A Variant Detection Pipeline for Inherited Cardiomyopathy–Associated Genes Using Next-Generation Sequencing Théo G.M. Oliveira, Miguel Mitne-Neto, Louise.

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Presentation transcript:

A Variant Detection Pipeline for Inherited Cardiomyopathy–Associated Genes Using Next-Generation Sequencing Théo G.M. Oliveira, Miguel Mitne-Neto, Louise T. Cerdeira, Julia D.C. Marsiglia, Edmundo Arteaga-Fernandez, José E. Krieger, Alexandre C. Pereira The Journal of Molecular Diagnostics Volume 17, Issue 4, Pages 420-430 (July 2015) DOI: 10.1016/j.jmoldx.2015.02.003 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Workflow of sample processing in the validation design. Pipeline was designed in CLC Genomics Workbench 6.5 (CLCbio, Boston, MA). Quality-based variant call was tested for five parameter sets of coverage and variant allele frequencies (VAFs) (30× of coverage with 35%, 25%, and 20% of VAF; 10× of coverage with 35% and 20% of VAF). BAM, .bam file; BED, .bed file; dbSNP, Single Nucleotide Polymorphism database; FDR, false-discovery rate; GTF, .gtf file; HCM, hypertrophic cardiomyopathy; PGM, Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA); PPV, positive predictive value; Q, Phred value (Q score); QC, quality control; SNV, single-nucleotide variant; VCF, .vcf file. The Journal of Molecular Diagnostics 2015 17, 420-430DOI: (10.1016/j.jmoldx.2015.02.003) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Histogram based on the mean coverage frequency of target regions. Bins are plotted in a width of 10. The black bar indicates 28 target regions with a mean coverage in the range of 0 to 10×, which were considered blind spots in the assay. The Journal of Molecular Diagnostics 2015 17, 420-430DOI: (10.1016/j.jmoldx.2015.02.003) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Boxplot showing the coverage distribution of all samples sequenced. Groups with interleaving colors denote different runs in which each sample was processed. Boxes represent interquartile ranges (IQRs); horizontal line, the median coverage; whiskers are in Tukey format and represent data points that are <1.5 × IQR from first/third quartiles. The Journal of Molecular Diagnostics 2015 17, 420-430DOI: (10.1016/j.jmoldx.2015.02.003) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Interassay (A) and intra-assay (B) reproducibility of NA12878 triplicates in 20× coverage/10% variant allele frequencies. The Journal of Molecular Diagnostics 2015 17, 420-430DOI: (10.1016/j.jmoldx.2015.02.003) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 FastQC visual plot of read linear quality. Plot was generated from 62,566,137 sequences from 60 sequenced samples. The Journal of Molecular Diagnostics 2015 17, 420-430DOI: (10.1016/j.jmoldx.2015.02.003) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S2 Read quality distribution. Plot was generated from 62,566,137 sequences from 60 sequenced samples. The great majority of reads are of Phred value (Q score) >20, and the mean point of quality scores is around Q29. The Journal of Molecular Diagnostics 2015 17, 420-430DOI: (10.1016/j.jmoldx.2015.02.003) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions