SNP capture of an M. suaveolens F1 population
Fresh leaf tissue was harvested and frozen. An internal protocol, similar to Qiagen DNeasy Plant Kit, was used for DNA extraction. Sample quality was assessed using agarose gel electrophoresis and quantified using SYBR Green I. Illumina genomic library preparation input was 0.5-1 g. Fragmentation was performed using a Covaris E220 sonicator at the factory settings for 300 bp in 130 l. The fragmented DNA was purified using in-house magnetic beads. Libraries were then constructed using the Roche KAPA Hyper Kit (catalog #KK8514) following the manufacturers instructions and enriched for 6 cycles. Libraries were quantified using SYBR Green I and combined in various pool sizes (23-48 libraries per pool) in equimolar amounts. Pools were bead-cleaned and size-selected with agarose gel-extraction targeting 200-500 bp. Samples were further concentrated with magnetic bead cleanup and 500 ng of input was eluted to 7 l. Capture libraries were made using the MyBaits Hybridization Capture for Targeted NGS (Arbor Biosciences) following the manufacturers instructions with a few exceptions. The hybridization temperature was reduced to 63oC for 16 hours and we substituted Block C and Block O with Roche EZ Developer Mix, plant repeat blockers (# 06684335001). Capture enrichment was done for 10-12 cycles. Final capture libraries were purified with magnetic beads, quantified with the Qubit dsDNA HS Assay Kit (Invitrogen), and pooled in equimolar amounts. Pooled library quality was assessed using the Agilent Bioanalyzer 2100 DNA Kit and qPCR. Sequencing was done on one lane of PE150 Illumina HiSeq4000 and on one lane of PE150 Illumina NovaSeq sequencers. Quality assessment and sequencing was carried out by the DNA Technologies and Expression Analysis Core at the UC Davis Genome Center, supported by NIH Shared Instrumentation Grant 1S10OD010786-01.