Germany

2Department of Dermatology, University Hospital, West German Cancer Center, University Duisburg-Essen, and German Consortium for Translational Cancer Research (DKTK), D-45147 Essen, Germany

3Department for Translational Skin Cancer Research, University Duisburg-Essen, and German Consortium for Translational Cancer Research (DKTK), D-45141 Essen, Germany

4Departments of Surgery and Pathology, Technical University Dresden, D-01307 Dresden, Germany

Correspondence to:

Susanne Horn, email: Susanne.Horn@uk-essen.de

Mathias Stiller, email: m.stiller@dkfz-heidelberg.de

Keywords: high-throughput sequencing, library preparation, formalin-fixed paraffin embedded (FFPE) tissue, cancer, whole exome sequencing

Received: April 27, 2016     Accepted: June 30, 2016     Published: July 24, 2016

ABSTRACT

DNA derived from formalin-fixed and paraffin-embedded (FFPE) tissue has been a challenge to large-scale genomic sequencing, due to its low quality and quantities. Improved techniques enabling the genome-wide analysis of FFPE material would be of great value, both from a research and clinical perspective.

Comparing a single-strand DNA library preparation method originally developed for ancient DNA to conventional protocols using double-stranded DNA derived from FFPE material we obtain on average 900-fold more library molecules and improved sequence complexity from as little as 5 ng input DNA. FFPE DNA is highly fragmented, usually below 100bp, and up to 60% of reads start after or end prior to adenine residues, suggesting that crosslinks predominate at adenine residues. Similar to ancient DNA, C > T substitutions are slightly increased with maximum rates up to 3% at the ends of molecules. In whole exome sequencing of single-strand libraries from lung, breast, colorectal, prostate and skin cancers we identify known cancer mutations. In summary, we show that single-strand library preparation enables genomic sequencing, even from low amounts of degraded FFPE DNA. This method provides a clear advantage both in research and clinical settings, where FFPE material (e.g. from biopsies) often is the only source of DNA available. Improving the genetic characterization that can be performed on conventional archived FFPE tissue, the single-strand library preparation allows scarce samples to be used in personalized medicine and enables larger sample sizes in future sequencing studies.


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