Whole-exome sequencing of Canis lupus familiaris: genomic DNA
Extracted DNA was fragmented on a Covaris S2 System in a 130 l volume (aim: 400 bp fragments, settings: duty cycle: 10%, intensity: 4, cycles per burst: 200, time: 55s). After shearing, the DNA yield was measured with Quant-iTTM Picogreen  dsDNA Assay (Life Technologies). Depending on the yield after DNA-extraction, between 500 ng and 1  g of the fragmented DNA was used as input for the library preparation. Samples were end repaired, A-tailed and ligated with TruSeq adapters using the reagents from the NEBNext Ultra DNA Library Prep Master mix set for Illumina (New England Biolabs) according to the manufactur- ers protocol. Size selection was performed on a 2% E-Gel (Invitrogen Life Technologies) (G4010-02), fragments were selected with an insert size around 300 bp. One  l of the ligated product was subse- quently amplified in an enrichment PCR (10 cycles) for library quality assessment as recommended in the SeqCap EZ Library SR Users Guide (Nimblegen, Roche). Thereafter, the pre-capture LM-PCR was performed on the samples for 8 cycles as prescribed in the SeqCap EZ library protocol. The concentration of each PCR product was determined using Quant-iTTM Picogreen  dsDNA Assay (Life Technologies).Four times four samples were equimolarly pooled to obtain a total DNA input of 1250 ng. The pooled library was hybridized for 67-68 hours with the baits (SeqCap Developer Library). The hybridized library was washed and the captured and pooled DNA was recovered. After a final amplification (LM-PCR, 18 cycles), the quality of the library was checked using the High Sensitivity DNA chip (Agilent).