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Protocol publication

[…] tom Agilent SureSelectXT Kit to capture genomic sequences of coding exons, splice junctions, 5′ UTR and 3′ UTR regions in 765 selected genes with indexing barcodes for each sample. We sequenced a total target length of 5.28Mb including 1.76 MB of coding exons. We isolated the hybridized library fragments, quantitated by qPCR and sequenced, and then sequenced paired-end reads with the Illumina HiSeq2000™ sequencing platform. We required sequencing data for each sample to have over 10X coverage at greater than 90% targeted regions and over 20X coverage at greater than 80% targeted regions., We aligned sequence reads in each individual to the human reference genome (NCBI build 37.3, hg19) with BWA (v0.59). We called the consensus genotypes in the target regions with The Genome Analysis Toolkit (GATK, v2.18) with the workflow and parameters recommended in the best practice variant detection with GATK v4,. We applied GATK duplicate removal, indel realignment, base quality score recalibration, and performed multi-sample SNP and indel discovery and genotyping across all samples simultaneously using variant quality score recalibration (VQSR). Other than high quality variants assigned “PASS” by VQSR, we also included only those variants in lower tranches with truth sensitivity between 99.0-100 that were also separately recorded in the exome sequencing project (ESP) database of 6500 samples. We annotated variants with snpEff (v2.05)., We further excluded SNPs failing Hardy-Weinberg test in controls (p<10−6). We also excluded alleles from further analysis that had high missing genotype data (>1%), likely due to systematic low coverage or difficulty mapping reads across many samples. We also excluded samples with high missing genotype data (>1%) for common alleles with >1% frequency in our data set. For burden testing, we only tested those genes on autosomes that obtained >10X coverage at an average of >90% of the targeted region and had rare coding variants (n=681)., We sequenced the CFI gene in individuals that were carriers of rare CFI variants at Beckman Coulter Genomics. We designed p […]

Pipeline specifications

Software tools BWA, GATK, SnpEff
Diseases Retinal Degeneration