Laboratory Methods

To get the most out of DISCOVAR and DISCOVAR de novo you need to generate appropriate data. To help you do this, we are happy to share details on some of the laboratory methods used here at the Broad Institute.

PCR-free library construction
250 base read sequencing

PCR-free library construction

Illumina PCR-free fragment shotgun libraries were prepared using the ‘with-bead pond library’ construction protocol described by Fisher et al. [PMID: 21205303] with the following modifications:

500 ng of genomic DNA, in a volume of 50 μl, was sheared to a size of ~400 bp using a Covaris E210 instrument (Covaris) using Illumina’s TruSeq PCR-free protocol shearing parameters (Illumina, Part # 15036187 A): Duty cycle = 10%, intensity = 5, cycles per burst = 200, time = 45 seconds. Fragmented DNA was then cleaned up with 0.6x Agencourt AmPure XP SPRI beads and eluted in 40 μl Tris-HCl pH8.0, following manufacturer’s recommendations (Beckman Coulter). DNA fragments were then further cleaned up with 3.0x Agencourt AmPure XP SPRI beads, following manufacturer’s recommendations (Beckman Coulter), but DNA was not eluted from the SPRI beads. Then using the KAPA Library Preparation Kit reagents (KAPA Biosystems, Catalog # KK8241), DNA fragments bound to the SPRI beads were subjected to end repair, A-base tailing and Illumina ‘PCR-free’ TruSeq adapter ligation (Illumina, Catalog FC-121-3001) following manufacturer’s recommendations (KAPA Biosystems). A second 0.7x SPRI clean up was performed following adapter ligation to remove adapter dimers and library fragments below ~150 bp in size. No library PCR amplification enrichment was performed. Sequence ready Illumina PCR-free library was then eluted off the SPRI beads following manufacturer’s recommendations (Beckman Coulter). Libraries were quantified with quantitative PCR using KAPA Library Quant kit (KAPA Biosystems, Catalog # KK4824) and an Agilent Bioanalyzer High Sensitivity Chip (Agilent Technologies) following the manufacturer’s recommendations.

250 base read sequencing

Libraries were sequenced with 250 base paired-end reads on an Illumina HiSeq 2500 instrument in Rapid Run Mode, with the following modifications. Reagents from two 200 cycle TruSeq Rapid SBS Kit v1 (Illumina, catalog # FC-402-4001) were combined and run using a 500 cycle run. To enable a 500 cycle run the <SBSMAXCycleRR> value in the HiSeqControlSoftware.Options.cfg file was changed to 500 cycles i.e. <SBSMAXCycleRR>500</SBSMaxCycleRR>. According to Illumina it is also possible to define the number of cycles in the HiSeq Control Software under the Run Configuration tab, however entering non-supported read length will result in a warning message. Currently Illumina does not support read lengths greater than 150 bases on the HiSeq 2500 with the v1 chemistry, however they plan to do so with the next release.