Similar protocols

To access compelling stats and trends, optimize your time and resources and pinpoint new correlations, you will need to subscribe to our premium service.

Subscribe

Pipeline publication

[…]  − 110 (GenTegra LLC, Pleasanton, CA), according to manufacturer’s protocol. An additional blood contamination purification step was done using the QIAamp Blood DNA Mini Kit (QIAGEN, Hilden, Germany). A commercially available exome capture kit (Illumina Nextera Rapid Exome Capture kit, Illumina Inc., San Diego, CA) was used for exome enrichment and capture, followed by rapid exome sequencing using the MiSeq system (Illumina Inc., San Diego, CA). Though the kit is designed to capture human exomes, there is enough sequence homology between human and dog genomes to ensure a successful capture of canine exomes with a large coverage area []., The quality of the raw exome reads was analyzed using FastQC [], and Trimmomatic [] was used to filter out bad reads. The retention criteria were: leading bases with quality higher than 25, trailing not less than 20, four base sliding window cutoff of 25 and reads over 50 bases long. Each sample exome was then mapped to the reference dog genome CanFam3.1 (Broad CanFam3.1/canFam3 Assembly, September 2011) using Bowtie2 [] with default parameters, followed by variant calling using SAMtools [] and BCFtools []. Then SnpEff [] was used to annotate each called variant, generating a variant call file (VCF). Another round of annotation was carried out to remove variants not called in all 8 samples, creating a second VCF file. A commercially available sequence annotation software package (Golden Helix SNP and Variation Suite, Golden Helix Inc., Bozeman, MT) was used to differentiate between the LS negative and LS positive dogs. Two separate selection settings were used to identify variants between the LS negative and LS positive groups. First setting: unaffected = “a/a”, affected = “b/b”. Second setting: unaffected = “a/a”, affected = “a/b” or “b/b” (where “a” = wild/reference allele; “b” = affected/alternate allele; “a/a” and “b/b” = homozygous for wild and alternate alleles, respectively; and “a/b” = heterozygous). The 2nd setting was intended to account for variant allele having an effect on LS in both homozygous and heterozygous states. Exonic variants were identified by aligning the […]

Pipeline specifications

Software tools FastQC, Trimmomatic, Bowtie2, SAMtools, bcftools, SnpEff, SVS
Organisms Canis lupus familiaris, Homo sapiens
Diseases Proteostasis Deficiencies, Constriction, Pathologic