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Clinical Trial
. 2012;13(7):8933-8942.
doi: 10.3390/ijms13078933. Epub 2012 Jul 17.

Exome enrichment and SOLiD sequencing of formalin fixed paraffin embedded (FFPE) prostate cancer tissue

Roopika Menon  1 , Mario Deng  1 , Diana Boehm  1 , Martin Braun  1 , Falko Fend  2 , Detlef Boehm  3 , Saskia Biskup  3 , Sven Perner  1
Affiliations
Clinical Trial

Exome enrichment and SOLiD sequencing of formalin fixed paraffin embedded (FFPE) prostate cancer tissue

Roopika Menon et al. Int J Mol Sci. 2012.

Abstract

Next generation sequencing (NGS) technologies have revolutionized cancer research allowing the comprehensive study of cancer using high throughput deep sequencing methodologies. These methods detect genomic alterations, nucleotide substitutions, insertions, deletions and copy number alterations. SOLiD (Sequencing by Oligonucleotide Ligation and Detection, Life Technologies) is a promising technology generating billions of 50 bp sequencing reads. This robust technique, successfully applied in gene identification, might be helpful in detecting novel genes associated with cancer initiation and progression using formalin fixed paraffin embedded (FFPE) tissue. This study's aim was to compare the validity of whole exome sequencing of fresh-frozen vs. FFPE tumor tissue by normalization to normal prostatic FFPE tissue, obtained from the same patient. One primary fresh-frozen sample, corresponding FFPE prostate cancer sample and matched adjacent normal prostatic tissue was subjected to exome sequencing. The sequenced reads were mapped and compared. Our study was the first to show comparable exome sequencing results between FFPE and corresponding fresh-frozen cancer tissues using SOLiD sequencing. A prior study has been conducted comparing the validity of sequencing of FFPE vs. fresh frozen samples using other NGS platforms. Our validation further proves that FFPE material is a reliable source of material for whole exome sequencing.

Keywords: SOLiD4; exome sequencing; next-generation sequencing; prostate cancer.

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Figures

Figure 1
Figure 1
Venn diagrams representing single nucleotide variation (SNV) profiles. The SNV analysis showed a total of 6853 SNVs for FFPE normal prostatic, 5445 SNVs for FFPE tumor and 7707 SNVs for fresh frozen tumor. (a) FFPE tumor vs. fresh frozen tumor tissue: The total number of common SNVs between FFPE tumor and fresh frozen tumor were 4618. 84.9% of the FFPE SNVs were common to the fresh frozen sample. (b) FFPE tumor vs. FFPE normal prostatic tissue: Upon normalization with FFPE normal prostatic, the tumor specific SNVs for FFPE tumor were 864. There was a 84.1% overlap between the fresh frozen tumor and FFPE non prostatic tissue. (c) Fresh-frozen tumor vs. FFPE normal prostatic tissue: Upon normalization with FFPE normal prostatic, the tumor specific SNVs for fresh frozen tumor were 2151. There was a 72.1% overlap between FFPE tumor and FFPE normal prostatic tissue.
Figure 2
Figure 2
Copy number variation analysis of exome sequenced fresh frozen tumor and FFPE tumor after normalization with FFPE normal prostatic tissue. Copy number variation analysis of fresh frozen tumor and FFPE tumor versus FFPE normal prostatic on three chromosomes. For each window the ratio between normal and tumor tissue has been calculated. Furthermore, the log2 has been applied to these ratios. The genomic position is represented on the x-axis. The log2 ratios of the fragments are represented on the y-axis. The average is depicted by the red line.
Figure 2
Figure 2
Copy number variation analysis of exome sequenced fresh frozen tumor and FFPE tumor after normalization with FFPE normal prostatic tissue. Copy number variation analysis of fresh frozen tumor and FFPE tumor versus FFPE normal prostatic on three chromosomes. For each window the ratio between normal and tumor tissue has been calculated. Furthermore, the log2 has been applied to these ratios. The genomic position is represented on the x-axis. The log2 ratios of the fragments are represented on the y-axis. The average is depicted by the red line.
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