Preparing Libraries for 454 FLX and Illumina Sequencing

Libraries for 454 FLX Titanium sequencing were constructed using random DNA shearing with a Bioruptor (Diagenode Denville, NJ, USA). Fragments were polished and blunt-end ligated (NEBNext DNA Library Prep Kit, New England Biolabs, Ipswich, MA, USA) to in-house Multiplex Identifier barcode oligos (IDT, Coralville, IA, USA), with small fragments removed by magnetic beads (Beckman Coulter, Danvers, MA, USA). The libraries were quantified using a digital PCR quantified standard curve (White III et al., 2009 (link)), diluted, and pooled for 454 pyrosequencing with Titanium chemistry (The Centre for Applied Genomics, SickKids Hospital, Toronto, ON, Canada).
For Illumina library construction, DNA was sheared by ultrasonication (Covaris M220 series, Woburn, MA, USA), and the fragments end-paired, A-tailed (NxSeq DNA Sample Prep Kit, Lucigen, Middleton, WI, USA) and ligated to TruSeq adapters (IDT, Coralville, IA, USA); small fragments were removed twice using magnetic beads (Beckman Coulter, Danvers, MA). The resulting libraries were pooled, and sequenced using both 250 bp and 100 bp paired-end sequencing on the MiSeq (UCLA Genotyping & Sequencing Core, Los Angeles, CA, USA) and HiSeq (McGill University and Génome Québec Innovation Centre, Montreal, QC, Canada) platforms, respectively.