Abstract B65: Targeted resequencing of the EphB2 gene in colorectal cancer for SNP discovery using next generation sequencing

Abstract

Abstract Colorectal cancer (CRC) remains a leading cause of cancer morbidity and mortality in the United States. CRC is the third most common cause of deaths in the U. S. with the highest incidence observed in the African American (AA) population compared to other American populations. Our group was the first to implicate the EphB2 tyrosine kinase receptor as a tumor suppressor in prostate cancer (PC), where we reported somatic inactivating mutations occurring in 5%-10% of sporadic tumors. With its role in maintaining normal epithelial architecture and functional data suggestive of a tumor suppressor in both prostate and colon cancer. Recent advances in next-generation sequencing (NGS) afford new opportunities for large scale SNP discovery using a targeted resequencing approach. Several methodologies have been developed for targeted resequencing. Among these methods is the RainDance picoliter massive single-plex PCR assay, which provides the capability to assess up to 10,000 independent amplicons in a single reaction. We designed a RainDance PCR assay containing 700 amplicons, encompassing the entire 225,000bp genomic region of the EphB2 gene. These individual pools of amplicons from each sample were then used as template for NGS and barcoded to generate pools of 16 unique individuals using the Life Technologies SOLiD version 3-Plus system. Utilizing this method we screened 20 colorectal cancer cases and 27 sporadic prostate cancer cases. Thus far we have identified both known and novel variants within the EphB2 gene region among of which are several common coding variants including the 2055A>T nonsense mutation (K1019X) in three of 10 female CRC cases. We will provide data on the number of novel and known single nucleotide polymorphisms (SNPs) for all CRC and PC cases as well as quality assessment and overall performance of the RainDance method and performance metrics for SOliD NGS along with a comparison of utilizing several different analyses pipelines to identify the novel and known SNPs. These data will provide us with unprecedented details of the genetic variation of the EphB2 gene in African Americans. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B65.