Computational protocol: Whole exome sequencing in an Indian family links Coats plus syndrome and dextrocardia with a homozygous novel CTC1 and a rare HES7 variation

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Protocol publication

[…] Whole exome sequencing (WES) was carried out in three members of this family comprising the proband and both the parents. For exome sequencing, 2 μg of DNA was used for fragmentation, and DNA library preparation was carried out according to Illumina DNA sample prep protocol.v2. Exonic regions were captured and enriched using Truseq Illumina exome capture kit.v2 protocol. Cluster generation using cBOT was carried out for exome enriched libraries on Illumina flow cell.v3 followed by 100 bp paired end sequencing on Hiseq2000 using Illumina SBS kit.v3 protocol. Nearly 12 Gb raw data were generated for each sample. The base calls for all the sample reads were analyzed on CASAVA and subsequent pipeline for data processing is described in the following section. Whole exome sequencing analysis was performed as described before [] with slight modifications at the level of raw read clipping (for details see Additional file : Table S2 and Text). From WES data, variation data (known and novel variations) for 15 candidate genes involved in telomere length maintenance activity (TERF2IP, POT1, TERF1, OBFC1/STN1, CTC1, TERT, DKC1, WRAP53, TINF2, TPP1, TERF2, TEN1, TERC, NHP2 and NOP10) (Additional file : Table S3) and 28 genes associated with dextrocardia/situs-inversus phenotype were initially analyzed (Additional file : Tables S4 and S5). Following candidate gene analysis, the WES data was analyzed independently to obtain any likely disease associated candidate variation. The variant filtering protocol was performed essentially as described earlier [].In short, total variations (passing quality filters) → protein deleterious variations → Novel variants (absent in dbSNP/TGP) → homozygous/compound heterozygous variations → In silico predicted damaging variations. [...] The combined variant calling file (vcf) of WES data of all the samples was analyzed for homozygosity mapping using the web based homozygosity mapper tool with default settings (http://www.homozygositymapper.org/). […]

Pipeline specifications

Software tools BaseSpace, HomozygosityMapper
Application WES analysis
Organisms Homo sapiens
Diseases Bone Diseases, Metabolic, Bone Marrow Diseases, Brain Diseases, Dextrocardia, Hypertension, Telangiectasis, Fractures, Bone, Leukoencephalopathies
Chemicals Amino Acids