Computational protocol: Identification of a novel mutation in the EXT1 gene from a patient with multiple osteochondromas by exome sequencing

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Protocol publication

[…] Raw read sequencing quality was assessed following completion of exome sequencing, to ensure that low quality and contaminated reads were removed. Reads with too many N bases (>10%) or low base quality (>50% bases with base quality <5) were discarded. Burrows-Wheeler Aligner (BWA) software (version hg19, build 37.1, was then used to align clean reads to the (University of California Santa Cruz (UCSC) human reference genome. Evaluation of the capture experiment was executed at the same time. Based on the BWA alignment results, Short Oligonucleotide Analysis Package snp software (version 1.03; and SAMtools software (version 0.9.41; were used to search for single nucleotide variant (SNV) and insertion and deletion (indel) mutations, respectively. Once base polymorphisms of the target region were obtained, information of interest was aligned to data from the following databases by the Huada Gene Research Institute: the National Center for Biotechnology Information Database of Short Genetic Variation (dbSNP) (, the International HapMap Project (, the 1000 Genomes Project (, the Exome Sequencing Project (ESP; esp6500siv2), the Exome Aggregation Consortium (ExAC; version 0.3) and the Kaviar database (version no. 160,204) (), which include various ethnic groups. Mutations of interest were screened, marked and analysed for gene function. Quality control, carried out before analysis for clean data and after data processing, was present throughout this pipeline in order to obtain clean data and alignments. [...] Confirmatory Sanger sequencing was carried out in the proband and his sister (II5) to validate the candidate mutation that was identified by targeted exome sequencing. Target sequences from 200 unrelated healthy controls also underwent Sanger sequencing to estimate population frequencies and the pathogenicity of the candidate mutation. Sanger sequencing was performed using the standard protocol (). Primers used were as follows: 5′-CAGTCCGGATCATTTCTGGCC-'3 and 5′-ACTGAGGTGACAACTGGTCTC-'3. Sequence comparisons and analyses were performed using DNAman 8.0 (Lynnon Biosoft, San Ramon, CA, USA) and Chromas 2.0 (Technelysium Pty, Ltd., South Brisbane, Australia), respectively. […]

Pipeline specifications

Software tools BWA, SAMtools, DNAMAN
Databases dbSNP International HapMap Project Kaviar
Application WES analysis
Organisms Homo sapiens