title: CV10_Heavy_2_16S
design: To analyse changes in the composition and diversity of the bacterial communities in the SIP culture treatments, we set up parallel incubations with 15N-N? (treatment) to trace N?-fixing bacteria and with 14N-N? (natural abundance, control) as a negative control. DNA samples from the SIP heavy and light fractions were used as templates to amplify the V4V5 hypervariable regions of the 16S rRNA gene using prokaryotic primers 515F (5-GTGCCAGCMGCCGCGGGTAA-3) and 907R (5?-CCGTCAATTCMTTTRAGTTT-3?) . The resulting amplicons were then sequenced on a NovaSeq 6000 platform in paired-end 250 bp mode (PE250). Raw sequences of the 16S rRNA gene were controlled for quality using QIIME 2 (v2024.10; Northern Arizona University, Center for Applied Microbiome Science, Flagstaff, USA; https://github.com/qiime2/), with steps including filtering, deduplicating, denoising, merging, and removing chimeras . Amplicon sequence variants (ASVs) were generated using the DADA2 pipeline in QIIME 2 . The truncating and trimming parameters of DADA2 were set to -p-trim-left-f 0, -p-trim-left-r 0, -p-trunc-len-f 220, and -p-trunc-len-r 205. The feature table generated by DADA2 was filtered to remove ASVs with frequencies <2, and the remaining ASVs were classified using the naive Bayesian classifier in QIIME 2. The classifier was trained based on 99% of the OTUs in the SILVA rRNA database (v152; Max Planck Institute for Marine Microbiology, Microbial Genomics and Bioinformatics Research Group, Bremen, Germany; https://www.arb-silva.de/) .
identifiers.namespace: SUB15630868
identifiers.id: CV10_Heavy2_16S