Nextera adaptor primers

From 200 mg of stools, DNA was extracted by QIAmp Fast DNA Stool mini kit (Qiagen, Germany), following the manufacturer’s instructions. The V3–V4 variable region (~ 460 bp) of 16S rRNA was amplified following the MiSeq rRNA Amplicon Sequencing protocol (Illumina, San Diego, CA). The DNA amplifications were set up using a 2 × KAPA Hifi HotStart ready Mix (KAPA Biosystems Inc., Wilmington, MA, USA) following the manufacturer’s protocol. The DNA amplicons were cleaned up by AMPure XP beads (Beckman Coulter Inc., Beverly, MA, USA). Illumina Nextera adaptor-primers were used to barcode each sample. The final library was quantified by Quant-iT™ PicoGreen® dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA). Samples were sequenced on an Illumina MiSeq™ platform according to the manufacturer’s specifications to generate paired-end reads of 300 base-length [ 35].

The general workflow included isolation of genomic DNA from stool samples, followed by amplification and sequencing of the variable region from the 16S rRNA gene, as described by Botticelli et al. [17 (link)]. DNA extraction was carried out using the QIAmp Fast DNA Stool mini kit from Qiagen, Hilden, Germany. The variable region V3-V4 from the 16S rRNA gene (~460 bp) was amplified according to the MiSeq rRNA Amplicon Sequencing workflow (Illumina, San Diego, CA, USA). For the amplicons purification, AMPure XP beads (Beckman Coulter Inc., Beverly, MA, USA) were used. A second round of amplification was conducted with a unique set of Illumina Nextera adaptor primers followed by purification to obtain the final library. The quantification was performed using Quant-iT™ PicoGreen^® dsDNA Assay Kit (Termo Fisher Scientifc, Waltham, MA, USA). The library was finally diluted to 4 nM, pooled and sequenced using the Illumina MiSeqDX. The Qiime v1.8. (http://qiime.org/1.4.0/) pipeline was followed [41 (link)], sequences were organized into operational taxonomic units (OTUs) and alignments were conducted using PyNAST v.0.1 software (https://biocore.github.io/pynast/) [42 (link)] against the Greengenes 13_08 database [43 (link)] with a 97% threshold of similarity.