Agilent 2100 bioanalyzer trace

16S ribosomal RNA gene amplicon libraries were prepared using a primer pair sequences for the V3–V4 region following the Illumina protocol “16S Metagenomic Sequencing Library Preparation for Illumina MiSeq System” (#15044223 rev. B). Bacterial 16S rRNA gene amplicons were generated from 50 ng of microbial genomic DNA in 25 μL PCR using Platinum^® Taq DNA Polymerase High Fidelity kit (Thermo Fisher Scientific, Italy) and tailed forward and reverse primer Pro341F (5′-CCTACGGGNBGCASCAG -3′) and Pro805R (5′-GACTACNVGGGTATCTAATCC -3′) selected by Takahashi et al. [41 (link)]. The expected size on Agilent 2100 Bioanalyzer trace after the amplicon PCR step was ~550 bp. The entire procedure for 16S rRNA gene library preparation and sequencing is described in Rimoldi et al. [10 (link)]. Briefly, Illumina paired-end adapters with unique Nextera XT indexes were ligated to 16S amplicons using Nextera XT Index Kit (Illumina, San Diego, CA, USA). All libraries were then subjected to quality control using qPCR using KAPA Library Quantification Kits Illumina^® Platforms (Kapa Biosystems Ltd., London, UK), pooled at equimolar concentrations, and diluted to 6 picomolar. Pooled libraries were then multiplexed and sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) with paired-end 2 × 300 bp sequencing chemistry.

Methodology applied for 16S rRNA gene library preparation and sequencing have been described in Rimoldi et al. [47 (link)] and Terova et al. [48 (link)]. The Illumina protocol “16S Metagenomic Sequencing Library Preparation for Illumina MiSeq System” (#15044223 rev. B) was applied for library preparation. The V3-V4 region was amplified from 50 ng of microbial genomic DNA using Platinum® Taq DNA Polymerase High Fidelity kit (Thermo Fisher Scientific, Italy) and tailed using forward and reverse primers Pro341F (5′-CCTACGGGNBGCASCAG -3′) and Pro805R (5′-GACTACNVGGGTATCTAATCC -3′) selected by Takahashi et al. [49 (link)]. The amplicon length was checked on Agilent 2100 Bioanalyzer trace and the expected size was ~550 bp.
Illumina paired-end adapters with unique Nextera XT indexes were ligated to 16S amplicons using Nextera XT Index Kit (Illumina, San Diego, CA). Libraries were purified and normalized using the SequalPrep ™ Normalization Plate kit (Thermo Fisher), pooled at equimolar concentrations and diluted at 6 pM prior to sequencing on the Illumina MiSeq platform (Illumina). Libraries were sequenced with v3 chemistry on 300PE MiSeq runs.

16S ribosomal RNA gene amplicon libraries were prepared using a pair of primers specific for the V3-V4 region applying the Illumina protocol “16S Metagenomic Sequencing Library Preparation for IlluminaMiSeq System” (#15044223 rev. B). Amplicons of 16S rRNA gene were generated starting from 10 μL of microbial genomic DNA by PCR using Platinum®Taq DNA Polymerase High Fidelity (Thermo Fisher Scientific, Italy) and tailed forward and reverse primer Pro341F (5′-CCTACGGGNBGCASCAG-3′) and Pro805R (5′-GACTACNVGGGTATCTAATCC-3′) selected by [38 (link)] The expected size of PCR products on Agilent 2100 Bioanalyzer trace was ~550 bp. The entire procedure for 16S rRNA gene library preparation and sequencing is described in [18 (link)] In brief, Illumina paired-end adapters with unique Nextera XT indexes were ligated to 16S amplicons using Nextera XT Index Kit (Illumina, San Diego, CA, USA). A quality control of all libraries was then performed by qPCR using KAPA Library Quantification Kits Illumina® Platforms (KapaBiosystems Ltd, UK). Libraries were then pooled at equimolar concentrations and diluted to 6 pM. Pooled libraries were then multiplexed and sequenced on an Illumina MiSeq platform (Illumina) with paired-end 2 × 300 bp sequencing chemistry.