Targeted high throughput sequencing in clinical cancer settings: formaldehyde fixed-paraffin embedded (FFPE) tumor tissues, input amount and tumor heterogeneity.

Author(s): Kerick M, Isau M, Timmermann B, Sültmann H, Herwig R, Krobitsch S, Schaefer G, Verdorfer I, Bartsch G, Klocker H, Lehrach H, Schweiger MR

Publication: BMC Med Genomics, 2011, Vol. 4, Page 68

PubMed ID: 21958464 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine the effects of preservation method and amount of DNA template on sequencing success and single nucelotide polymorphism (SNP) analysis results in prostate cancer specimens.

Conclusion of Paper

Specimens that were embedded in Optimal Cutting Temperature compound (OCT) and snap frozen or formalin-fixed and paraffin-embedded were successfully sequenced with similar coverage profiles and a high degree of correlation of coverage per exon. The coefficient of variation was higher between snap frozen and formalin-fixed specimens than between two specimens preserved by the same method. Accuracies above 98% were achieved for both formalin-fixed and snap frozen specimens for genotype calls using the Affymetrix SNP arrays 6.0. 1.2% and 1.75% of loci investigated with at least 20-fold coverage were discordant for single nucleotide variation (SNV) detection or insertions and deletions (InDels), respectively, between formalin-fixed and snap frozen specimens. The majority of the discordance was due to either an SNV or InDels found in formalin-fixed tissue but not snap frozen tissue (false positives) and was eliminated by using a cut-off of 80-fold coverage. Enrichment efficiency after targeted resequencing of 3.9 Mb of independent DNA regions, as opposed to the 52 Mb whole exome target region, was similarly successful whether 500 ng, 1500 ng, or 3000 ng of DNA was included as template. A slightly broader range of variant/reference ratios for SNVs was observed when less template DNA was included indicating less complexity of these samples, but overall reproducibility of calls was high, regardless of DNA input amount.

Studies

  1. Study Purpose

    The purpose of this study was to determine the effects of method of preservation and DNA template amount on sequencing success and the detection of SNVs and InDels in prostate cancer specimens. Tumor tissue and paired benign tissue were macro-dissected from the differently preserved specimens. DNA extraction was performed using the EZI DNA tissue kit procedure for snap frozen and formalin-fixed specimens but with modifications included for the formalin-fixed specimens.

    Summary of Findings:

    Specimens that were embedded in OCT and snap frozen or formalin-fixed and paraffin-embedded were successfully sequenced with more than 99% of regions captured by at least one read. Both methods of preservation had similar coverage profiles and a high degree of correlation of enrichment per exon. The coefficient of variation was higher between snap frozen and formalin-fixed specimens than between two specimens preserved by the same method (either formalin or snap frozen). An insignificant shift in the GC-dependent coverage profile was seen between formalin-fixed and snap frozen specimens with snap frozen specimens showing better coverage of exons with high GC content and formalin-fixed specimens showing better coverage of exons with low GC content. Accuracies above 98% were achieved for both formalin-fixed and snap frozen specimens for genotype calls with Affymetrix SNP 6.0 arrays. 1.2% and 1.75% of loci investigated with at least 20-fold coverage were discordant for SNV detection or InDels, respectively, between formalin-fixed and snap frozen specimens. The majority of the discordance was due to either an SNV or InDels found in formalin-fixed tissue but not snap frozen tissue (false positives). When 80-fold coverage was used as a more stringent cutoff, no discordance between SNVs in differently preserved specimens was seen. Likewise, when 40-fold coverage was used as a cutoff, no InDels discordance remained between differently preserved specimens. Enrichment efficiency after targeted resequencing of 3.9 Mb of independent DNA regions, as opposed to the 52 Mb whole exome target region, was similarly successful whether 500 ng, 1500 ng, or 3000 ng of DNA was included as template. A slightly broader range of variant/reference ratios for SNVs was observed when less template DNA was included indicating less complexity of these samples, but overall reproducibility of calls was high, regardless of DNA input amount.

    Biospecimens
    Preservative Types
    • OCT
    • Formalin
    Diagnoses:
    • Neoplastic - Carcinoma
    • Neoplastic - Benign
    Platform:
    AnalyteTechnology Platform
    DNA Spectrophotometry
    DNA DNA sequencing
    DNA Real-time qPCR
    DNA PCR
    DNA SNP assay
    Morphology H-and-E microscopy
    Protein Immunohistochemistry
    DNA Bioanalyzer
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
    OCT
    PCR Specific Template amount 500 ng DNA
    1500 ng DNA
    3000 ng DNA

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