Abstract Recent genome-wide association studies (GWAS) have identified genetic loci associated with lung cancer risk. Direct sequencing is regarded as the ultimate approach for moving beyond genetic mapping to identify causal mutations and define disease candidate genes. We conducted a pilot next-generation sequencing (NGS) of all exons of TERT and CLPTM1L genes within the 5p15 locus from our NSCLC GWAS, replicating previous studies. We performed an amplicon capture-NGS on 138 samples, including 82 NSCLC cases and 56 controls. These results were highly consistent across samples, with the majority covering 85-95% of bases at 100-fold and with only 5 samples failing to achieve 100-fold coverage for at least 80% of bases. We identified 11 SNPs in TERT and 5 SNPs in CLPTM1L. Using our SKAT/SKAT-O method, we found that both genes were associated with NSCLC (TERT, p=0.006; and CLPTM1L, p=0.04). When the SNPs of the two genes were combined, we saw a greater joint effect (p=0.001). In a subgroup analysis focusing on functional SNPs, including 7 and 4 nonsynonymous SNPs in TERT and CLPTM1L, respectively, we found a stronger association with TERT (p=0.0005), as well as an increased joint effect (p=0.0001). Moreover, none of the sequenced subjects had functional SNPs in both TERT and CLPTM1L genes. Furthermore, most nonsynonymous variations seemed clustered either in the cases or in the controls. Bioinformatic annotation of these nonsynonymous SNPs revealed that those clustered in the controls had only benign effects on the encoded protein, whereas those overrepresented in the NSCLC cases had possible damage to the encoded protein. Using Sanger dideoxy-terminator sequencing at an independent core facility and Taqman genotyping, all of the 11 functional SNPs identified by amplicon capture-NGS were confirmed. In summary, the results seem to support the hypotheses that aggregated functional variations, but not the neutral ones, contribute to the risks of NSCLC development. However, the initial finding is not conclusive, as this pilot project was limited by a small sample set and structural variations were not examined. Citation Format: Zhaoxi Wang, Yang Zhao, Li Su, Seunggeun Lee, Xihong Lin, David C. Christiani. Targeted sequencing of 5p15 locus defined by non-small cell lung cancer (NSCLC) GWAS using next-generation sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2547. doi:10.1158/1538-7445.AM2013-2547