Amplicon Library Preparation for Illumina Sequencing

Amplicon libraries were prepared, as per Illumina instructions, by a 25-cycle PCR. PCR primers were the same as those used for qPCR but flanked with Illumina Nextera overhang sequences. A unique combination of Nextera XT Indices (Illumina, Cambridge, UK) were added to PCR products from each sample, via an 8-cycle PCR. PCR products were quantified using a Quant-iT™ PicoGreen™ dsDNA Assay Kit (ThermoFisher Scientific, Dartford, UK) and pooled in equimolar concentrations. Quantification of the amplicon libraries was determined via NEBNext^® Library Quant Kit for Illumina (New England BioLabs Inc., Hitchin, UK), prior to sequencing on the Illumina MiSeq^® platform (Illumina, Cambridge, UK), using a MiSeq^® 600 cycle v3 reagent kit and 20% PhiX sequencing control standard. Raw sequence data have been submitted to the European Nucleotide Archive database under accession number PRJEB37243. Sequence output from the Illumina MiSeq platform were analysed within R [50 ] using an ASV (amplicon sequence variants) bioinformatics pipeline within the DADA2 package [51 (link)]. The DADA2 pipeline included primer trimming, quality filtering, error correction and sample inference (using the DADA2 algorithm at default parameters and pooling of samples), and removal of chimeras. Finally, taxonomy assignments for ASVs were obtained using the RDP (Ribosomal Database Project) classifier [52 (link)].