ASAP pipeline

Description of Method

The amplicon sequence data was analyzed using an internal pipeline Amplicon Sequencing Analysis Pipeline (ASAP, version 1.4). The MiSeq sequences (2 * 251 bp) were first subjected to quality check with FastQC (version 0.11.5). The pair-end sequences were then merged based on the 3’ overlap using PEAR (version 0.9.10) [1] with a quality score cutoff of 20, minimum assembled length of 200, maximum assembled length of 400 and minimum overlap length of 50 bp. The program of QIIME packages (version 1.9.1) [2] was used to assign reads to samples (demultiplexing) based on the barcodes with the maximum barcode error of 0 and trimming quality score cutoff of 20. Primer sequences (forward and reverse) were trimmed. Sequences of library splitting of multiple sequencing rounds (two rounds in this study) were merged. Dereplication was performed using USEARCH (version 9.2.64) [3] with the command fastx_uniques (with the option of -sizeout for sequence abundance output). Operational Taxonomic Units (OTUs) were clustered using UPARSE (command -cluster_otus of USEARCH) [4] with OTU identity threshold of 0.97 and singletons and chimeric sequences were removed during this process. OTU table was made using command of -usearch_global of USEARCH. The representative sequences of OTUs were classified using RDP Classifier (16S: training set 16, June 2016, ITS: trainset fungalits_warcup, July 2016) [5] with confidence cutoff of 0.8. OTUs assigned to Chloroplast (at Order level) were removed (for ITS, delete this sentence). The representative sequences of OTUs were used to construct the phylogenetic tree. Sequences were aligned using MAFFT (version 3.8.31) [6] and alignments were filtered using Gblocks (version 0.91b) [7] with option of -t=d, -b4=3 and -b5=h. FastTree [8] was used to construct phylogenetic tree with the filtered alignment. The phylogenetic tree and OTU table was used in calculation of alpha diversity (phylogeny based indexes) and beta diversity (UniFrac distance) using programs of QIIME.

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