Computational protocol: Understory Dwarf Bamboo Affects Microbial Community Structures and Soil Properties in a Betula ermanii Forest in Northern Japan

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Protocol publication

[…] Raw data formatted as FASTQ were analyzed with FLASH version 1.2.11 to merge paired-end 300-bp reads from next-generation sequencing experiments (). The CD-HIT-OTU program ( was used to identify operational taxonomic units (OTUs) (). Three-step clustering was performed with (1) raw read filtering and trimming; (2) error-free read selection; and (3) OTU clustering at distant cut-offs (0.03). The taxonomy assignment of bacterial and fungal sequences was based on the full alignments of 16S rRNA and ITS genes, respectively. Cluster sequences were uploaded to the Silva SSU database for 16S rRNA genes and the UNITE database for ITS genes. Quantitative insights into microbial ecology (QIIME) software version 1.9.1 were used to perform alpha diversity analyses (Chao1, Shannon Index, Simpson Index) on bacterial and fungal communities (). Differences in the number of OTUs and alpha diversity estimates were tested using a one-way ANOVA, followed by Tukey’s honest significant difference test (HSD).Significant differences in soil physicochemical properties between the SI and SR plots on the five sampling dates were tested using generalized linear mixed models, followed by Tukey’s HSD test. Generalized linear mixed models were also used to examine the relationships between the number of OTUs of microbial communities and soil physicochemical properties in the SI and SR plots. Differences between the relative abundance of microbial communities were tested with the Kruskal–Wallis method (). All statistical calculations were performed in R version 3.2 (). Values p<0.05 were considered to be significant. A redundancy analysis (RDA) is a constrained ordination technique that was used to test whether the occurrence of microbial communities matches specific environmental source profiles (, ), which was performed with abundance and environmental data using Canoco version 4.5 (). […]

Pipeline specifications

Software tools CD-HIT-OTU, QIIME
Application 16S rRNA-seq analysis
Organisms Fungi, Bacteria
Chemicals Carbon, Nitrogen