Ion universal library quantitation kit

For 16S rRNA amplicon sequencing, similar methods were followed to those previously reported (20 (link)). Twelve microliters of DNA extracted from each sample was used to perform the libraries, and the Ion GeneStudio™ S5 System (Life Technologies, Carlsbad, CA, USA) was used. The 16S hypervariable regions were amplified with two sets of primers, v2-4-8 and v3-6,7-9, and libraries were then constructed by using the Ion 16S™ Metagenomics Kit (Life Technologies) and the Ion Xpress™ Plus Fragment Library Kit (Life Technologies). Libraries containing equal amounts of PCR products pooled with a barcode were prepared by using the Ion Xpress™ Barcode Adapters Kit (Life Technologies). Then, these libraries were quantified by using the Ion Universal Library Quantitation Kit (Life Technologies). Next, 10 pM of each library was pooled and loaded on an Ion OneTouch™ 2 System (Life Technologies), which automatically performs template preparation and enrichment. Template-positive ion sphere particles were enriched with Dynabeads™ MyOne™ Streptavidin C1 magnetic beads (Invitrogen, Carlsbad, CA, USA) by using an Ion One Touch ES instrument. Finally, an Ion 520™ chip (Life Technologies) was loaded with the samples on an Ion GeneStudio™ S5 System sequencer using the Ion 520™ and Ion 530™ Loading Reagents supplied in the OT2-Kit (Life Technologies).

For 16S rRNA amplicon sequencing, 2 μL of DNA extracted from each sample was used to construct the libraries, and the Ion GeneStudio^TM S5 System (Life Technologies, Carlsbad, CA, USA) was used. For this purpose, the 16S hypervariable regions were amplified with two sets of primers, v2–4–8 and v3–6, 7–9, and the libraries were prepared by using the Ion 16S^TM Metagenomics Kit (Life Technologies) and the Ion Xpress^TM Plus Fragment Library Kit (Life Technologies). Libraries containing equal amounts of PCR products pooled with a barcode were prepared by using the Ion Xpress^TM Barcode Adapters Kit (Life Technologies). Then, these libraries were quantified by using the Ion Universal Library Quantitation Kit (Life Technologies). Next, 10 pM of each library was pooled and loaded on an Ion OneTouch™ 2 System (Life Technologies), which automatically performs template preparation and enrichment. Template-positive ion sphere particles were enriched with Dynabeads™ MyOne™ Streptavidin C1 magnetic beads (Invitrogen, Carlsbad, CA, USA) by using an Ion One Touch ES instrument. Finally, an Ion 520 TM chip (Life Technologies) was loaded with the samples on an Ion GeneStudioTM S5 System sequencer using the Ion 520™ & Ion 530™ Loading Reagents supplied in the OT2-Kit (Life Technologies).

The sequencing procedure was described in detail in the previous publication [6 (link)]. The DNA library was constructed using the Ion Plus Fragment Library Kit (Life Technologies, Carlsbad, CA, USA) and purified using the Agencourt AMPure XP Reagent (Beckman Coulter, Brea, CA, USA) and then quantified by the Ion Universal Library Quantitation Kit and a real-time PCR instrument—Quant Studio 5 (Life Technologies, Carlsbad, CA, USA). The library was coated onto beads and sequenced using an Ion PGM System using the Ion PGM™ Hi-Q™ View Sequencing Kit on an Ion 316™ Chip Kit v2 BC (Life Technologies, Carlsbad, CA, USA).