Computational protocol: Combination of Whole Genome Sequencing, Linkage, and Functional Studies Implicates a Missense Mutation in Titin as a Cause of Autosomal Dominant Cardiomyopathy With Features of Left Ventricular Noncompaction

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Protocol publication

[…] SNP array genotyping was performed using the Illumina HumanCytoSNP-12v1 BeadChip (Illumina, San Diego, CA), containing nearly 300 000 genetic markers, according to the manufacturer’s protocols. A refined subset of roughly 24 000 SNPs in approximate linkage equilibrium was generated using the software PLINK v1.07 and the HapMap genotype file available from the PLINK website ( Linkage analysis of the SNP subset was performed using MERLIN v1.1.2, specifying an autosomal dominant disease model. Genomic intervals with logarithm of the odds scores >0, compatible with segregation of variants in these regions, were selected for downstream analyses.WGS was performed on genomic DNA extracted from peripheral blood as part of the WGS500 project as described previously.Sequence reads from the affected individuals were mapped to the human reference genome (hs37d5 version of build 37) using STAMPY. Duplicate reads were removed with PICARD ( The software Platypus (version 0.8.1, default parameters) was used jointly on the two.bam files to call SNPs and short (<50 bp) indels across both samples.All the 5 946 161 identified variants were annotated with an in-house pipeline based on the Variant Effect Predictor Ensembl framework (version 77). Several additional databases were used to integrate the information provided by Variant Effect Predictor (Table I in the Data Supplement). Known associations with diseases were screened using HGMD ( and ClinVar.Variants were filtered by in-house Python scripts based on criteria outlined in Table I in the Data Supplement (steps 1–10), followed by manual inspection (steps 11–13). The variants remaining after step 10 are documented in Results and in Tables II and III in the Data Supplement. Confirmatory Sanger sequencing was performed with the primers listed in Table IV in the Data Supplement.Both SNP and WGS data were interrogated also for clinically relevant copy number variants using Nexus Copy Number 7.5.2 Discovery Edition (BioDiscovery, Hawthorne, CA; see Methods in the Data Supplement). […]

Pipeline specifications

Software tools PLINK, Merlin, Stampy, Picard, Platypus, VEP
Databases ClinVar HGMD
Applications WGS analysis, GWAS
Organisms Homo sapiens, Dipturus trachyderma
Diseases Cardiomyopathies