alias: HW_0180_exon
title: Target enrichment of Hybosida_sp_HW_0180 using designed exon-based probes.
design: Libraries were prepared and enriched following protocols in Faircloth, et al. (2015), but following modifications in Blaimer, et al. (2015). Between 5-100 ng of DNA were sheared by sonication, dried, rehydrated, and used as input for DNA library preparation (Kapa Hyper Prep Library kit, Kapa Biosystems). After ligation of universal stubs, a 0.7X SPRI bead clean was done (Kapa Pure Beads, Kapa Biosystems). For adapter ligation, we used TruSeq-style adapters (Faircloth and Glenn 2012) and the following PCR conditions: 15 _L post ligation library, 25 _L HiFi HotStart polymerase (Kapa Biosystems), 2.5 _L each of Illumina TruSeq-style i5 and i7 primers, and 5 _L double-distilled water (ddH2O). We used the following thermal protocol (Kapa Biosystems): 98C for 45 s; 13 cycles of 98C for 15 s, 65C for 30 s, 72C for 60 s, and final extension at 72C for 5 m. We generated two PCR amplified libraries from each post-ligation library, with each PCR containing a different i5-i7 adapter combination. This allowed one DNA sample to be equally divided into two separate enrichment reactions (UCE & exon). Samples were cleaned using 1.0X SPRI bead clean (Kapa Pure Beads, Kapa Biosystems), and libraries were divided into enrichment pools containing 8 libraries combined at equimolar ratios. The duplicated PCR products formed two different groups of pools, one for UCE enrichment and one for exon enrichment. We enriched one set of pools for UCE using the Arachnida 1.1Kv1 probe set (Arbor Biosciences). The duplicate set of pools with different indices was enriched using the custom designed exon probes (Arbor Biosciences). For both enrichments, we followed version 3.02 of the mybaits protocol (Arbor Biosciences). Hybridization reactions were incubated for 24 hours at 65C, subsequently all pools were bound to streptavidin beads (MyOne C1; Life Technologies) and washed. We combined 15 _L of streptavidin bead-bound, washed, enriched library with 25 _L HiFi HotStart Taq (Kapa Biosystems), 5 _L of Illumina TruSeq primer mix (5 _M forward and reverse primers) and 5 _L of ddH2O. Post-enrichment PCR used the following thermal profile: 98C for 45 s; 18 cycles of 98C for 15 s, 60C for 30 s, 72C for 60 s; and a final extension of 72C for 5 m. We purified resulting reactions using 1.0X bead clean using Kapa Pure Beads (Kapa Biosystems), and resuspended the enriched pools to 22 _L. Pools were quantified and combined at an equimolar concentration of 30 nM, and size-selected for 250600 bp with a BluePippin (SageScience). We sequenced the pooled libraries in a single lane of a paired-end run on an Illumina HiSeq (2x150bp rapid run).
identifiers.namespace: SUB4144322
identifiers.id: HW_0180_exon