ena-STUDY-ESTACION BIOLOGICA DONANA-27-11-2014-13:01:18:473-1850
Capture and resequence of dog and wolf samples using Agilent SureSelect and Illumina
Mouth swab samples were collected from 5 mongrel dogs (dogs of unknown ancestry, except one pure breed German shepherd) and blood or tissue samples from 22 dead wolves from widespread geographic locations (British Columbia, Canada, n=2; Finland, n=3; Italy, n=3; North Western Territories, Canada, n=3; Spain, n=4; Sweden, n=4; United States, n=2). Except for the wolves from the United States, which were captive and from which blood samples were obtained, all other wolves were wild and died for reasons unrelated to this study. We extracted DNA using the QIAgen DNeasy kit and prepared Illumina paired-end libraries for each sample using the Agilent protocol for indexed paired-end Illumina libraries and Agilent SureSelect capture system. Briefly, DNA was sheared with a Covaris S2 device (Covaris, Inc. Woburn, MA, USA), end-repaired, A-tailed, ligated with Illumina's indexing-specific paired-end adaptors and PCR amplified for 5 cycles. We enriched for 16 chromosomal regions, each containing a locus associated with a distinct phenotypic character (morphological or behavioral) in dogs in a central position, and 100-150 kb upstream and downstream (totalling ca. 200-300 kb in length). We enriched with a custom Agilent SureSelect RNA oligo kit. The oligo-targeted regions added up to 2.48 Mbp (repetitive regions excluded) and encompassed approximately 3.96 Mbp of the dog genome. Libraries were then Illumina indexed/barcoded in a PCR of 13 cycles. A Nanodrop spectrophotometer and a Bioanalyzer instrument were used to assess both quality and quantity of the samples at various steps during the laboratory procedures. Libraries were validated using real-time quantitative PCR, pooled and then 90 or 100-bp paired-end sequenced on 4 lanes of an Illumina GAIIx machine, yielding 84,682,789 of paired-end reads. For read alignment and SNP calling we followed the Broad Institute Best Practice Variant Detection guide (http://www.broadinstitute.org/gatk/guide/best-practices).
Mouth swab samples were collected from 5 mongrel dogs (dogs of unknown ancestry, except one pure breed German shepherd) and blood or tissue samples from 22 dead wolves from widespread geographic locations (British Columbia, Canada, n=2; Finland, n=3; Italy, n=3; North Western Territories, Canada, n=3; Spain, n=4; Sweden, n=4; United States, n=2). Except for the wolves from the United States, which were captive and from which blood samples were obtained, all other wolves were wild and died for reasons unrelated to this study. We extracted DNA using the QIAgen DNeasy kit and prepared Illumina paired-end libraries for each sample using the Agilent protocol for indexed paired-end Illumina libraries and Agilent SureSelect capture system. Briefly, DNA was sheared with a Covaris S2 device (Covaris, Inc. Woburn, MA, USA), end-repaired, A-tailed, ligated with Illumina's indexing-specific paired-end adaptors and PCR amplified for 5 cycles. We enriched for 16 chromosomal regions, each containing a locus associated with a distinct phenotypic character (morphological or behavioral) in dogs in a central position, and 100-150 kb upstream and downstream (totalling ca. 200-300 kb in length). We enriched with a custom Agilent SureSelect RNA oligo kit. The oligo-targeted regions added up to 2.48 Mbp (repetitive regions excluded) and encompassed approximately 3.96 Mbp of the dog genome. Libraries were then Illumina indexed/barcoded in a PCR of 13 cycles. A Nanodrop spectrophotometer and a Bioanalyzer instrument were used to assess both quality and quantity of the samples at various steps during the laboratory procedures. Libraries were validated using real-time quantitative PCR, pooled and then 90 or 100-bp paired-end sequenced on 4 lanes of an Illumina GAIIx machine, yielding 84,682,789 of paired-end reads. For read alignment and SNP calling we followed the Broad Institute Best Practice Variant Detection guide (http://www.broadinstitute.org/gatk/guide/best-practices).