BSE-Seq of Triticum aestivum
Total 4.5 mg of genomic DNA was fragmented into 300 bp using the Bioruptor UCD-200 sonicator (Diagenode, Denville, NJ). Pre-libraries were constructed using the Kapa Hyper DNA library prep kit for Illumina HiSeq. Fragmented DNA (1 mg) was end repaired with an end-repair enzyme and a deoxyadenosine was added to the 30 end of the fragments. Kapa barcoded DNA and Kapa indexed adapters were ligated to the sample libraries. The adapter-ligated libraries were selected for an average insert size of 350 bp using next-generation sequencing cleanup and size select (NucleoMag, Macherey-Nagel, Duren, Germany) according to the manufacturers instructions. The pre-capture amplification was performed using the NumbleGen protocol with eight PCR cycles. Equal amounts of products from six libraries were pooled to obtain a total of 1 mg of DNA for the hybridization. Hybridization of sample libraries was performed at 47C for 60h~72 h using the SeqCap EZ library (Roche/NimbleGen, Madison, WI), probe custom designed with IWGSC RefSeq V1.0 genome. The hybrids were captured using the capture beads from the SeqCap EZ pure capture bead kit, washed, and amplified by ligationmediated PCR. The quality of the captured libraries was assessed using the Bioanalyzer 4200 instrument (Agilent Technologies, Santa Clara, CA). The libraries were quantified by quantitative real-time (qRT)-PCR and then sequenced using the Illumina HiSeq Nova platform to generate 150-bp paired-end reads.