title: WGS of wild annual Helianthus samples
design: Genomic DNA was extracted from leaf tissue of Helianthus spp. using either a modified CTAB protocol (based on Murray and Thompson, 1980). DNA was sheared to an average fragment size of 350 bp using a Covaris M220 ultrasonicator (Covaris, Woburn, Massachusetts, USA), following the manufacturers recommendations. 750 ng of sheared DNA were used as starting material to prepare paired-end whole-genome shotgun Illumina libraries. End-repairing of the sheared DNA fragments was performed using the NEBNext End Repair Module (NEB, Ipswich, Massachusetts, USA). The fragments were then A-tailed using Klenow Fragment (3-->5exo-) from NEB and then ligated to 24-bp-long, non-barcoded adapters with a 3 T-overhang using the Quick Ligation Kit from NEB. After each enzymatic step, the reactions were purified using 1.6 volumes of paramagnetic SPRI beads, prepared according to Rohland et al. 2012. An enrichment step was then performed using KAPA HiFi HotStart ReadyMix (Roche, Basel, Switzerland) and short, non-indexed primers that do not extend the adapters. The reactions were then purified using 1.6 volumes of SPRI beads. In order to reduce the representation of repetitive sequences, the enriched libraries were treated with a Duplex-Specific Nuclease (DSN; Evrogen, Moscow, Russia), following the protocols reported in Shagina et al. 2010 and Matvienko et al. 2013, with modifications (Todesco et al., in preparation). The fragments were then further amplified using Kapa HiFi HotStart ReadyMix and primers that completed the adapters and added a six-bp index to the P7 adapter. The sequence of the completed adapters is identical to that Illuminas TruSeq adapters. After amplification, the libraries were purified twice with 1.6 volumes of SPRI beads, quantified using a QuBit dsDNA Broad Range Assay Kit (Invitrogen, Carlsbad, California, USA) and analyzed on a 2100 Bioanalyzer instrument using a High Sensitivity DNA Analysis Kit (Agilent, Santa Clara, California, USA). All libraries were sequenced at the Genome Quebc Innovation Center a HiSeq 4000 instrument (Illumina, San Diego, CA, USA), with 2x150bp reads.
identifiers.namespace: SUB6257079
identifiers.id: HI.4141.003.Index_8.PET0392