Agilent high sensitivity assay kit

Peripheral blood samples were collected in Vacutainer EDTA tubes (BD, Franklin Lakes, NJ) and stored at 4 °C before plasma isolation within 3 days. Plasma was separated from whole blood by centrifuge at 1350×g for 10 min at 4 °C and stored at − 80 °C. Plasma cfDNA was extracted using QIAamp Circulating Nucleic Acid Kit (QIAGEN, Germantown, MD) following manufacturer’s instructions. The cfDNA was quantified using the Qubit Fluorometer with dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA) and Bioanalyzer 2100 with Agilent High Sensitivity Assay Kit (Agilent Technologies, Santa Clara, CA).

Amplicons of the V6–V8 variable region of the bacterial 16S rDNA gene were generated in triplicate per DNA sample by PCR using the primers F968 (5′ tagged with Roche B adaptor) and R1401 (5′ tagged with the Roche A adaptor and MID barcode tags specific for each sample as suggested by Roche) as described by Huws and colleagues (2011 (link)), except that 30 cycles of amplification were used. All PCR products were initially verified by electrophoretic fractionation on a 1.0% agarose gel for 1 h, 120 V and 80 MA in 1% TAE (Tris base, acetic acid and EDTA) buffer before pooling of triplicate amplifications. The pooled PCR products (30 μl each sample) were subsequently run on a 2.0% agarose gel for 2 h, 120 V and 80 MA in 1% TAE buffer before bands were viewed and cut on a dark reader transilluminator (Clare Chemical Research, Colorado, USA). Amplicons were retrieved from cut bands using the Isolate II PCR and Gel Kit (Bioline, London, UK). Purified amplicons were verified and quantified using the Agilent High Sensitivity Assay Kit (Agilent Technologies, California, USA) prior to pyrosequencing using Titanium chemistry on a Roche GS-FLX 454 sequencer (Roche Diagnostics, West Sussex, UK) using the manufacturer's guidelines. These sequence data have been submitted to the short read archive under accession number SRP036181.