Computational protocol: Whole Exome Sequencing for a Patient with Rubinstein-Taybi Syndrome Reveals de Novo Variants besides an Overt CREBBP Mutation

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Protocol publication

[…] Blood samples were drawn from the patient and both biological parents. Genomic DNA was extracted using the DNeasy Blood & Tissue kit (QIAGEN, Valencia, CA, USA). Raw reads in FASTQ format from exome sequencing were aligned to the hg19 reference genome from the UCSC genome browser using the Burrows–Wheeler Aligner (BWA) [] with default parameters. Aligned reads were processed and polymerase chain reaction (PCR) duplicates were removed with SAMtools []. Single-nucleotide variants (SNVs) and insertions/deletions (indels) were identified using the Genome Analysis Toolkit (GATK) []. Regions near short indels were realigned using the IndelRealigner function in GATK according to default parameters. SNVs and indels affecting coding sequences or splicing sites were annotated by an in-house custom-made annotation system. All genomic changes were filtered against the Single Nucleotide Polymorphism Database (dbSNP; build 141) and the in-house control database comprising 54 Korean individuals.We classified the de novo variants using the recommendations of Ambry Genetics (Scheme for AD and XD Mendelian disorders) into five categories: Benign, VLB (Variant, Likely benign), VUS (Variant, Unknown significance), VLP (Variant, Likely pathogenic), and Pathogenic mutation. Variants classified as VUS, VLP, and Pathogenic mutation were also confirmed using an IGV browser. To evaluate the mutations in the general population, we also applied Sanger sequencing for these mutations to 80 normal subjects belonging to the Korean ethnic group. […]

Pipeline specifications

Software tools BWA, SAMtools, GATK, IGV
Databases UCSC Genome Browser
Applications WES analysis, Genome data visualization
Organisms Homo sapiens
Diseases Rubinstein-Taybi Syndrome, Limb Deformities, Congenital