QIIME Script Setting up usearch: qiime@qiime-VirtualBox:~$ cd qiime_software/usearch qiime@qiime-VirtualBox/qiime_software/usearch:~$ chmod 755 usearch qiime@qiime-VirtualBox/qiime_software/usearch:~$ cd qiime@qiime-VirtualBox:~$ source ./.bashrc qiime@qiime-VirtualBox:~$ export PATH=/home/qiime/qiime_software/usearch/:$PATH qiime@qiime-VirtualBox:~$ usearch --version qiime@qiime-VirtualBox:~$ svn co https://pycogent.svn.sourceforge.net/svnroot/pycogent/trunk /home/qiime/qiime_software/pycogent qiime@qiime-VirtualBox:~$ echo "export PYTHONPATH=/home/qiime/qiime_software/pycogent:$PYTHONPATH" >> ~/.bashrc Pick OTUs based on sequence similarity: * pick_otus.py Ði seqs.fna Ðm usearch -s 0.98 Ðg 2 Ðword_length 64 Ðreference_chimera_detection Ðo picked_otus/ Pick representative sequences for each OTU: * pick_rep_set.py Ði picked_otus/combined_seqs_otus.txt Ðf combined_seqs.fna Ðo rep_set.fna Assign taxonomy for OTUs using BLAST method: * assign_taxonomy.py Ði rep_set.fna Ðm blast Ðr taxonomy.fasta Ðt id_taxonomy.txt Assign taxonomy for OTUs using RDP method: * assign_taxonomy.py Ði rep_set.fna Ðr taxonomy.fasta Ðt id_taxonomy.txt Align OTUs: * align_seqs.py Ði rep_set.fna Ðm muscle Ðo muscle_alignment/ Create phylogenetic tree: * make_phylogeny.py Ði muscle_alignment/rep_set_aligned.fasta Ðo rep_phylo_fasttree.tre Create OTU table: * make_otu_table.py Ði picked_otus/seqs_otus.txt Ðt id_taxonomy.txt Ðo otu_table.biom Create OTU table while excluding samples where there was only 1 bat sample per OTU: * make_otu_table.py Ði picked_otus/seqs_otus.txt Ðt id_taxonomy.txt Ðo otu_table.biom2 -e 1MOTUbat.txt ##the next steps are the same for either set of OTUs, the only difference is the input/output file. Only scripts using the full set of OTUs is provided below. Performing multiple rarefactions: * multiple_rarefactions.py Ði otu_table.biom Ðm 5 Ðx 95 Ðs 10 Ðn 10 Ðo rare_5-95 Determining alpha diversity: * alpha_diversity.py Ði rare_5-95/ -o alpha_rare/ -t rep_phylo_fasttree.tre Ðm observed_species,chao1,shannon,simpson,PD_whole_tree * collate_alpha.py Ði alpha_rare/ -o alpha_collated/ Generate rarefaction plots * make_rarefaction_plots.py Ði alpha_collated/ -m Map.txt Ðb BatSp Determining beta diversity * jackknifed_beta_diversity.py Ði otu_table.biom Ðo jackknifed_beta_diversity/ -e 60 Ðm Map.txt Ðt rep_phylo_fasttree.tre Ðp qiime_parameters.txt