Computational protocol: Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing

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Protocol publication

[…] We performed duo-based WES (two affected family members) in a cohort of 84 RD patients from 42 families, while in four cases only one exome was performed. For one family, WES was performed for three affected family members (pedigree LCA70 is depicted in ). Eight cases had been previously analyzed by our RD panel. Exomes were enriched using the SureSelect XT Human All Exon 50 Mb kit, versions 4 or 5 (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed on HiSeq 2500 systems (Illumina, San Diego, CA, USA). Reads were aligned against the human assembly hg19 (GRCh37) using Burrows—Wheeler Aligner version 0.7.5 []. We performed variant calling using SAMtools version 0.1.18 [], PINDEL version 0.2.4t [] and ExomeDepth version 1.0.0 []. Subsequently, variants were filtered using the SAMtools varFilter script and custom scripts. Shortly, only SNVs and indels in coding regions (nonsense, missense and canonical splice site variants as well as frameshift indels) having a potential effect on protein function in silico (assessed using predictions from PolyPhen-2 [http://genetics.bwh.harvard.edu/pph2/], SIFT [http://sift.bii.a-star.edu.sg/] and CADD [http://cadd.gs.washington.edu/]) were considered. From those, only private variants or those with a minor allele frequency <1% in a cohort of more than 66000 control individuals (ExAC Browser [http://exac.broadinstitute.org/]; and 6742 in-house exomes) were kept for subsequent analyses. […]

Pipeline specifications

Software tools BWA, SAMtools, Pindel, ExomeDepth, PolyPhen, SIFT, CADD
Databases ExAC browser
Applications WGS analysis, WES analysis
Organisms Homo sapiens
Diseases Retinal Dystrophies