Abstract 211: The performance characteristic of the low input tagmentation-based whole genome sequencing in high quality somatic variant calling

Abstract

Abstract Whole genome sequencing (WGS) is widely used for cancer diagnostic and therapeutic applications in clinical practice and clinical trials. Currently, the Truseq PCR-free library preparation methods are routinely used in genomics laboratories. However, microgram amounts of DNA input is a limitation due to limited materials often from clinically-derived specimens. Here, we evaluated a novel low input (100-300 ng) PCR-free tagmentation (TAG) based library preparation for WGS. Replicates of TAG- and ligation-based (Illumina TruSeq) sequencing libraries were prepared from 3 pairs of breast cancer-derived tumors (HCC1195, HCC1143 and HCC1187) and matched B-lymphocyte-derived normal (HCC1195BL, HCC1143BL and HCC1187BL) cell lines. Libraries were sequenced on Illumina Novaseq 6000 platforms to target 30x and 90x mean coverage for normal and tumor samples respectively. Raw sequencing data were aligned to the hg38 by Isaac Aligner before variant analysis by Strelka2. Technical sequencing metrics demonstrated high similarity between TAG- and ligation-based workflows, including passing filter reads, Q30%, aligned reads, mean coverage. Germline variants of HCC1395BL showed greater than 98% precision and sensitivity using ligation-based variants as a reference. For somatic mutation calling from TAG-based libraries, HCC1395, HCC1143 and HCC1187, 88%, 85% and 86% precision and 81%, 70%, 68% sensitivity was observed, respectively, compared to the ligation-based method. Furthermore, using the SEQC2 Consortium high-confidence mutation set, TAG- and ligation- based variant calls of HCC1395 had 90% and 93% precision and 74% and 73% sensitivity, respectively with this high confidence reference. Further investigation showed that a high fraction of false negative calls was associated with low variant allele frequency (<10%). Compared to an available reference dataset for benchmarking somatic calling pipelines from New York Genome Center, TAG- and ligation-based variant calls of HCC1143 yielded 85% and 83% precision and 63% and 63% sensitivity, respectively; TAG- and ligation-based variant calls of HCC1187 resulted in 91% and 92% precision and 81% and 79% sensitivity, respectively. In summary, the low input TAG-based WGS protocol produced highly reproducible variant calls with highly concordant somatic variant calls compared to the commonly-used PCR-free ligation-based methods and to reference callset. Further evaluation is warranted to broaden clinical application from minimal starting material.The views expressed in this abstract are solely of the authors and do not reflect the official policy of the Departments of Army/Navy/Air Force, Department of Defense, USUHS, HJF, or U.S. Government. Citation Format: Liqun Jiang, Xijun Zhang, Camille Alba, Gauthaman Sukumar, Elizabeth A. Rice, Matthew D. Wilkerson, Clifton L. Dalgard. The performance characteristic of the low input tagmentation-based whole genome sequencing in high quality somatic variant calling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 211.