Abstract Insight into the cancer genome has recently increased in an unprecedented way due to high throughput (HT) sequencing technology to examine the genome, exome, and transcriptome. We sought new genetic biomarkers for cancer by using HT sequencing to examine highly characterized primary sporadic tumors and tumor-derived cell lines. Several tumors/cell lines represented the most common subtype of kidney cancer, clear cell renal cell carcinoma, as well as NCI-60 panel cell lines from breast (MCF7) and prostate (PC3). Incorporation of NCI-60 panel cell lines was advantageous due to extensive prior characterization of these reagents, including gene expression, comparative genome hybridization, SNPs, and drug cytotoxicity. The study was performed using three major platforms (454, Illumina, ABI) to compare sequencing quality, cost, quantity, and computational amenability. Different methodologies were examined including exome, transcriptome, and whole genome sequencing. Alignment of sequence to a consensus genome (hg 18) allowed identification of novel and previously documented genomic variants. Various filters were implemented to sort the large number of variants detected, including dbSNP, personal genome variants, and 1000 genomes data. An average of 65 million 35-50 base pair sequences were generated from transcriptomes of two primary clear cell RCC tumors. Over 2 million 375 base pair sequences were generated from each of four cell line exomes. Differences were observed in extent and depth of coverage of a given target region by various experimental approaches. SNP array genotyping showed 1% discordance with sequencing. Previously characterized mutations were confirmed in HT sequences. We identified multiple regions of homozygosity at an unprecedented resolution. For example, in the clear cell renal cancer cell line, 786-0, a known mutation in VHL (311delG) was identified, and LOH at SNP positions across the entire length of chromosome 3 was confirmed. Alignment to a reference genome (hg 18) permitted identification of 2583-5307 genomic sequence variants, including 92-297 novel non-synonymous variants in the exomes of each cell line. Variants include frameshift and nonsense mutations. Novel non-synonymous variants were identified in cancer-related genes and these are being examined in additional tumors. Potential functions of newly identified mutated genetic loci suggest perturbation of signaling pathways and are candidates for targeted therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 2198.