Similar protocols

Pipeline publication

[…] for the final analysis. In order to correct for the multiple testing situation, we determined an empirical significance threshold by performing 100,000 permutations of the dataset with arbitrarily assigned phenotypes., We used the dog CanFam 3 genome assembly derived from a Boxer as reference genome sequence. All numbering within the canine CUBN gene corresponds to the accessions NM_001003148.1 (mRNA) and NP_001003148.1 (protein)., We prepared a fragment library with 300 bp insert size and collected one lane of illumina HiSeq2000 paired-end reads (2×100 bp). We obtained a total of 125,457,731 paired-end reads or roughly 10× coverage. We mapped the reads to the dog reference genome with the Burrows-Wheeler Aligner (BWA) version 0.5.9-r16 with default settings and obtained 243,130,060 uniquely mapping reads. After sorting the mapped reads by the coordinates of the sequence with Picard tools, we labeled the PCR duplicates also with Picard tools (http://sourceforge.net/projects/picard/). We used the Genome Analysis Tool Kit (GATK version 0591, ) to perform local realignment and to produce a cleaned BAM file. Variants calls were then made with the unified genotyper module of GATK. Variant data for each sample were obtained in variant call format (version 4.0) as raw calls for all samples and sites flagged using the variant filtration module of GATK. Variant calls that failed to pass the following filters were labeled accordingly in the call set: (i) Hard to Validate MQ0≥4 & ((MQ0/(1.0 * DP)) >0.1); (ii) strand bias (low Quality scores) QUAL <30.0 || (Quality by depth) QD <5.0 || (homopolymer runs ) HRun >5 || (strand bias) SB >0.00; (iii) SNP cluster window size 10. The snpEFF software together with the CanFam 3.1 annotation was used to predict the functional effects of detected variants., We used Sanger sequencing to confirm the illumina sequencing results and to perform targeted genotyping for selected variants. For these experiments we amplified PCR products using AmpliTaqGold360Mastermix (Applied Biosystems). PCR products were directly sequenced on an ABI 3730 capillary sequencer (Applied Biosystems) after treatment with exonuclease I and shrimp alkaline phosphatase. We analyzed the sequence data with Sequencher 4.9 (GeneCodes)., The authors are grateful to referring veterinarians and to all dog owners and breeders who donated blood samples and shared pedigree data of their dogs. We thank Brigitta Colomb and Muriel Fragnière for expert technical assistance, the NCCR Genomics Platform in Geneva for SNP genotyping, and the Next Generation Sequencing Platform of the University of Bern for performing the whole genome sequencing experiment.] […]

Pipeline specifications

Software tools BWA, Picard, GATK, SnpEff, Sequencher