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[…] ing kit designed to capture and generate indexed Illumina-compatible libraries enriched for a 250 kb region of 5p15.33. Genomic capture and library construction using the Agilent SureSelect system was performed as recommended by the manufacturer with a hybridization capture time of 72 hours., Captured and indexed libraries were multiplexed into 8 pools and sequenced on 8 lanes of an Illumina HiSeq instrument. Due to variations in pooling, under-performing libraries were re-sequenced on an additional lane. Sequencing was carried out as described by the manufacturer (Illumina) generating 2×101 bp indexed pair-end reads., We called bases and generated demultiplexed fastq files using Illumina's CASAVA pipeline. Reads were aligned using Novoalign V2.07.06 ( and any reads with a mapping quality less than 30 were deemed to be not uniquely aligned and were discarded. Next, we locally realigned reads using version 1.0.5083 of the Genome Analysis Tool Kit . Any read with more than 2 mismatches was discarded. We did not remove potential PCR duplicates because each pool contained reads from multiple individuals and it was important to maintain the relative allele frequencies present in the data. Detection of PCR bias was handled later by a metric built into the variant calling step., Samtools and downstream filtering were used to call single nucleotide variants (SNVs) in each individual pool based only on high quality (q> = 30) bases at positions that did not fall into UCSC's repeat mask or self chaining tracks. Any position where there were fewer than 200 reads available or when there was potentially a start point bias (a score of less than 1.25 using our metric, which is described next) was marked as having insufficient coverage and was excluded in the calls for the pool. The depth and start point bias cutoffs were selected by using the percentage of variants called that were reported in dbSNP as an indication of our true positive rate., Our start point bias metric […]

Pipeline specifications