Computational protocol: The effects of pastoral intensification on the feeding interactions of generalist predators in streams

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Protocol publication

[…] Sequence processing was performed in Galaxy (, Blankenberg et al., ; Giardine et al., ; Goecks, Nekrutenko, Taylor, & Team, ). Sequences were split by forward and reverse MIDs and adaptors, primers and MIDs were removed before filtering sequences by length (260–300 bp). Sequences from each individual were collapsed into unique haplotypes, and rare haplotypes (<2 copies) were excluded. The remaining sequences from all individuals were combined and clustered into molecular operational taxonomic units (MOTU) using the usearch algorithm in Qiime (usearch61; Edgar, ). MOTU clustering was repeated with similarity thresholds decreasing in increments of 0.01 from 0.97 to 0.87. For each similarity value, representative sequences were selected from the resultant MOTUs and “BLASTed” directly at the NCBI website ( using nucleotide blast (Zhang, Schwartz, Wagner, & Miller, ) optimized for very similar sequences (megablast) on the nucleotide collection (nr/nt) using default parameters. The output from the blast alignment was imported into MEGAN (MEtaGenomics ANalyzer; Huson, Auch, Qi, & Schuster, ), which assigns taxonomy to each MOTU at the lowest level that encompasses the top blast hits. The optimal similarity threshold was the value that resulted in the lowest number of species (excluding chimeras) with multiple MOTUs allocated to them, whilst retaining the majority of species assignments (0.89). […]

Pipeline specifications

Software tools Galaxy, USEARCH, QIIME, BLASTN, MEGAN
Application Metagenomic sequencing analysis
Organisms Dinocras cephalotes, Rhinophrynus dorsalis