Gs flx titanium sequencing kit xl

Whole genome shotgun libraries were generated from 1 µg genomic DNA using the GS FLX Titanium Rapid Library Preparation Kit (Roche Applied Science, CA) according to the manufacturer's protocol. Rapid library MID Adaptors MID10, MID11 and MID12 (Roche Applied Science, CA) were ligated to the CPCRI-1, CPCRI-2 and CPCRI-3 libraries, respectively. The quality of the libraries (library size ∼1,600 bp) was assessed using Bioanalyzer High sensitivity DNA chip (Agilent, CA). The libraries were titrated by emulsion titrations and based on the percent enrichment, appropriate amount of the libraries were used to set up the large volume emulsion PCRs for each of the individual libraries. The beads containing clonally amplified DNA were enriched and the sequencing primer was annealed. Finally, half of the beads containing the CPCRI-2 libraries were mixed with CPCRI-1 library beads and the other half with the CPCRI-3 library beads. Each set of bead mix was then loaded on a picoTiter plate (half the plate) and sequenced using the GS FLX Titanium Sequencing Kit XL+ (Roche Applied Science, CA). Upon sequencing and processing of the raw data, demultiplexed data were assembled using GS de novo assembler version 2.6 (Roche Applied Science, CA).

Two random shotgun ‘genomic’ libraries were generated via fragmentation of 500 ng each of pear genomic DNA employing the GS FLX+ Series XL+ Rapid Library preparation kit, following the manufacturer's recommendations (Roche, Indianapolis, IN, USA). Three 2 kb and two 7 kb paired-end libraries were constructed from pear genomic DNA using the GS FLX+ Series XLR70 Paired End Rapid Library preparation kit following the manufacturer's recommendations (Roche). Five and 15 µg of double-stranded genomic DNA was randomly fragmented via hydrodynamic shearing to an average size of 2,000 and 7,000 bp using the HydroShear apparatus (DigiLab, Marlborough, MA, USA). The libraries were quantified by quantitative PCR using the 454 Kapa Library Quantification Kit (Kapa Biosystems, Boston, MA, USA). Long sequencing reads from shotgun ‘genomic’ libraries and paired-end sequencing reads were produced by the GS FLX+ Series, using the GS FLX Titanium Sequencing Kit XL+ (Roche), according to the manufacturer's recommendations.