Nextera xt dna library kit

Equal volumes of each of the nine DNA extracts were pooled together and used to prepare a single shotgun library using the Nextera XT DNA library kit (Illumina, San Diego, USA). ESS was performed using a NextSeq® 500/550 v2 high output 300 cycle kit on an Illumina NextSeq system located in the Trace and Environmental DNA (TrEnD) Laboratory at Curtin University. Sequences with an average Q score ≤25 that contained no ambiguous nucleotides and were 151 base pairs (bp) in size (corresponding to the length of a DNA fragment sequenced uni-directionally), were compared to the National Center for Biotechnology Information (NCBI) nucleotide database using BLASTN on the Magnus Cray XC40 system located in the Pawsey Supercomputing Centre at Technology Park in west Australia. Assignment of sequences to taxa at a particular taxonomic level was assessed using the software MEGAN 5.11.3^{70 (link)} using a Min Score of 100 and a Top Percent of 10 under the LCA Parameters.

As previously described, we used an mNGS protocol optimized in our lab [43 (link)]. Briefly, after thawing all the samples were supplemented with MS2 bacteriophage (Levivirus genus) from a commercial kit (MS2, IC1 RNA internal control; r-gene, bioMérieux) to check the validity of the process. Only the RNA internal control MS2 was added because it validates all the steps of our protocol (including RT stage during amplification) in contrast to a DNA internal control. A no-template control (NTC) consisting of RNase free water was implemented to evaluate the contamination during the process. An additional negative control consisting of viral transport medium was added. For sample viral enrichment, a 3-step method was applied to 220 μL of vortexed sample spiked with MS2 (low-speed centrifugation, followed by the filtration of the supernatant and then Turbo DNase treatment), as described in detail in Bal et al. [43 (link)]. After viral enrichment, the total nucleic acids were extracted using one of the three methods selected for the study described above. After random nucleic acid amplification using modified whole transcriptome amplification (WTA2, Sigma-Aldrich, Darmstadt, Germany), libraries were prepared using the Nextera XT DNA Library kit and sequenced with Illumina NextSeq 500 ™ using a 2 x 150 PE high-output flow cell (Illumina, San Diego, CA, USA).

Total RNA was extracted from cells using RNeasy Micro Kit (QIAGEN 74004). For mRNA sequencing cDNA synthesis was performed using SMART-Seq Ultra Low Input RNA Kit for Sequencing (Takara 634889) from approximately 500 pg of total RNA. cDNA was validated using the High Sensitivity NGS Fragment Analysis Kit (Agilent formerly AATI DNF-474-0500) on a 12-Capillary Fragment Analyzer. Quantification was determined using the Quant-iT dsDNA Assay Kit, high sensitivity (Thermo Fisher Q33120), and 100 pg of cDNA was tagmented and ligated using the Nextera XT DNA Library Kit (Illumina FC-131-1024) at $\frac{1}{2}$ volumes to produce sequencing libraries. The resulting libraries were validated using the High Sensitivity NGS Fragment Analysis Kit on a 12-Capillary Fragment Analyzer and quantified using the Quant-iT dsDNA Assay Kit, high sensitivity. Equal concentrations of libraries were pooled, denatured, diluted, and subjected to paired-end sequencing using the Mid Output v2.5 kit (Illumina FC-404-2001) on a NextSeq550 following the manufacturer’s instructions.

All samples were frozen without additives in 1.7 mL Eppendorf tubes at −40°C. At one point during storage, the freezer had a technical failure, during which the temperature of the samples may have briefly exceeded 0°C (duration ≤12 h).
Sequencing was performed at the Genomics Support Center Tromsø (GSCT) at UiT the Arctic University of Norway. DNA isolation was performed on a QIAcube instrument (QIAgen, Hilden, Germany) using a QIAamp Fast DNA Stool Mini Kit (QIAgen). DNA concentration was measured on a Qubit 3 instrument (Thermo-Fisher, Massachusetts, USA). Library preparation was done with Nextera XT DNA library kit (Illumina, USA) using an input of 1 ng DNA. The samples were then sequenced on an Illumina NextSeq550 instrument with a NextSeq 500/550 High output v2 kit (300 cycles) (Illumina), generating 2*150 base-pair reads. The sequencing generated a mean of 27 271 415 (range 2 933 179 − 74 426 851) reads per sample.

DNA extraction was performed as previously described by the National Institute of Public Health and Environmental Protection (RIVM), Bilthoven, The Netherlands (Isolation of Genomic DNA from Mycobacteria Protocol), or according to the source state laboratory’s internal protocol. DNA was quantified with the Qubit 2.0 dsDNA Broad Range Assay. Genomic libraries were prepared using the Illumina Nextera XT DNA Library Kit using manual normalization and sequenced on the Illumina MiSeq Platform with v3 Chemistry for 300 bp paired-end reads.

cDNA from each sample was generated from 5 ng of total RNA using SMARTer Ultra Low Input RNA kit v4 (Clontech). The cDNA was amplified by 9 PCR cycles, followed by QC analysis on BioAnalyzer 2100 (Agilent). Sequence libraries were produced from 150 pg of cDNA using Nextera XT DNA library kit (Illumina), cleaned up with AMPure XP beads, and QC checked with Caliper LabChip GX. Paired-end sequencing data were generated on an Illumina HiSeq 2500, at a depth of 20 million reads per sample, with read length 50b × 2.