title: Illumina HiSeq 3000 paired end sequencing; Targeted resequencing of phenology genes in a diverse panel of barley varieties
design: Targeted resequencing of phenology genes in a diverse panel of barley varieties
library_construction_protocol: Samples were collected from leaves of a single two-week old barley plant per variety. Each variety was grown in the glasshouse at Murdoch University. Each variety was grown in pots filled with soil at a glasshouse at the Western Australian State Agricultural Biotechnology Centre (Murdoch University) until seedlings were two weeks old. Genomic DNA was extracted from leaves of a single two-week old barley plant per variety using the cetyl-trimethyl-ammonium bromide (CTAB) method (Murray, M. G., & Thompson, W. F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8 (19), 4321-4326 (1980)). DNA concentration was determined using a nanodrop spectrophotometer (Thermo Scientific) according to the manufacturer's protocol. DNA quantity and purity was further checked on 0.5% agarose gel (100V, 0.5xTBE, 60 min).  Physical shearing of genomic DNA was performed with an average size of 275 bp using a Covaris S2 Focused-Ultrasonicator. DNA was cleaned using the Agencourt AMPure XP SPRI beads (Beckman Coulter, Australia), followed by end repair and A-tailing of the fragments using unique Illumina 9 nt p5 and p7 barcode adaptors (IDT xGen Custom Universal Blocking Oligos) to generate dual-indexed libraries. Library preparation was conducted with KAPA Hyper Prep Kit (KAPA Biosystems, Wilmington, MA). We performed preliminary tests to compare different scales of KAPA sample library reactions for target capture efficiency. Half-scale KAPA sample library reactions were the best compromise in terms of target capture efficiency and coverage of target genes as the proportion of on-target reads for half-scale libraries was comparable to the full-scale libraries. Equimolar amounts of each amplified library were pooled in reactions of twelve indexed samples each (total of 500ng DNA per 12-plex pool) using a Biomek FXP laboratory automation workstation liquid handling system (Beckman Coulter, Australia). Hybridization of customised RNA baits with capture pools was performed at 65°C for 24 hours.  Dynabeads® MyOne™ Streptavidin C1 magnetic beads (Thermo Fisher Scientific, Australia) were used to isolate biotinylated DNA, and amplification of bead-bound enriched libraries (9-10 cycles) was performed using KAPA HiFi HotStart Ready Mix (KAPA Biosystems, Wilmington, MA, USA) and adapter-specific primers. Concentration of all enriched libraries was determined with a Qubit fluorometer (Thermo Fisher Scientific, Australia), and library size with Tapestation (Agilent, Santa Clara, CA, USA) for subsequent library titration and next-generation sequencing. Libraries were titrated with KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA).
identifiers.namespace: Western Barley Genetics Alliance, Western Australian State Agricultural Biotechnology Centre, School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia, Australia
identifiers.id: E-MTAB-73621542365086:P412_p