Rna prep with enrichment kit

The saliva samples were sequenced with two protocols: (1) shotgun DNA metagenomics (SDM) (Illumina), and (2) Illumina Respiratory Viral Oligo Panel (RVOP) sequencing (Illumina). DNA/RNA extraction was performed with the ZymoBIOMICS DNA/RNA Miniprep Kit. Quality control of SDM and RVOP libraries was performed using a Thermo Fisher Qubit 3.0 High Sensitivity DNA kit and an Agilent 2100 Bioanalyzer High Sensitivity DNA kit.
For the SDM, libraries were prepared, along with a ZymoBIOMICS Microbial Community DNA standard, using a Nextera XT DNA Library Prep kit following Illumina’s recommended guidelines (Illumina). Libraries were sequenced as paired-end, 2×150 bp, using a NextSeq 500 High-Output kit, with a 1% phi X sequencing control spike-in.
For the RVOP, libraries were prepared using an Illumina RNA Prep with Enrichment kit and enriched using the Respiratory Virus Oligos Panel v2 at 3-plex. Libraries were normalized by mass for three-plex enrichment but were not normalized by viral load as the viral copy number of each sample was unknown (Illumina). Libraries were sequenced as paired-end, 2×75 bp, using a NextSeq 500 Mid-Output kit, with a 1% phi X sequencing control spike-in.

The cDNA generated prior to whole genome amplification was used as input into the RNA Prep with Enrichment kit (Illumina). Second-strand cDNA synthesis, cDNA tagmentation, library construction, clean-up, and normalisation were performed according to manufacturer’s instructions. Individual libraries were then combined in 3-plex reactions for probe hybridisation. The RVOP v2 (Illumina) was used for probe hybridisation with the final hybridisation step held at 58 °C overnight. Hybridised probes were then captured and washed according to manufacturer’s instructions and amplified as follows: initial denaturation 98 °C for 30 s, 14 cycles of: 98 °C for 10 s, 60 °C for 30 s, 72 °C for 30 s, and a final 72 °C for 5 min. Library quantities and fragment size were determined using a Qubit 1× dsDNA HS assay and Agilent HS Tapestation and sequenced using 2 × 76-bp runs on the iSeq (Illumina).

Diluted culture RNA extracts were used as input into the RNA Prep with Enrichment kit (Illumina). RNA denaturation, first- and second-strand cDNA synthesis, cDNA tagmentation, library construction, cleanup, and normalization were performed according to manufacturer’s instructions. Individual libraries were then combined in 3-plex reactions for probe hybridization. The Respiratory Viral Oligo panel v2 (Illumina) was used for probe hybridization with the final hybridization step held at 58°C overnight. Hybridized probes were then captured and washed according to manufacturer’s instructions and amplified as follows: initial denaturation 98°C for 30 s, 14 cycles of: 98°C for 10 s, 60°C for 30 s, 72°C for 30 s, and a final 72°C for 5 min. Library quantities and fragment size were determined using a Qubit 1× dsDNA HS assay and Agilent HS Tapestation and sequenced using 2 × 76-bp runs on the Illumina MiniSeq. using BWA-mem version 0.7.17. SAMtools v1.10 was used to curate BAM files with an average mapping quality threshold (MAPQ) of ≥60 and an average read depth of ≥10 and to calculate average genome coverage for each reference sequence.