Abstract 436: Comparative analysis of PCR-free and bead-linked transposome libraries for somatic mutation detection by whole genome sequencing

Abstract

Abstract PCR-free DNA whole genome sequencing libraries are a standard in population-scale genomics, with high uniformity of coverage, reproducible addressable space and minimal amplification bias. However, high input requirements (>1µg) can be challenging for small volume biopsy, thin scroll, or low-weight tumor sources. Nextera DNA Flex is a newly developed, bead-linked transposome-based DNA library workflow that requires minimal amplification. It features a simplified single-tube technical process and could be an alternative for low input (~100ng) DNA samples. Here, we assessed library and sequencing metrics for both the TruSeq DNA PCR-free and the Nextera DNA Flex workflows using a reference genomic DNA (NA12878), and human normal-tumor lung adenocarcinoma pairs. PCR-free and Flex libraries were profiled equally on the Illumina HiSeq X, either at >30x or >90x depth for germline and tumor, respectively. Read sequences were aligned to the human reference genome using the Isaac aligner. Genomic alterations were identified by Starling, Strelka, and Canvas algorithms. The PCR-free workflow at 1µg input produced libraries with a >10nM yield and 550bp peak size, while the Flex workflow using a 10-fold reduced input of 100ng resulted in >25nM, 600bp libraries, with a wider fragment size distribution. Single lane output on HiSeq X yielded equivalent Picard coverage for both workflows (>30x). In contrast, tumor libraries generated 122x and 108x coverage for PCR-free and Flex workflows respectively. This 11% decrease in coverage was associated with a higher duplicate rate from smaller insert size observed in the Flex workflow. Furthermore, there was high variant (SNP and INDEL) concordance of >98% and >97% for germline and tumor, respectively. Additionally, CNV coverage was highly correlated between Flex and PCR-free samples (average r=0.952 per chromosome). Across tumor-normal pairs, somatic mutations were detected in both workflows with a >79% concordance using a 0.15 allele fraction cutoff. Moreover, somatic mutation calls are observed in PCR-free only and Flex only groups at equal ratios (~10% of total calls). In addition, tumor purity and mutant allele fractions between matched samples were in high agreement, providing another measure of genomic concordance between workflows. Finally, review of canonical mutations identified COSMIC annotated KRAS mutations in both workflows. In summary, germline and somatic alterations identified by the Flex workflow achieved overall high agreement on matched assays of the PCR-free workflow. Nextera Flex may be a promising alternative for low-input tissue scenarios. Citation Format: Coralie Viollet, Xijun Zhang, Harvey B. Pollard, Matthew D. Wilkerson, Clifton L. Dalgard. Comparative analysis of PCR-free and bead-linked transposome libraries for somatic mutation detection by whole genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 436.