V2-3_ITS
For metabarcoding analyses, polymerase chain reactions (PCRs) were performed to amplify the V3-V4 region of the 16S ribosomal RNA (16S rRNA) gene and for the internal transcribed spacer 2 (ITS2) region of ribosomal RNA, representing bacterial and fungal diversity, respectively. The primers used for the 16S rRNA gene (~464 bp) were NexR-16SFWD: 5-CCTACGGGNGGCWGCAG-3 and NexR-16SREV: 5-GACTACHVGGGTATCTAATCC-3 (Klindworth et al., 2013). For ITS2 (~330 bp), primers ITS3: 5-GCATCGATGAAGAACGCAGC-3 and ITS4: 5-TCCTCCGCTTATTGATATGC-3 were used (White, 1990). Illumina overhang adaptors were added to the locus-specific primers, as described in Kozich et al. (2013). The 16S rRNA PCR reactions were conducted using a T100 Thermal Cycler (Bio-Rad, USA) in a total volume of 30 L, including 15 L of MyFi Mix (Bioline, United Kingdom), 0.75 L of each 10 M primer (final concentration of 250 nM), 11 L of DNA-free water (DNase-/RNase-Free Distilled Water, Invitrogen, USA), and 3 L of template DNA. For ITS2, the reaction volume was also 30 L, with 15 L of MyFi Mix, 1.5 L of each 10 M primer (final concentration of 500 nM), 9 L of DNA-free water, and 3 L of template DNA. The thermocycling conditions for 16S rRNA were as follows: 95C for 1 minute, followed by 35 cycles of 95C for 15 seconds, 50C for 15 seconds, 72C for 15 seconds, and a final extension at 72C for 7 minutes. For ITS2, the conditions were: 94C for 5 minutes, followed by 35 cycles of 94C for 30 seconds, 55C for 30 seconds, 72C for 40 seconds, and a final extension at 72C for 10 minutes. Negative controls (no template control - NTC) were included for all PCR reactions. The amplification products were purified and normalized using the SequalPrep Normalization Kit (Thermo Fisher Scientific, USA) to ~1 ng/L. The cleaned amplicons were indexed with the xGen Amplicon UDI Primers (Integrated DNA Technologies, USA) kit and underwent high-throughput sequencing on an Illumina NextSeq 2000 platform. The library pool, diluted to a final loading concentration of 600 pM with a 30% PhiX spike, was sequenced using the NextSeq Reagent X-LEAP SBS kit P1-600 cycle (2 301 bp) (Illumina, USA).
SUB15706288
MEF-24-01-096