16s metagenomics kit

Stool samples (2 g) were collected per sampling by using a small sterilized spoon provided with the container and stored at −80 °C within 4 h of defecation until further analysis. The stool samples were thawed, and DNA was extracted using a Nucleo Spin DNA Stool Kit (MACHEREY-NAGEL, Düren, Germany). The seven hypervariable regions of the DNA, excluding v1 and v5 of the 16S rRNA region, were amplified using a 16S metagenomics kit following the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). After refinement, a library was generated using the Ion Plus Fragment Library Kit and Ion Xpress Barcode Adapters Kit (Thermo Fisher Scientific, Waltham, MA, USA). The barcoded library was quantified and pooled to generate the final concentration of 50 pM per target using the Ion Universal Library Quantification Kit with the Quant Studio 5 system (Thermo Fisher Scientific, Waltham, MA, USA). The Ion Chef Instrument and the corresponding kit were used to achieve target concentrations for template preparation and emulsion PCR, and sequence analysis was performed using the Ion Gene Studio S5 System and Ion 530 chip (Thermo Fisher Scientific, Waltham, MA, USA). The sequence data were analyzed using Ion Reporter Software with the Metagenomics 16S w1.1 v5.16 workflow (Thermo Fisher Scientific, Waltham, MA, USA).

Ribosomal 16S rRNA gene sequences were amplified using the 16S Metagenomics Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA), consisting of primer pools to amplify multiple variable regions (V2-4–8 and V3–6) [7 (link),8 (link),9 (link)] of the 16S rRNA. The libraries were created using the Ion Plus Fragment Library Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Barcodes were added to each sample using the Ion Xpress Barcode Adapters kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Emulsion PCR and sequencing of the amplicon libraries were performed on an Ion 520 chip (Ion 520^TM Chip Kit) via the Ion Chef System and Torrent S5^TM system, respectively, using the Ion 520^TM/530^TM Kit-Chef (Thermo Fisher Scientific Inc., Waltham, MA, USA). Base calling and run demultiplexing were performed using Torrent Suite^TM Server software (Thermo Fisher Inc., Waltham, MA, USA), version 5.4.0, with default parameters for 16S Target Sequencing (bead loading ≤ 30, key signal ≤ 30, and usable sequences ≤ 30).

Ribosomal 16S rRNA gene sequences were amplified from cDNA using the 16S Metagenomics Kit (Thermo Fisher Scientific, Monza, Italy). The kit included two primer sets that selectively amplify the corresponding hypervariable regions of the 16S region in bacteria: primer set V2–4–8 and primer set V3–6, 7–9. Libraries were created using the Ion Plus Fragment Library Kit (Thermo Fisher Scientific, Monza, Italy). Barcodes were added to each sample using the Ion Xpress Barcode Adapters kit (Thermo Fisher Scientific, Monza, Italy). Emulsion PCR and sequencing of the amplicon libraries were performed on an Ion 520 chip (Ion 520TM Chip Kit), using the Ion Torrent S5TM system and the Ion 520TM/530TM Kit-Chef (Thermo Fisher Scientific, Monza, Italy) according to the manufacturer’s instructions. After sequencing, the individual sequence reads were filtered using Ion Reporter Software V4.0, to remove low quality and polyclonal sequences.

According to the manufacturer’s protocol, at least 3 ng of total DNA was used for 16S and DNA library preparation. The bacterial population was analyzed by amplifying the 16S rRNA genes using a 16S Metagenomics Kit (Thermo Fisher Scientific) with V2-4-8 and V3-6,7-9 primers, targeting the seven hypervariable regions (V2, V3, V4, V6-7, V8, and V9) of bacterial 16S rRNA genes. Amplicon lengths of V2, V3, V4, V6-7, V8, and V9 hypervariable regions were 250, 215, 288, 260, 295, and 209 bp, respectively. The targeted 16S sequencing libraries were amplified using the Ion Plus Fragment Library Kit and Ion Xpress Barcode Adapters Kit (Thermo Fisher Scientific). The amplified DNA was purified using Agencourt AMpure XP beads (Beckman Coulter, USA) on DynaMag-2 Magnet (Thermo Fisher Scientific). The concentration of purified library was measured on an Agilent 2100 Bioanalyzer using an Agilent High Sensitivity DNA Kit (Agilent Technologies, USA). Then, the library was diluted into a final concentration of 100 pM. The Ion Chef instrument was used to perform clonal amplification of the pooled library on ion sphere particles by emulsion PCR, bead enrichment, and chip loading using the Ion Torrent 510 & 520 & 530 Kit Chef Template Preparation System (Thermo Fisher Scientific). Load ion 530 chips were sequenced using the Ion Torrent S5 System (Thermo Fisher Scientific) with 850 flows.

For microbial community profiling, 16S-rRNA gene sequences were amplified from cDNA using the 16S Metagenomics Kit (Thermo Fisher Scientific, Madrid, Spain), which includes two primer sets of the 16S hypervariable regions in bacteria: primer set V2–4–8 and primer set V3–6, 7–9. PCR products were purified using Agencourt AMPure XP DNA purification beads (Beckman Coulter Genomics GmbH, Bernried, Germany) to remove primer dimers. The concentration and the average size of each amplicon were determined using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen), and the amount of DNA fragments per microliter was calculated. Libraries were prepared using the Ion Plus Fragment Library Kit, and samples were labeled using the Ion Xpress Barcode Adapters 1–16 kit. Library concentrations were determined using the Ion Universal Library Quantification Kit. Emulsion PCR and sequencing of the amplicon libraries were carried out using the Ion Torrent S5 system and the Ion 520/530 Kit-Chef. After sequencing, the individual sequence reads were filtered using the Ion Reporter Software V4.0 to remove low-quality and polyclonal sequences. All Ion platforms and reagents were from Thermo Fischer Scientific.

Sequencing was performed with the use of an Ion Torrent Personal Genome Machine (PGM) platform (Life Technologies, Carlsbad, CA, USA) and 16S Metagenomics Kit (Life Technologies; Carlsbad, CA, USA) as described previously [90 (link)]. A maximum of 26 barcoded 16S samples were sequenced on an Ion 318 v2 chip (Life Technologies, Carlsbad, CA, USA) using the Ion PGM Sequencing 400 Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol.
Sequencing data were analyzed and bacterial taxonomy was evaluated with the use Ion Reporter software (Thermo Fischer, Waltham, MA, USA) and fallowing databases: Curated MicroSEQ(R) 16S Reference Library v2013.1; Curated Greengenes v13.5. Data are presented as a percentage of bacterial taxa in each sample.

Partial 16S rRNA gene sequences were amplified from extracted DNA using the 16S Metagenomics Kit (Life Technologies, Monza, Italy) that is designed for rapid analysis of polybacterial samples using Ion Torrent sequencing technology. The kit includes two primer sets that selectively amplify the corresponding hypervariable regions of the 16S region in bacteria: primer set V2-4-8 and primer set V3-6, 7-9. The PCR conditions used were 10 min at 95 °C, 30 cycles of 30 s at 95 °C, 30 s at 58 °C, and 20 s at 72 °C, followed by 7 min at 72 °C. Amplification was carried out by using a SimpliAmp thermal cycler (Life Technologies, Monza, Italy). The integrity of the PCR amplicons was analyzed by electrophoresis on 2% agarose gel.