Validation of a Next-Generation Sequencing Pipeline for the Molecular Diagnosis of Multiple Inherited Cancer Predisposing Syndromes Paula Paulo, Pedro.

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Presentation transcript:

Validation of a Next-Generation Sequencing Pipeline for the Molecular Diagnosis of Multiple Inherited Cancer Predisposing Syndromes Paula Paulo, Pedro Pinto, Ana Peixoto, Catarina Santos, Carla Pinto, Patrícia Rocha, Isabel Veiga, Gabriela Soares, Catarina Machado, Fabiana Ramos, Manuel R. Teixeira The Journal of Molecular Diagnostics Volume 19, Issue 4, Pages 502-513 (July 2017) DOI: 10.1016/j.jmoldx.2017.05.001 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Comparison of variant call characteristics of the single-nucleotide variants (SNVs) and insertions/deletions (INDELs) present in the control series among the four analysis software programs. Variant coverage (A) and alternative variant frequency (B) for each SNV or INDEL. For simplification, only the mutated gene is indicated. Arrow indicates the TP53 mutation missed by MiSeq Reporter. The Journal of Molecular Diagnostics 2017 19, 502-513DOI: (10.1016/j.jmoldx.2017.05.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Analysis of the large NF1 INDEL c.7000-5_7031dup found in sample N1. A: Golden Helix GenomeBrowse images of the sequence alignments (.bam files) performed by the four alignment software programs: MiSeq Reporter, Isaac Enrichment, Burrows-Wheeler Aligner (BWA) Enrichment, and NextGENe. The number of reads with the variant and the coverage of the genomic position are shown. B: Electropherogram of the NF1 mutation c.7000-5_7031dup validated by Sanger sequencing (reverse strand) in N1 case (top panel) in comparison with a normal control sample (bottom panel). The duplicated nucleotides and the insertion site are indicated. The Journal of Molecular Diagnostics 2017 19, 502-513DOI: (10.1016/j.jmoldx.2017.05.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Coverage performance for the target regions encompassing the 23 genes queried in this study. A: Mean coverage variation by gene. Deviations are shown regarding the maximum and minimum values. B: Target regions showing recurrent (in >10% of the samples) coverage <20×. Bars (left y axis) show the fraction of the target region missed (dark gray) relative to the target region size (light gray), and dark line (right y axis) shows the percentage of samples, for each region. n = 32 (B). The Journal of Molecular Diagnostics 2017 19, 502-513DOI: (10.1016/j.jmoldx.2017.05.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Pipeline analysis for targeted NGS of multiple inherited cancer syndromes using the TruSight Cancer panel. In our analysis pipeline, .fastq files are used for alignment and variant calling in Isaac Enrichment, Burrows-Wheeler Aligner (BWA) Enrichment, and NextGENe software, and .vcf files are then imported to GeneticistAssistant for variant filtering. Supervised analysis of filtered variants will allow the identification of disease-associated variants and/or of pathogenic variants associated with cancer predisposing syndromes outside the clinical context (incidental finding), with both being supported (if needed) by visual inspection of .bam files in Golden Helix GenomeBrowse (GB). Variants called because of sequencing errors are discarded on visual inspection of .bam files in Golden Helix GB. 1000G, 1000 Genomes Project; ACMGG, American College of Medical Genetics and Genomics; Alt.var.freq., percentage of reads with alternative allele; CDS, coding sequence; EVS, Exome Variant Server; ExAC, Exome Aggregation Consortium; In-house freq., variant frequency among all of the samples in our in-house database; MAF, minor allele frequency. The Journal of Molecular Diagnostics 2017 19, 502-513DOI: (10.1016/j.jmoldx.2017.05.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 Golden Helix GenomeBrowse images of the sequence alignments (.bam files) of MiSeq Reporter for the missed variants. A: TP53 mutation c.818_827del in sample C3. B: NF1 mutation c.4092_4098del in sample N4. C: NF1 mutation c.2204_2213del in sample N8. The number of reads with the variant and the coverage of the genomic position are shown. Screenshots of the sequence alignments of Isaac Enrichment are shown for comparison. The Journal of Molecular Diagnostics 2017 19, 502-513DOI: (10.1016/j.jmoldx.2017.05.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S2 Family pedigrees of NF1 patients harboring the missense mutation c.1496T>G, p.Leu499Arg. Family pedigree of patient N9 is shown to the right and of patient N10 to the left. The Journal of Molecular Diagnostics 2017 19, 502-513DOI: (10.1016/j.jmoldx.2017.05.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S3 Average exon-level coverage of the 32 samples for each of the 23 genes included in the TruSight Cancer panel evaluated in this study. The y axis represents coverage; and the x axis, the coding exons. For each exon, maximum, minimum, and average coverage are shown. Exon 4 of BRCA1 and exons 2 and 3 of MUTYH do not exist. The Journal of Molecular Diagnostics 2017 19, 502-513DOI: (10.1016/j.jmoldx.2017.05.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S4 Venn diagram showing the number of genes from the Cancer Gene Census database (COSMIC-CGC) and from Rahman's46 list of cancer predisposing genes targeted by the TruSight Cancer panel. The Journal of Molecular Diagnostics 2017 19, 502-513DOI: (10.1016/j.jmoldx.2017.05.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions