Computational protocol: Next-generation sequencing identifies pathogenic and modifier mutations in a consanguineous Chinese family with hypertrophic cardiomyopathy

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Protocol publication

[…] Mapping of the sequencing reads to the human genome reference sequence (hg19) was performed with the burrows-wheeler alignment tool.[] The Short Oligonucleotide Analysis Package (SOAPsnp) and the Genome Analysis Toolkit were used to discover single-nucleotide polymorphism and insertion-deletion, respectively.[–] Gene related annotation was mainly done with ANNOVAR.[] The pathogenicity of a variant was determined based on frequency in the population and in silico prediction. We excluded common variants (frequency > 1%) present in the National Heart, Lung, and Blood Institute ESP (Exome Sequencing Project), the 1000 Genomes database, and exomes generated in house from 550 unaffected Chinese control samples. Variants annotated in the Human Gene Mutation Database (HGMD) were regarded as disease-causing. All nonsynonymous variants were subjected to in silico analysis, which included functional annotation algorithms such as SIFT, PolyPhen2, GERP++, and MutationTaster. All variants identified by NGS and deemed as pathogenic were verified by Sanger sequencing, using primers designed from Primer 3 online software (http://bioinfo.ut.ee/primer3–0.4.0/primer3/input.htm). […]

Pipeline specifications

Software tools SOAPsnp, GATK, ANNOVAR, PolyPhen, GERP, MutationTaster, Primer3
Applications WES analysis, qPCR
Organisms Homo sapiens
Diseases Cardiomyopathy, Hypertrophic, Cardiomyopathies
Chemicals Aldosterone, Calcium