Computational protocol: Identification of Novel Variants in LTBP2 and PXDN Using Whole Exome Sequencing in Developmental and Congenital Glaucoma

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Protocol publication

[…] WES was employed to identify the disease-associated genes in four subjects, belonged to three families. Genomic DNA was prepared from peripheral venous blood, and was randomly fragmented into 200–400 base pair (bp) fragments with the Covaris Acoustic System according to the manufacturer’s instructions (Covaris, Inc., Woburn, MA). Kapa Library preparation (Kapa Biosystems, Inc., Wilmington, MA) was performed on a Caliper Sciclone NGS workstation (Caliper Life Sciences, Hopkinton, MA) followed by capture using the Nimblegen SeqCap EZ V2 kit (Roche Nimblegen, Inc., Madison, WI). Each captured library was then loaded on an Illumina HiSeq2000 sequencer using Illumina TruSeq V3 chemistry (Illumina, Inc., San Diego, CA).Downstream analyses included demultiplexing (CASAVA software, Illumina), alignment to the human reference genome (GRCh37, UCSC hg19) using the Burrows-Wheeler alignment (BWA)[] tool. Alignments were sorted by Picard ( and subsequently processed by GATK [].Finally, the mean depth of coverage was determined using GATK, and Free mix values were estimated through verify BAMid []. Samples that passed technical QC metrics were genotyped to gVCF level through GATKs Haplotype Caller. Indels, SNVs, microinsertions and microdeletions were filtered separately using GATKs Variant-Quality Score Recalibration. Both the SNV and indel sets were annotated using ANNOVAR []. Only mapped reads were used for subsequent analysis. They were annotated with information from the University of California, Santa Cruz genome annotation database (, consensus coding sequence (, Ensembl (, RefSeq (, MirBase (, and EntrezGene ( variants obtained through WES were first filtered against several public databases for the minor allele frequency (MAF) < 0.5%, including dbSNP135, 1000 Genomes Project databases (, 1000g2012April_all version), NHLBI GO Exome Sequencing Project (ESP6500,, esp6500si_all version) and the Human Genetic Variation Database (HGVD, Subsequently, only coding nonsynonymous variants, frameshift, and splice site variants were analyzed. To predict the functional impact of the sequence variants on the encoded protein, the pathogenicity of missense variants was evaluated by publically available tools including PhyloP, Grantham, polymorphism phenotyping v-2 (PolyPhen-2) (version 2.1.0 r367;, MutationTaster, and sorting intolerant from tolerant (SIFT; validation and segregations analysis of variants identified by WES were carried out by performing standard PCR and Sanger sequencing using ABI BigDye chemistry (Applied Biosystems Inc, Foster City, California, USA), and was processed through an automated ABI 3730 Sequencer (Applied Biosystems, Inc). […]

Pipeline specifications

Software tools BaseSpace, BWA, Picard, GATK, ANNOVAR, PHAST, PolyPhen, MutationTaster, SIFT
Databases dbSNP CCDS miRBase HGVD
Application WES analysis
Organisms Homo sapiens
Diseases Glaucoma