Computational protocol: Targeted Sequencing of Large Genomic Regions with CATCH-Seq

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Protocol publication

[…] Pass filter sequence linked to each index and inline barcodes was demuxed from fastq files and assigned to individual libraries using software that was also used to design inline barcoded adapter sequences. Bordering adapter sequences were removed from reads using the AdapterRemoval software . Standard sequencing fastq files were aligned with BWA in paired end format to the human hg19 reference genome. PCR duplicates were removed and sam files were filtered by q20 mapping quality. For determination of CNV boundaries, read depth was extracted from wig files in non-overlapping 100 bp segments across the length of the target genomic coordinates and the fraction of total bases per segment was calculated. LogR values were determined across the target site by log2 of individual cases read fraction divided by the median read fraction of all control samples. For determination of CpG methylation across a target region, human hg19 reference forward and reverse strand sequences were each bisulfite converted in silico and a reference for mapping was built with Bismark. In conjunction with Bowtie2, Bismark was modified to function with local mode for read mapping. Duplicate reads were removed, and methylation values were extracted using Bismark. CpGs with less than a 20× minimum depth coverage were filtered and percent methylation values were used for further data analysis. Alignment files demonstrating coverage of one chr11 target by CATCH-Seq in comparison to WGS from 15 merged individuals from 1000 genomes data within this same target region, in addition to our CATCH-Seq repeat blocking analysis on another chr11 target have been submitted and are available from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) with BioProject accession number [SRP042633]. […]

Pipeline specifications

Software tools AdapterRemoval, BWA, Bowtie2
Application WGS analysis