Analyzing changes in bacterial community composition and diversity using 16S rRNA gene amplicon sequencing

To determine changes in bacterial community composition and diversity during composting all collected samples (each time point consisting of 5 replicates) were subjected to 16S rRNA gene amplicon sequencing using a NovaSeq6000 platform (Illumina, PE250 mode, Guangdong Magigene Biotechnology Co. Ltd, Guangzhou, China). Total genomic DNA for amplicon sequencing was extracted using a DNeasy PowerSoil kit (Qiagen, Hilden, Germany) following manufacturer’s instructions. The prokaryote (bacteria and archaea) primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) targeting the V4-V5 region of the 16S rRNA gene were used. The raw 16S rRNA gene sequences were processed using QIIME 2 (version 2019.7) [29] and quality filtered (i.e., filtered, dereplicated, denoised, merged, and assessed for chimaeras) to produce amplicon sequence variants (ASVs) using the DADA2 pipeline in QIIME2 [30]. The truncation and trimming parameters in DADA2 were set to –p-trim-left-f 0, –p-trim-left-r 0; and –p-trunc-len -f 248, –p-trunc-len-r 230. The DADA2-generated feature table was filtered to remove ASVs at a frequency less than two and the remaining ASVs were classified using the QIIME2 naive Bayes classifier trained on 99% operational taxonomic units available at the SILVA rRNA database (v 138) [31]. Microbial diversity was estimated using alpha diversity (Shannon index and observed OTU) and community composition using beta diversity (weighted UniFrac distance) based on the q2-diversity pipeline within QIIME2. To quantify the community assembly process, null model analysis was used to detect the community assembly mechanism by calculating the standard deviation between the observed ecological model and the randomly generated ecological model [32, 33].