title: Confidence-based somatic mutation evaluation and prioritization.
abstract: Next generation sequencing (NGS) has enabled high throughput discovery of somatic mutations. Detection depends on experimental design, lab platforms, parameters and analysis algorithms. However, NGS-based somatic mutation detection is prone to erroneous calls, with reported validation rates near 54% and congruence between algorithms less than 50%. Here, we developed an algorithm to assign a single statistic, a false discovery rate (FDR), to each somatic mutation identified by NGS. This FDR confidence value accurately discriminates true mutations from erroneous calls. Using sequencing data generated from triplicate exome profiling of C57BL/6 mice and B16-F10 melanoma cells, we used the existing algorithms GATK, SAMtools and SomaticSNiPer to identify somatic mutations. For each identified mutation, our algorithm assigned an FDR. We selected 139 mutations for validation, including 50 somatic mutations assigned a low FDR (high confidence) and 44 mutations assigned a high FDR (low confidence). All of the high confidence somatic mutations validated (50 of 50), none of the 44 low confidence somatic mutations validated, and 15 of 45 mutations with an intermediate FDR validated. Furthermore, the assignment of a single FDR to individual mutations enables statistical comparisons of lab and computation methodologies, including ROC curves and AUC metrics. Using the HiSeq 2000, single end 50 nt reads from replicates generate the highest confidence somatic mutation call set.
authors.affiliation: TRON - Translational Oncology at Johannes Gutenberg University of Mainz Medicine, Mainz, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Löwer
authors.forename: Bernhard Y
authors.initials: BY
authors.lastname: Renard
authors.forename: Jos
authors.initials: J
authors.lastname: de Graaf
authors.forename: Meike
authors.initials: M
authors.lastname: Wagner
authors.forename: Claudia
authors.initials: C
authors.lastname: Paret
authors.forename: Christoph
authors.initials: C
authors.lastname: Kneip
authors.forename: Ozlem
authors.initials: O
authors.lastname: Türeci
authors.forename: Mustafa
authors.initials: M
authors.lastname: Diken
authors.forename: Cedrik
authors.initials: C
authors.lastname: Britten
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kreiter
authors.forename: Michael
authors.initials: M
authors.lastname: Koslowski
authors.forename: John C
authors.initials: JC
authors.lastname: Castle
authors.forename: Ugur
authors.initials: U
authors.lastname: Sahin
mesh_terms: D000818:Animals; D016477:Artifacts; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000235:genetics*; D059472:Exome / Q000235:genetics*; D005189:False Positive Reactions; D008545:Melanoma / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004273:DNA, Neoplasm