Large insert library sequencing for Phytophthora nicotianae race 1
Library preparation was performed using standard Illumina protocols and Illumina paired-end adapters (Bentley et al., 2008). For small insert libraries, 5 µg genomic DNA was sheared into fragments, end repaired, A-tailed, and ligated to Illumina paired-end adapters (Illumina). The ligated fragments were selected by size at 2000 bp on 1.5% agarose gel and amplified by ligation-mediated PCR. The DNA fragments were end-repaired using biotinylated nucleotide analogues, size selected at 2 kb, and circularized by intramolecular ligation. Circular DNA molecules were sheared with Adaptive Focused Acoustic to an average size of 500 bp. Biotinylated fragments were purified with magnetic beads (Invitrogen), end-repaired, A-tailed and ligated to Illumina paired-end adapters, size-selected again and purified by ligation-mediated PCR. DNA libraries were sequenced on the Illumina HiSeq 2500 platform.