Genome-wide massively parallel sequencing of formaldehyde fixed-paraffin embedded (FFPE) tumor tissues for copy-number- and mutation-analysis.

Author(s): Schweiger MR, Kerick M, Timmermann B, Albrecht MW, Borodina T, Parkhomchuk D, Zatloukal K, Lehrach H

Publication: PLoS One, 2009, Vol. 4, Page e5548

PubMed ID: 19440246 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of cold ischemia time, formaldehyde fixation and paraffin-embedding, storage duration and DNA extraction method on detection of copy number and SNVs by whole genome sequencing of normal and breast tumor specimens. DNA was extracted from 3 um sections. After excision, specimens were stored in a humidified chamber at room temperature to investigate the effects of cold ischemia.

   Summary of Findings:

   The authors report that DNA extraction kit did not affect the rate of successful sequencing. There was a correlation of 0.82 between formaldehyde-fixed and frozen specimens for readcounts per unit, and this correlation increased to 0.97 when the GC content was 40%, but this dependence on GC content for high correlation decreased as readcounts per unit increased. Further, cold ischemia time and storage of formaldehyde-fixed blocks had little or no effect on the correlation of readcounts between formaldehyde-fixed and frozen specimens. Formaldehyde fixation had no significant effect on copy number alterations when compared with snap-frozen specimens. More SNVs were identified using formaldehyde-fixed than snap-frozen breast tumor specimens, particularly when the specimen was fixed for 72 h instead of 24 h, but the false positive rate of SNV detection in formaldehyde-fixed specimens could be partially attenuated by increasing the number of reads.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	DNA sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formaldehyde Snap frozen
   Biospecimen Preservation	Time in fixative	24 h 72 h
   Biospecimen Acquisition	Cold ischemia time	<20 min 1 h 3 h 6 h
   Storage	Storage duration	1 year 14 years 18 years
   Analyte Extraction and Purification	Analyte isolation method	QIAamp Arcturus PicoPure Macherey-Nagel

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