Nebnext dna sample preparation kit

Paired-end libraries were prepared using NEBNext DNA sample preparation kit following the manufacturer's protocol (New England Biolab). Briefly, DNA was fragmented using the Covaris E210 sonicator to generate double-stranded DNA fragments with a fragment size of 400–500 bp. The ends were repaired, phosphorylated, followed by 3′ end adenylation. Paired end DNA adaptors were ligated and the resulting constructs size selected for ∼500 bp fragments. The excised gel band was purified following manufacturer's protocol utilizing Qiagen Gel Extraction Kits. These fragments were enriched with 12 cycles of PCR. The concentration and size distribution of the libraries was determined on an Agilent Bioanalyzer DNA 1000 chip.
Libraries were loaded onto paired end flow cells and sequenced as 101 by 2 paired end indexed reads on Illumina HiSeq 2000 and base-calling performed using Illumina's RTA version 1.7.45.0.

Illumina paired-end libraries with mean fragment lengths of 140 bp and 550 bp were generated using the NEBNext DNA sample preparation kit (New England Biolabs) with TruSeq Illumina adaptors. Genomic DNA was fragmented by nebulization, and mate-pair libraries with mean insert sizes of 1.8 kb, 3.4 kb and 8.6 kb were prepared using the Illumina Mate Pair Library Kit (v2) according to the NEBNext instructions. Libraries were sequenced using an Illumina GAIIx (PE-75) or a HiSeq2000 instrument (PE-100). Eleven independent PacBio whole genome long-range shotgun sequencing libraries were prepared from 100 μg genomic DNA with insert size up to 150 kb and sequenced on a total of 181 SMRT cells with P6-C4 chemistry at the Genome Sequencing and Analysis Core Resource at Duke University (Durham, NC).

sWGA products (≥500 ng total DNA) were cleaned using Agencourt Ampure XP beads (Beckman Coulter) following manufacturer’s instructions. Briefly, 1.8 volumes of beads per 1 volume of sample were mixed and incubated for 5 min at room temperature. After incubation, the tube containing bead/DNA mixture was placed on a magnetic rack to capture the DNA-bound beads while the unbound solution was discarded. Beads were washed twice with 200 µl of 80% ethanol and the bound DNA eluted with 60 µl of EB buffer. Cleaned amplified DNA products (~05–1 µg DNA) were used to prepare a PCR-free Illumina library using the NEBNext DNA sample preparation kit (New England Biolabs) for high throughput sequencing. DNA libraries were sequenced at the Wellcome Trust Sanger Institute using Illumina HiSeq 2500 instruments and Illumina V.3 chemistry. Paired-end sequencing was performed with 100-base reads and an 8-base index read. 12-multiplex sample libraries were loaded to target at least 20 million reads per sample.

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.

The Illumina sequencing technology used to sequence Plasmodium spp has previously been described [20 (link)]. In brief, The SWGA amplicons were cleaned using Ampure XP beads and incubated for 5 min at 25°C. After which the bead/DNA mixture was placed on a magnetic rack to capture the DNA-bound beads. Beads were washed twice with 200 μL of 80% ethanol and the bound DNA eluted with 60 μL of elution buffer. Cleaned amplified DNA products (approx. 0.5–1 μg DNA) were used to prepare DNA library using the NEBNext DNA sample preparation kit (New England Biolabs) for high throughput sequencing.