PacBio circular consensus sequencing of NB-LRR sequence capture on barley cultivar CI16153
DNA sequencing libraries was enriched according to the MYbaits protocol (MYbaits User Manual version 2.3.1) and using MYbaits reagents (MYcroarray). Briefly, 500 ng of the prepped library was hybridized in hybridization buffer (10x SSPE, 10X Denhardts solution, 10 mM EDTA, 0.2% SDS) to the biotinylated RNA baits for 20 h at 65C on a thermocycler. After hybridization bound DNA was recovered using magnetic streptavidin-coated beads as follows: the hybridization mix was added to 30 L Dynabeads MyOne Streptavidin C1 (Invitrogen, Life Technologies) that had been washed 3 times and resuspended in binding buffer (1 M NaCl; 10 mM Tris-HCl, pH 7.5; 1 mM EDTA). After 30 m at 65C, beads were pulled down and washed three times at 65C for 10 m with 0.02% SSC/0.1% SDS followed by resuspension in 30 L of nuclease-free water. Library was then PCR amplified (26 cycles) using Kapa HiFi HotStart Ready Mix (Kapa Biosystems) and Illumina P5 and P7 primers. The amplified library was size fractionated with the Sage Scientific Electrophoretic Lateral Fractionator (SageELF, Sage Science) using a 0.75% SageELF agarose gel cassette. Fractions with size distribution between 3 and 4 kb were pooled and purified with AMPure PB beads (Pacific Biosciences). Then, library was assembled for PacBio sequencing using the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences) according the 2-kb Template Preparation and Sequencing protocol (www.pacificbiosciences.com/support/pubmap/documentation.html). PacBio RSII sequencing using C4-P6 chemistry was performed at the Earlham Institute (Norwich, UK), using three SMRT cells for the barley cultivar CI 16153. The barley capture library TSLMMHV1 is composed of 99,421 100 mer baits with 2x coverage over the target space. Targeted sequences include repeat masked Mla locus from Morex2, all cloned alleles of Mla7, the Mlo locus30, the Rpg1 locus31, and the rpg4/Rpg5 locus32. In addition, the capture library targets the barley NLR gene space identified in genomic sequence from barley accessions Barke, Bowman, and Morex, full length cDNA derived from barley cultivar Haruna Nijo, and transcriptomes of barley cultivars Abed Binder 12, Baronesse, CI 16153, CIho 4196, Manchuria, Pallas, Russell, and SusPtrit.