Agentcourt ampure xp system

Libraries of 16S DNA were constructed based on PCR amplification of 7 of the 9 hypervariable regions of the 16S rDNA gene (V2, V3, V4, V6–V9). Amplification was performed in two independent reactions using the 16S Metagenomics™ system according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA) in a Verity™ thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). An equimolar mixture using the amplification products was prepared, and 50 nanograms were used to construct the 16S rDNA libraries with the Ion Plus Fragment Library commercial system and the Ion Xpress barcode adapters (Thermo Fisher Scientific). Library purification was carried out using the Agentcourt AMPure XP system according to the manufacturer’s instructions (Beckman Coulter, Brea, CA, USA) and quantified with a highly sensitive DNA commercial system and the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Library concentration was adjusted to 26 pM followed by PCR amplification of the PCR emulsion using a volume of 25 µL of the equimolar mixture for all samples (One-Touch 2, Thermo Fisher Scientific, Waltham, MA, USA) and enriched with the OneTouch Enrichment system (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was carried out using the Ion S5™ system (Thermo Fisher Scientific, Waltham, MA, USA).

The construction of 16S DNA libraries started with the PCR-based amplification of 7 of the 9 hypervariable regions of the 16S rDNA gene (V2, V3, V4, V6–V9), achieved in two independent reactions throughout the use of the 16S metagenomics system following the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA) in a SelectCycler device (Select BioProduct, Life Science Research, Waltham, MA, USA). Afterwards, 50 nanograms of the equimolar mixture prepared from the amplification products were used to generate the 16S rDNA libraries with the Ion Plus Fragment Library commercial system and the Ion Xpress barcode adapters (Thermo Fisher Scientific). Libraries were purified with the Agentcourt AMPure XP system according to the manufacturer’s instructions (Beckman Coulter, Brea, CA) and quantified using a highly sensitive DNA commercial system and the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Concentration was adjusted to 26 pM followed by PCR-amplification of the PCR emulsion using a volume of 25 µL of the equimolar mixture of all samples (One-Touch 2, Thermo Fisher Scientific) and enriched with the OneTouch Enrichment system (Thermo Fisher Scientific). Sequencing was performed using the Ion S5™ system (Thermo Fisher Scientific).

PCR-based amplification of 7 of the 9 hypervariable regions of the 16S rRNA gene (V2, V3, V4, V6-7, V8, and V9) was performed in two independent reactions using the 16S metagenomics system according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA) in a SelectCycler device (Select BioProduct, Life Science Research, Waltham, MA, USA). Subsequently, 50 nanograms of an equimolar mixture prepared from the amplification products were used to generate the 16S rRNA libraries by using the Ion Plus Fragment commercial system and Ion Xpress barcode adapters (Thermo Fisher Scientific). Purification was performed in each of the steps with the Agentcourt AMPure XP system according to the manufacturer’s instructions (Beckman Coulter, Brea, CA, USA). Libraries were quantified using the highly sensitive DNA commercial system and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and the concentration was adjusted to 26 pM. PCR-amplification of the emulsion (PCR emulsion) was carried out in a 25 µL volume from an equimolar mixture of all samples (One-Touch 2, Thermo Fisher Scientific) and enriched in the OneTouch Enrichment system (Thermo Fisher Scientific). Sequencing was performed on the PGM-Ion Torrent (Personal Genome Machine) platform.