Agencourt ampure xp magnetic particles

We used the methodology previously described by Pisarenko et al. [33 (link)]. The preparation of a genomic library with a 400-bp read length was performed using the Ion Xpress Plus Fragment Library Kit reagent kit (Life Technologies, USA) in accordance with the manufacturer’s protocol. DNA library fragments were separated using a ready-to-use commercial kit 2% E-Gel SizeSelect agarose gel (Invitrogen, USA). The finished library of DNA fragments was purified using Agencourt AMPure XP magnetic particles (Beckman Coulter, USA). Library quality and concentration were determined using the Experion™ Automated Electrophoresis System and Experion DNA 1 K Reagents and Supplies and Experion DNA Chips kits (Bio-Rad, USA). Monoclonal amplification on microspheres was performed using Ion PGM Hi-Q View OT2 Kit reagents (Life Technologies, USA) in accordance with the manufacturer’s protocol. Microsphere enrichment was performed using Dynabeads MyOne Streptavidin C1 magnetic particles (Invitrogen, Life Technologies, USA). The effectiveness of the enrichment process was evaluated using the Ion Sphere Quality Control Kit (Life Technologies, USA). Genome sequencing was performed using an Ion Torrent PGM sequencer and Ion 316 Chips Kit V2 chips (Life Technologies, USA).

The preparation of genomic libraries with a 400 bp read length was performed using the Ion Xpress Plus Fragment Library Kit reagent kit (Life Technologies, USA) in accordance with the manufacturer’s protocol. DNA library fragments were separated using 2% E-Gel SizeSelect agarose gel (Invitrogen, USA). The finished libraries of DNA fragments were purified using Agencourt AMPure XP magnetic particles (Beckman Coulter, USA). Libraries quality and concentration were determined using the Experion™ Automated Electrophoresis System and Experion DNA 1 K Reagents and Supplies and Experion DNA Chips kits (Bio-Rad, USA). Monoclonal amplification on microspheres was performed using Ion PGM Hi-Q View OT2 Kit reagents (Life Technologies, USA) in accordance with the manufacturer’s protocol. Microsphere enrichment was performed using Dynabeads MyOne Streptavidin C1 magnetic particles (Invitrogen, Life Technologies, USA). The effectiveness of the enrichment process was evaluated using the Ion Sphere Quality Control Kit (Life Technologies, USA). Genome sequencing was performed using an Ion Torrent PGM sequencer and Ion 318 Chips Kit V2 chips (Life Technologies, USA).

To study the uncultivated metagenome, total DNA from aerosol and swab samples was isolated using a PureLink™ Microbiome DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA). Since the amount of DNA in the samples was low, the number of amplification cycles was increased to 30 according to the manufacturer’s instructions.
Amplification of the seven most variable regions of the 16S rRNA gene (V2, V3, V4, V6, V7, V8, and V9) was performed using the Ion 16S™ Metagenomics Kit (Thermo Fisher Scientific, Waltham, MA, USA). To test the amplification of the required specific regions, electrophoresis in 2% agarose gel was performed with visualization with ethidium bromide. A library cleaning purification was performed using Agencourt AMPure XP magnetic particles (Beckman Coulter, Brea, CA, USA). Amplicon barcoding was performed using IonCode Barcode Adapters (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Sequencing was performed on an Ion S5XL sequencer (Thermo Fisher Scientific, Waltham, MA, USA) using Ion 520 and Ion 530 Chips (Thermo Fisher Scientific, Waltham, MA, USA). The sequence data were deposited in the NCBI Sequence Read Archive under accession number PRJNA788451.