Additional file 1: Figure S1. of Replicate exome-sequencing in a multiple-generation family: improved interpretation of next-generation sequencing data
Systematic approach to study exome capture variability in exome-sequencing (A) Three-generation pedigree in which two individuals have an undiagnosed disease that segregated as an autosomal dominant disorder and a de novo variation arose in the second generation. (B) Model of individual subject sample blood DNA processing and sequencing. A sample of blood went through DNA isolation, and independent libraries (in triplicate) were sequenced to appropriate comparable depth and analyzed for various quality control parameters, target coverage, read depth and nucleotide variation detection. (C) Schematic illustration of sequencing read depth vs. targeted genomic region in relation to exome sequencing in replicate. Listed are also the main approaches taken in this study to analyzed exome replicate data. (D) Two main hypotheses tested using replicate exome data: (i) Biases in sequence capture resulting in poor coverage are addressable through repetition (ii) Library replication is beneficial to overall interpretation of sequence variation data. Figure S2. Titration of percentage targeted exome sequenced as a function of depth of sequencing thresholds in all three replicates per sample. Error bars show standard error for replicate sequencing. As expected, higher depth of sequencing thresholds (x-axis) result in higher-coverage (y-axis) variability in replicate exome data. Table S2.Titration of percentage target exome sequenced as a function of depth of sequencing thresholds (attached excel file). Table S3. Primers used for and results of Sanger sequencing analysis for resolution of replicate discordances in NGS data. Table S4. Primers used for and results of Sanger sequencing validation of de novo variants detected using NGS. (Concordant NGS and Sanger genotypes are highlighted in yellow). Figure S3. Box-plot of GC-content distribution in all first-exons (blue) and high-GC content (>70% GC; >=50 bp length). Table S5. Evaluation of coverage of targeted exons with high GC content (attached excel file). Table S6. Quote from Illumina for exome enrichment kits. Quotes in red indicate costs when the study was undertaken. Nextera prices, and other kit prices (in white) reflect current costs per sample (see last column). (DOC 1 mb
