Abstract
BackgroundCancer re-sequencing programs rely on DNA isolated from fresh snap frozen tissues, the preparation of which is combined with additional preservation efforts. Tissue samples at pathology departments are routinely stored as formalin-fixed and paraffin-embedded (FFPE) samples and their use would open up access to a variety of clinical trials. However, FFPE preparation is incompatible with many down-stream molecular biology techniques such as PCR based amplification methods and gene expression studies.Methodology/Principal FindingsHere we investigated the sample quality requirements of FFPE tissues for massively parallel short-read sequencing approaches. We evaluated key variables of pre-fixation, fixation related and post-fixation processes that occur in routine medical service (e.g. degree of autolysis, duration of fixation and of storage). We also investigated the influence of tissue storage time on sequencing quality by using material that was up to 18 years old. Finally, we analyzed normal and tumor breast tissues using the Sequencing by Synthesis technique (Illumina Genome Analyzer, Solexa) to simultaneously localize genome-wide copy number alterations and to detect genomic variations such as substitutions and point-deletions and/or insertions in FFPE tissue samples.Conclusions/SignificanceThe application of second generation sequencing techniques on small amounts of FFPE material opens up the possibility to analyze tissue samples which have been collected during routine clinical work as well as in the context of clinical trials. This is in particular important since FFPE samples are amply available from surgical tumor resections and histopathological diagnosis, and comprise tissue from precursor lesions, primary tumors, lymphogenic and/or hematogenic metastases. Large-scale studies using this tissue material will result in a better prediction of the prognosis of cancer patients and the early identification of patients which will respond to therapy.
Highlights
The advance of second generation sequencing techniques (454 sequencing, the Illumina Genome Analyzer (Solexa), the SOLiD platform, the Polonator and the HeliScope Single Molecule Sequencer technology) enables us to investigate many ‘‘-omes’’ of patients material, such as genomes, transcriptomes, epigenomes and miRNAomes [1,2,3]
For the comparison of snap frozen and formalinfixed and paraffin-embedded (FFPE) tissue DNA we prepared breast tissue samples from one patient, divided the tissue in seven equal amounts and preserved them either as snap frozen or as FFPE tissue with ischemic times ranging from 20 min. to over 6 hours and fixation times from 24 to 72 hours (Table 1)
Overall fragment numbers, aligned fragments and chromosome-wise coverage are similar for snap frozen and FFPE tissues, proving that fresh FFPE tissues can be used for high throughput re-sequencing approaches
Summary
The advance of second generation sequencing techniques (454 sequencing, the Illumina Genome Analyzer (Solexa), the SOLiD platform, the Polonator and the HeliScope Single Molecule Sequencer technology) enables us to investigate many ‘‘-omes’’ of patients material, such as genomes, transcriptomes, epigenomes and miRNAomes [1,2,3]. The sequencing techniques are based on the amplification of single DNA molecules and thereby provide, in a sense, ‘digital’ information [4,5,6]. They are considerably less errorprone to the ‘contamination’ between normal and diseased tissues and are especially advantageous for the analysis of tumor tissues [7]. To take advantage of preserved patient tissue we investigated the sample quality requirements of formaldehyde-fixed and paraffin-embedded (FFPE) tissues and established a protocol for the application of massively parallel short-read sequencing approaches. We analyzed normal and tumor breast tissues using the Sequencing by Synthesis (Illumina Genome Analyzer) technology to simultaneously localize genome-wide copy number alterations and to detect genomic variations such as substitutions and point-deletions and/or insertions in FFPE tissue samples
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