Abstract 1160: Whole genome amplification and high-throughput sequencing of formalin-fixed paraffin-embedded colorectal cancer

Abstract

Abstract Formalin-fixed paraffin-embedded (FFPE) tumors represent a valuable resource for translational cancer genomic research. However, genetic analysis of the FFPE material is challenging, as archived tissues are often small biopsies and the fixation process damages DNA. In researching a solution to this, we have evaluated the performance of different whole genome amplification methods for obtaining DNA samples from archived tumors and their use as a template in high-throughput sequencing. Two whole genome amplification methods were applied using 20 μm sections of FFPE colorectal cancer tissue and normal human genome DNA samples. Genomic DNA was amplified using Multiple Displacement Amplification (MDA) method and a modified version of Degenerate Oligonucleotide PCR (DOP) assay. Non-amplified human genomic DNA was used as a reference when genomic coverage, linearity and uniformity of the amplified products were assessed. Paired-end sequencing libraries were prepared from the DOP amplified templates while the MDA samples underwent single read library preparation. Sequencing was performed using an Illumina Genome Analyzer II. 2.5 million 54-mer reads mapping to the human genome were generated for each sample. Whole genome amplification was shown to produce artifacts when compared to non-amplified genomic DNA sequences. The modified DOP method preferentially amplified non-repetitive sequences in the human genome. Using the MDA-based method, genomic sequence coverage was generally more even between regions of repetitive and non-repetitive DNA. However, spread of the sequence read counts was greater in the MDA method and MDA amplification offered less dynamic range to identify copy number aberrations. Using whole genome amplification methods with the FFPE colorectal tumor sample we were able to generate 130 Mb of genomic sequence data and identify DNA copy number gains in chromosomes 13 and 20q. Whole genome amplification and high-throughput sequencing can be used to generate sequence data from minute amounts of FFPE material and identify DNA copy number aberrations in the archived tumors. Nonetheless, whole genome amplification produces artifacts by introducing bias towards specific regions of the genome, generating artificial and random DNA fragment assemblies (MDA) and introducing universal primer sequence in the ends of the amplified DNA fragments (DOP). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1160.