Discovering single nucleotide polymorphisms (SNPs) in 454 sequence data from Ophiognomonia clavigignenti-juglandacearum, an Ascomycete with an uncharacterized genome
The pooled genomic DNA was incubated for 10 min at 37 ºC after addition of 10 units of Riboshredder (Epicentre Biotechnologies) followed by removal of degraded RNA using 3 washes of 1 ml of 1xTE through a YM-30 Microcon spin column (Millipore). Library construction was performed as described in Roche’s General Library Preparation Method Manual. Briefly, the pool was concentrated to 100 µl and fragmented using a nebulizer for 1 min at 30 PSI, then fractionated away from any residual proteins, low molecular weight nucleic acids and other solutes using MinElute (Qiagen) columns. The resulting eluate, containing roughly 1.5 µg of sheared genomic DNA as assayed on a NanoDrop 1000 (Thermoscientific), and those fragments between 550 and 1100 bp agarose gel isolated. Roche Titanium PA and PB adaptors were ligated to these fragments after they were end-blunted using a mixture of T4-PNK and T4 DNA polymerase provided in the Roche fragment library construction kit. After capture by magnetic streptavidin-conjugated bead of the biotinylated B-adaptor-containing construct and nick translation, ssDNA DNA molecules were eluted from the beads in 18 ?l and emulsion PCR performed to generate templated beads. Beads were enriched, and deposited in a 1/4th PicoTiterPlate (PTP) region and run using Roche Titantium chemistry. The Roche assembly engine, gsAssembler was used to assemble the resulting sequence data.