Abstract 3619: Copy number estimation of cancer samples with genome, exome and targeted panel next generation sequencing
Authors: Shams Soheil , O’Hara Andrea , Che Zhiwei 
Published: July 2016

Next-generation sequencing (NGS) is mainly used to obtain sequence variants (SNVs). However, obtaining copy number results from NGS has gained momentum in both research and clinical applications. While microarray has traditionally been the gold standard for copy number analysis, NGS is rapidly gaining momentum as the first line analysis for tumor samples. The genomic content captured may vary from batch to batch, where targeted regions may include the full genome, all exonic regions or may be limited to exonic regions in specific genes that have a clear diagnosis, are actionable with prescription drugs or compounds in clinical trials, or have known impacts on prognosis or outcome. Thus, obtaining copy number information from a range of NGS methods is needed. As a result, we have developed a method called BAM (pooled reference) which only requires the loading of BAM files and the NGS design file to generate copy number results. BAM (pooled reference) can derive copy number results from Whole Genome Sequencing (WGS), Whole Exome Sequencing (WES), and targeted panel NGS data. This method builds a reference file out of a pool of BAM files that are either from normal controls or from unrelated experimental samples, then generates copy number calls for each experimental sample. In combination with the VCF files that contain the sequence mutations, an integrated analysis of both events can be easily carried out. To test this algorithm, five colon adenocarcinoma whole exome sequencing samples were processed through the BAM (pooled reference) algorithm in Nexus Copy Number 8.0. GC correction schemes based on a range of window size and presence or absence of GC probe content were applied to the data and assessed for overall quality. Differences in overall read-depth resulted in variable sample quality across the cohorts, however most sample quality was adequate for copy number estimation and a quality threshold was assessed. Among the samples tested, the best quality after GC correction comes from the 50kb region size with or without the probes. Next, the copy number profiles of the TCGA COAD samples from WES and microarray were compared for accuracy. Using microarray results as a reference for assessing calls greater than 5 MB, no false positive and one false negative call were observed; the single false negative call was attributable to low-level mosaicism in the tumor sample. Results indicate that relative copy number can be estimated and is comparable to the results achieved with microarray for the same targeted regions. This analysis series was then repeated using a secondary cohort of unrelated samples subjected to microarray and targeted panel NGS to validate results. The BAM (pooled reference) method has been tested in a variety of cancer samples. This is an ideal tool for copy number estimation with NGS results in cancer samples because it provides a way for non-pair matched analysis with genome, exome and targeted NGS.

Citation Format: Soheil Shams, Andrea O’Hara, Zhiwei Che. Copy number estimation of cancer samples with genome, exome and targeted panel next generation sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3619.