Microbial DNA enrichment and host DNA depletion was performed on pooled oesophageal brushings using the MolYsis Basic5 kit (Molzym, Bremen, Germany) according to the manufacturer’s instructions, with the resulting cell pellet stored at −20 °C until DNA extraction. Saliva samples were always collected prior to the collection of oesophageal brushings and stored at 4 °C in 1 : 1 DNA/RNA shield Solution (Zymo Research) for 24–48 h before DNA extraction. DNA was extracted from saliva and oesophageal brushings using the QIAmp DNA Mini Kit according to manufacturer’s instruction (Qiagen, Hilden, Germany), with DNA fractions eluted in 50 µl of dH2O and stored at −20 °C. All oesophageal brush samples were processed within 1–3 h of collection.
DNA quantification was performed using a Qubit 3.0 fluorometer (Invitrogen, CA) and double-stranded DNA (dsDNA) HS assay kit. Pooled Illumina sequencing libraries were constructed according to methods previously described by Ravi and colleagues [21 (link)]. Paired-end metagenomic sequencing was performed on the Illumina Novaseq 6000 platform yielding 2×250 bp paired-end sequencing reads.
DNA quantification was performed using a Qubit 3.0 fluorometer (Invitrogen, CA) and double-stranded DNA (dsDNA) HS assay kit. Pooled Illumina sequencing libraries were constructed according to methods previously described by Ravi and colleagues [21 (link)]. Paired-end metagenomic sequencing was performed on the Illumina Novaseq 6000 platform yielding 2×250 bp paired-end sequencing reads.
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