Somatic Point Mutation Calling in Low Cellularity Tumors

Abstract

Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.

Whole–genome characterization of chemoresistant ovarian cancer

“…Somatic SNV variant detection. High confidence somatic substitutions were identified as the intersect of the post filtered output of the mutation detection tools qSNP 53 and the Unified Genotyper 54 (GATK). In addition, supporting evidence for variants was sought in matched independent and/or orthogonal data sets such as long mate pair sequencing data produced on Life Technologies SOLiD platform and HiSeq RNA transcriptome sequencing.…”

Diff-Quik Cytology Smears from Endobronchial Ultrasound Transbronchial Needle Aspiration Lymph Node Specimens as a Source of DNA for Next-Generation Sequencing Instead of Cell Blocks

“…Sequencing of 48 genes was performed using the TruSeq Amplicon Cancer Panel (Illumina, San Diego, CA, USA) on a MiSeq system, yielding 80% of sequenced bases at a median coverage of ×936 for 54 smears (range 545–3,076) and ×513 for 41 FFPE cell blocks (range 133–1,253). Point mutations were called by qSNP [33], annotated with allele frequency in the general population ExAC and gnomaAD version 2.0.2 [34] and gene consequence using SnpEff 4.0e (build 2014-09-13). Mutations were considered high confidence and used in subsequent analysis if the variant position had a read depth > 50, the alternative allele was present in > 10% of the reads, and the variant had an allele frequency in the general population of < 1%.…”

“…Various factors influence the confidence in a somatic mutation call, including sequence depth in tumour, base qualities of alleles, number of mutant reads, and mutant allele ratio. This published somatic mutation calling strategy [33] has been designed to maximise sensitivity in specimens with low tumour purity. Postprocessing manual checks to verify calls were performed using Integrative Genomics Viewer to control the false discovery rate.…”

Long Noncoding RNAs CUPID1 and CUPID2 Mediate Breast Cancer Risk at 11q13 by Modulating the Response to DNA Damage

“…15 Duplicate aligned reads were marked with Picard (v.1.141), and aligned data were sorted by coordinates with SAMtools (v.1.3). Somatic substitutions were detected by a dual calling strategy, and substitutions were detected with qSNP 16 and the HaplotypeCaller module of the Genome Analysis Toolkit. 17 High-confidence somatic substitutions used in downstream analysis passed variant-characteristic filtering: (1) minimum coverage depth of 8 reads for control data and 12 reads for tumor data; (2) at least 5 variant-supporting reads with individual start positions and for which the variant was not within the first or last 5 bases, was supported by reads in both sequencing directions, and was more than 5 bp from a mononucleotide run of 7 or more bases in length; and (3) somatic variants had <3% variant evidence identified in the control sample.…”