Hiseq 2500 dna sequencer

Total RNA samples were submitted to the North Carolina State Genomic Sciences Laboratory (Raleigh, NC, USA) for Illumina RNA library construction and sequencing. The amplified library fragments were purified and checked for quality and final concentration by using an Agilent 2100 Bioanalyser with a High Sensitivity DNA chip (Agilent Technologies, USA). The final quantified libraries were pooled in equimolar amounts for sequencing on one lane, according to the MiSeq protocol, and single libraries on four lanes of an Illumina HiSeq 2500 DNA sequencer, utilising a 125 bp single end sequencing flow cell with a HiSeq Reagent Kit v4 (Illumina, USA). Flow cell cluster generation for the HiSeq2500 was performed using an automated cBot system (Illumina, USA). The Real Time Analysis (RTA), version 1.18.64 software package was used to generate raw bcl, or base call files, which were then de-multiplexed by sample into fastq files for data submission using bcl2fastq2 software version v2.16.0. The raw fastq files were deposited in the Sequence Read Archives (SRA) of the National Center for Biotechnology Information (NCBI) under accession number SRP070144 of Bioproject PRJNA311553 and Biosample SAMN04485566.

RNA sequencing was essentially performed as described previously [27 (link)]. Briefly, total RNA was quantified using Qubit RNA Assay Kit (Thermo Fisher Scientific) and quality-controlled using Agilent RNA6000 Nano Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies Japan, Ltd., Tokyo, Japan). PolyA-RNA was selected from 300 ng of total RNA, and cDNA was generated, followed by PCR amplification. cDNA Library for RNA sequencing was prepared using a NEBnext Ultra II Directional RNA Library prep with Beads (New England Biolabs Japan, Inc.). The library was quality-controlled using the Agilent 2100 Bioanalyzer and quantified using a Kapa Library Quantification Kit (NIPPON Genetics CO., Ltd.) and subjected to paired-end sequencing of 101-bp fragments using a TruSeq PE Cluster Kit v3HS (FC401-3001) on HiSeq2500 DNA sequencer (Illumina).

Detailed P. falciparum primers designed to identify P. falciparum genes have been published (Oyola et al., 2016 (link)). Selective whole genome amplification (sWGA) reaction was performed in a 50 μL reaction volume containing at least 5 ng of template DNA, 1× BSA, 1 mM dNTPs, 2.5 μM of each amplification primer, 1× Phi29 reaction buffer and 30 units of Phi29 polymerase enzyme (New England Biolabs). Isothermal conditions were used for sWGA. The conditions were 35 °C for 5 min, 34 °C for 10 min, 33 °C for 15 min, 32 °C for 20 min, 31 °C for 30 min, 30 °C for 16 h then heating at 65 °C for 15 min to inactivate the enzymes prior to cooling to 4 °C. Subsequent to sWGA, the products were cleaned using Ampure XP beads after which the bead/DNA mixture was placed on a magnetic rack to capture the DNA-bound beads. Beads were washed twice with 80% ethanol and the bound DNA eluted with elution buffer. DNA libraries were prepared with the cleaned DNA products using NEBNext DNA sample preparation kit (New England Biolabs). DNA libraries were sequenced using Illumina HiSeq 2500 DNA Sequencer.