Bioanalyzer 2100 hs dna chip

Preliminary testing with three restriction enzymes (PstI, MspI and ApeK1) for fragment size range led to selection of ApeKI for GBS library construction. Three GBS libraries were constructed at the USDA-ARS National Clonal Germplasm Repository (NCGR) and one was constructed at Clemson University according to the procedure previously described (Elshire et al., 2011 (link)) for 96 samples using DNA (100 ng per sample) digested with 4 U of ApeKI (New England Biolabs, Ipswich, MA, USA). The annealed and normalized unique and four-nucleotide-barcoded adaptors were obtained from Clemson University Genomics Institute (CUGI) and from the Oregon State University (OSU) Center for Genome Research and Biocomputing (CGRB) core facility. Two libraries were sequenced at the CGRB, one at CUGI, and one at the North Carolina State University Genomic Sciences Laboratory (Table S1). At each of these labs, libraries were quantitated with a Qubit^® fluorometer (Invitrogen, Carlsbad, CA, USA), checked for adequate size distribution (150–350 bp) with the Bioanalyzer 2100 HS-DNA chip (Agilent Technologies, Santa Clara, CA, USA), and sequenced with the Illumina HiSeq2000 (101 bp, single-end).

Approximately 1–5 ng of purified dsDNA was fragmented and tagged with adapter sequences in a single step using Nextera XT DNA Library Preparation Kit (Illumina Inc., San Diego, CA, USA) at 55 °C for 5 min, followed by incubation at 10 °C and sample neutralization, according to the manufacturer’s instruction. Tagmented libraries were then used as inputs in PCR reactions using 14 cycles of PCR amplification. PCR adds the Index 1 (i7), Index 2 (i5), and full adapter sequences to the tagmented DNA. The index adapters and Nextera PCR Master Mix were added directly to the 25 μL of tagmented dsDNA from the previous step. PCR reactions were cleaned up with Agencourt Ampure XP (Beckman Coulter, Brea, CA, USA) beads following the manufacturer’s protocol, eluting with 15 μL RNAse-free water. No direct size selection was performed on the resulting adapter-ligated library. Libraries were assayed for quality using an Agilent Bioanalyzer 2100 HS DNA chip. Libraries were then sequenced on an Illumina MiSeq platform following the manufacturer’s standard cluster generation and sequencing protocols.