Quick t4 dna ligase

All sequencing libraries were prepared as PCR-free. Whole-genome amplified or unamplified genomic DNA (1.5 μg in 75 µl of TE buffer) was sheared using a Covaris S2 (Covaris, Inc., Woburn, MA, USA) to obtain a fragment-size distribution of ∼300 to ∼600 bp. The sheared DNA fragments were end-repaired and A-tailed using the NEBNext DNA sample preparation kit (NEB), following an Illumina sample preparation protocol. Pre-annealed paired-end Illumina PCR-free adapters were ligated to the A-tailed fragments in a 50-µl reaction containing 10 µl of DNA sample, 1× Quick T4 DNA ligase buffer, 10 µl of PCR-free PE-adapter mixture, 5 µl of Quick T4 DNA ligase (NEB) and incubated at 20°C for 30 min. The ligation reaction was cleaned twice using Agencourt Ampure XP beads (Beckman Coulter). Cleaned DNA was eluted with 20 µl of buffer EB. Aliquots were analysed using an Agilent 2100 Bioanalyzer (Agilent Technologies) to determine the size distribution and to check for adapter contamination. Samples were sequenced using either Illumina Hiseq 2500 or Miseq technologies (San Diego, CA, USA) with 75 bp read length and the paired-end read options. Corresponding WGA and non-WGA samples were run in the same lanes, using different multiplex tags. This strategy reduces potential confounding artefacts relating to sequencing chemistry.