A pool of 48 libraries contained 39 starling samples, two house sparrow (Passer domesticus) samples (to compare bait performance in any conserved regions because we planned to use this bait supplier and protocol with another study on house sparrows), and seven replicate libraries. Eight superb starling samples were used in a previously unpublished DNA methylation study that used RRBS. Three of these eight were included in duplicate libraries and one in triplicate libraries, and the house sparrow samples were also run in duplicate. The remaining 31 additional superb starling samples were included to assess target capture performance when scaling up to a pool of 48 libraries.
We followed the myBaits v4.01 protocol, but lowered the default hybridization and clean up temperature from 65°C to 63°C as myBaits recommends for fragmented DNA. We note, however, that a temperature of 65°C works similarly well with EM-seq converted libraries [24 ]. Hybridization beads bound to the pooled library-blocker mix were cleaned with three washes, and the resuspended bead-bound DNA was then amplified with 16 PCR cycles in a KAPA HiFi reaction using a 60°C annealing temperature and a one minute extension step at 72°C. The amplified capture pool was subsequently cleaned up with AMPure XP beads and sequenced at 2x150 bp reads with 5% PhiX spike-in in one lane (110 Gb) of an Illumina HiSeq 4000 at the Novogene Corporation, Sacramento, CA.
We followed the myBaits v4.01 protocol, but lowered the default hybridization and clean up temperature from 65°C to 63°C as myBaits recommends for fragmented DNA. We note, however, that a temperature of 65°C works similarly well with EM-seq converted libraries [24 ]. Hybridization beads bound to the pooled library-blocker mix were cleaned with three washes, and the resuspended bead-bound DNA was then amplified with 16 PCR cycles in a KAPA HiFi reaction using a 60°C annealing temperature and a one minute extension step at 72°C. The amplified capture pool was subsequently cleaned up with AMPure XP beads and sequenced at 2x150 bp reads with 5% PhiX spike-in in one lane (110 Gb) of an Illumina HiSeq 4000 at the Novogene Corporation, Sacramento, CA.
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