16S rRNA Gene Amplification and Sequencing

The V4 hypervariable region of the 16S rRNA genes was amplified using primers 515F and 806R (Apprill et al., 2015 (link); Parada et al., 2016 (link)). PCR was performed using high fidelity AccuPrime^TMTaq DNA Polymerase (Invitrogen, Cat. No. 12346086), including 35 cycles with an annealing temperature of 50°C. PCR clean-up and removal of small fragments was done with the Nucleo Spin Gel and PCR Clean-up Kit (Macherey-Nagel, Cat. No. 740609.250). Quantification of extracted PCR products was performed using PicoGreen assay (QuantIT, Thermo Fisher Scientific, Cat. No. P11496). Thereafter, pooling and sequencing of sample-specific libraries were performed by following the Illumina MiSeq protocol. Data processing was done using the IMNGS platform (Lagkouvardos et al., 2016 (link)), applying the UPARSE analysis pipeline (Edgar, 2013 (link)) with the following settings: Number of allowed mismatches in the barcode: 1, Min fastq quality score for trimming of unpaired reads: 20, Max rate of expected errors in paired sequences: 2, Minimum relative abundance of Operational Taxonomic Units (OTU) cutoff (0-1): 0.25%. The taxonomy of OTUs clustered at 97% sequence identity was determined using SILVA¹ (Pruesse et al., 2012 (link)). The data were submitted to the Sequence Read Archive and are available under the accession number PRJNA514431.