Computational protocol: A novel PRKAG2 mutation in a Chinese family with cardiac hypertrophy and ventricular pre-excitation

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Protocol publication

[…] Genomic DNA was extracted from peripheral blood leukocytes using the QIAamp DNA Blood Midi Kit (QIAGEN, Hilden, Germany), according to standard protocols. The proband had previously been screened using a targeted sequencing panel containing 64 candidate genes reported to be causative of inherited cardiomyopathy. No pathogenic mutations were detected. Next, three affected individuals (II-9, III-6, and III-8) and one unaffected individual (II-2) were selected for whole-exome sequencing. The Agilent SureSelect Human All Exon V5 capture kit (Agilent, Santa Clara, CA, USA) was used for exome capture. Samples from the family quartette were multiplexed on a single lane and 101-bp, paired-end sequencing was performed using Illumina’s HiSeq4000 platform (Illumina, Inc, San Diego, CA, USA) to an average depth of 141×.Sequence data were aligned to the human reference genome (GRCh37/hg19) with BWA followed by sorting and marking of duplicate reads using Picard (version 2.4, http://broadinstitute.github.io/picard/). Local realignment of insertions/deletions (indels) and base quality score recalibration were performed using GATK (version 3.6, https://www.broadinstitute.org/gatk/). GATK was also used to call and filter variants within a genome–wide region. The resultant variants were annotated with ANNOWAR and sequentially filtered using the following criteria: (1) variants with an East Asian minor allele frequency ≥0.01 in the databases for 1000 Genomes Project (http://browser.1000genomes.org) and Exome Aggregation Consortium (ExAC, http://exac.broadinstitute.org) were removed; (2) variants in exons and splicing sites were retained; (3) certain types of variants were retained, including nonsynonymous, frameshift, nonframeshift insertion/deletion, stopgain and unknown; (4) variants in genes expressed in the heart according to the Human Protein Atlas (www.proteinatlas.org); and (5) variants that passed manual confirmation using the Integrative Genomics Viewer (IGV, http://software.broadinstitute.org/software/igv) were retained. Effects of variants on splicing signals were evaluated using the Human Splicing Finder (version 3.0, www.umd.be/HSF3/HSF.html).Candidate variants were validated by Sanger sequencing. Three hundred unrelated healthy control samples were subjected to variant examination. […]

Pipeline specifications

Software tools BWA, Picard, GATK, IGV, HSF
Databases Human Protein Atlas
Applications WGS analysis, WES analysis
Organisms Homo sapiens
Chemicals Gadolinium