2.6. Data analysis
After 16S rRNA sequencing, the reads were sorted to the samples
according to barcodes, and the barcode and primers were removed.
The low-quality reads (i.e., quality score b 25) were discarded
using Trimmomatic (v0.30, http://www.usadellab.org/cms/?page=
trimmomatic). The clean reads were joined using ‘make.contigs’ in
the Mothur software package (v 1.34.0 (http://www.mothur.org).
Quality filtering on the sequences was performed, and sequences
that did not fulfill the necessary criteria (i.e., length between 400
and 480 bp and no ambiguous bases) were discarded.
The clean sequences were clustered into operational taxonomic
units (OTUs) with the UCLUST method at 97% similarity using Qiime
(http://qiime.org/tutorials/otu_picking.html). The most frequent OTUs
were plotted on a heatmap. One representative sequence for each OTU
was selected and assigned to taxonomic category using the Ribosomal
Database Program (RDP) classifier at a confidence threshold of 0.97.
The RDP classifier uses the Greengenes 16S rRNA gene database (13_8
release http://greengenes.secondgenome.com/), which has taxonomic
categories predicted down to the genus level.
Statistical analysis was conducted using SPSS Statistics 19 (IBM
Corp., Armonk, NY, USA). One-way ANOVA was used to detect the differences
in antioxidant enzyme activities and chlorophyll fluorescence
characteristics among different treatment systems, and two-way
ANOVA was used to evaluate the effects of TAP loading, sampling site
and season on plant photosynthetic parameters and enzyme activities
in the substrate.