Abstract 4742: Using 1ng of DNA to detect haplotype phasing and gene fusions from whole exome sequencing of cancer cell lines (2015)

Abstract We have used a new platform from 10X Genomics to obtain ultra-deep and long-range exome sequencing data from 1ng of cancer cell line DNA. While traditional Whole Exome Sequencing (WES) methods have inputs of ∼50-500ng and lack long-range data, we demonstrate that the 10X platform can successfully obtain both gene fusion and haplotype phasing information along with standard SNP and indel variant-calling from WES on an Illumina HiSeq using just 1ng of DNA. The 10X platform massively partitions long DNA into &amp;gt;100,000 individual reactions. Each partition generates sequencing libraries with a unique barcode; the integrated downstream analysis platform uses the barcodes to map Illumina's short reads back to original long DNA fragments. Because the barcoding is performed prior to exome enrichment, the long-range information conveyed by the barcodes is retained even after standard hybrid capture enrichment. Powerful algorithms are implemented using a custom informatics toolset that is self-contained and easy to install, and the resulting structural variant calls can be easily visualized and explored using 10X's haplotype-aware genome browser. We used the 10X platform to analyze cancer cell lines with previously reported gene fusions. In all cases described, the fusion breakpoints were in introns of &amp;gt; 5.9kbp, and would therefore not be detectable with standard WES. In lung cancer cell line NCI-H2228, we called two annotated fusions, EML4/ALK, and ALK/PTPN3. Two fusion genes (MCHR2/ANO4, and CR627240/SACS) were identified in glioblastoma cell line U-87MG, and at least four (CD44/FGFR2, PVT1/PDHX, PVT1/ATE1, PVT1/PPAPDC1A) in gastric cancer cell line SNU-16. Long-range information provided by the technology also enables haplotype phasing of exome samples. Our algorithms phased the vast majority of SNPs within genes, with phase blocks up to 1.25Mbp. We used this haplotype information to phase somatic structural variants, including large-scale deletions and amplifications. To achieve the sensitivity required to call somatic mutations from low purity tumor specimens, it is necessary to be able to attain high coverage depth. This can be a significant challenge for low inputs; however we demonstrate duplicate-removed depth of &amp;gt;200x from inputs as low as 1ng, and sensitivity and precision for variant calling from 1ng libraries that is equivalent to that from ligation-based libraries prepared from 200ng of DNA. It is becoming increasingly apparent that a complete understanding of tumor genomic profiles requires the ability to detect structural variation and phasing information from low input and high coverage sequencing libraries. With this study we demonstrate the ability of the 10X platform to enable such applications from ultra-deep targeted sequencing data using only 1ng of DNA. Citation Format: Mirna Jarosz, Michael Schnall-Levin, Grace X. Y. Zheng, Patrick Marks, Sofia Kyriazopoulou-Panagiotopoulou, Patrice Mudivarti, Kristina Giorda. Using 1ng of DNA to detect haplotype phasing and gene fusions from whole exome sequencing of cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4742. doi:10.1158/1538-7445.AM2015-4742