Miseq v2 chemistry

The V3–V4 hypervariable region of the bacterial 16S rRNA gene was sequenced using an Illumina MiSeq V2 chemistry (2 × 250 bp) after Illumina libraries prepared following the Illumina 16S Metagenomic Sequencing Library Preparation Guide (Part #15,044,223 Rev. B)^{31 (link)} and the Nextera XT Index Kit. The resulting data are registered in NCBI as Bioproject PRJNA944646.

A full list of sequenced samples is provided in additional Table 3. Vibrio-Sequin mix 1 was spiked to samples S25-S37, whereas samples S38-S47 were spiked with Vibrio-Sequin mix 2. Standards were spiked at 2% fractional abundance. Moreover, Vibrio-Sequins were sequenced pure and undiluted in triplicates (S1-S3 & S25-S27) to assess technical variation and sequencing errors. The Illumina DNA Prep Tagmentation Kit (Illumina, USA) was used to prepare DNA libraries according to manufacturer’s protocol with a standard DNA input of ~ 120 ng DNA (except for samples S28, S29, S37, S38, S39 and S46 (additional file 3). DNA libraries were quantified using the HS dsDNA Qubit Assay on a Qubit 4.0 (Life Technologies, USA) and fragment sizes were examined on a 2100 Bioanalyzer using the High Sensitivity DNA Kit (Agilent Technologies, USA). Subsequently, DNA libraries were pooled and denatured to a 16.5 pM combined library and sequenced on a Miseq system using Miseq v2 chemistry (Illumina, USA) with 2 × 201 bp reads. Raw sequencing data are available at NCBI’s Sequence Read Archive under the project number PRJNA914529.

The frozen plasma samples were shipped to Wright Labs, LLC. for 16S rRNA sequencing (V3–V4 region). Microbial DNA was extracted from samples using the DNA/RNA Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. After extraction, DNA purity and concentration were determined using Qubit 4 Fluorometer (Invitrogen, Carlsbad, CA, USA) and dsDNA HS assay kit (ThermoFisher Scientific, Waltham, MA, USA). PCR products were pooled, and gel-purified on a 2% agarose gel using Qiagen Gel Purification Kit (Qiagen, Frederick, MD, USA). After a quality check using 2100 Bioanalyzer and DNA 1000 chip (Agilent Technologies, Santa Clara, CA, USA), 16S rRNA sequencing was performed using an Illumina MiSeq v2 chemistry with paired-end 250 base pair reads as per the Earth Microbiome Project’s protocol [107 (link)]. One negative control was processed in parallel with the samples and sequenced as well.

Libraries were prepared using a Nextera XT DNA Sample Preparation Kit (Illumina, San Diego, CA, USA) with 1 ng of DNA according to the manufacturer’s protocol (Nextera XT protocol, Version October 2012) and sequenced using a MiSeq Personal Sequencer (Illumina, San Diego, CA, USA). Sequencing reactions were performed using MiSeq v2. Chemistry (Illumina, San Diego, CA, USA).

RNA was extracted from 500 μL of plasma using the NucliSENS easyMAG system (bioMérieux) into 30 μL of water, of which 5 μL was processed with the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) with published modifications to the manufacturer's protocol.36 A 500‐ng aliquot of the pooled library was enriched using the xGen Lockdown protocol (Rapid Protocol for DNA Probe Hybridization and Target Capture Using an Illumina TruSeq Library [v1.0]; Integrated DNA Technologies) with a comprehensive panel of HCV‐specific, 120‐nucleotide DNA oligonucleotide probes (Integrated DNA Technologies), designed using a published algorithm.31 The enriched library was sequenced using Illumina MiSeq v2 chemistry to produce paired 150‐bp reads.