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, Local realignment of insertions/deletions (indels) and base quality score recalibration were performed using GATK (version 3.6, 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 ( and Exome Aggregation Consortium (ExAC, 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 (; and (5) variants that passed manual confirmation using the Integrative Genomics Viewer (IGV, were retained. Effects of variants on splicing signals were evaluated using the Human Splicing Finder (version 3.0, 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