Fungi 18S

QIIME - Fungal 18S OTU picking, using BLAST

Download 18S reference database & decompress

wget ftp://ftp.microbio.me/qiime/tutorial_files/its_12_11_otus.tgz

tar -zxvf its_12_11_otus.tgz

gzip -d its_12_11_otus/rep_set/97_otus.fasta.gz

gzip -d its_12_11_otus/rep_set/99_otus.fasta.gz

gzip -d its_12_11_otus/taxonomy/97_otu_taxonomy.txt.gz

gzip -d its_12_11_otus/taxonomy/99_otu_taxonomy.txt.gz # move folder "its_12_11_otus/" to a permanent database directory, accessible for example:

/home/myname/data/its_12_11_otus/

Create OTU setting file

# create new parameter file for pick_open_reference_otus.py and add 4 setting lines (adapt correct database path)

> otu_18S_settings.txt

echo "pick_otus:enable_rev_strand_match True" >> otu_18S_settings.txt

echo "assign_taxonomy:assignment_method blast" >> otu_18S_settings.txt

echo "assign_taxonomy:id_to_taxonomy_fp /home/myname/data/its_12_11_otus/taxonomy/97_otu_taxonomy.txt" >> otu_18S_settings.txt

echo "assign_taxonomy:reference_seqs_fp /home/myname/data/its_12_11_otus/rep_set/97_otus.fasta" >> otu_18S_settings.txt

# check content of parameter file

cat otu_18S_settings.txt

pick_otus:enable_rev_strand_match True

assign_taxonomy:assignment_method blast

assign_taxonomy:id_to_taxonomy_fp /home/myname/data/its_12_11_otus/taxonomy/97_otu_taxonomy.txt

assign_taxonomy:reference_seqs_fp /home/myname/data/its_12_11_otus/rep_set/97_otus.fasta

Run OTU clustering and 18S annotation

# tell BLAST where to find the 18S reference sequence database

export BLASTMAT=/home/myname/data/its_12_11_otus/rep_set/

# run OTU picking

pick_open_reference_otus.py -i seqs.fna -r /home/myname/data/its_12_11_otus/rep_set/97_otus.fasta -o OTU_18S/ -p otu_18S_settings.txt --suppress_align_and_tree

# Result

OTU_18S/otu_table_mc2_w_tax.biom # OTU abundance table of all samples, including 18S taxonomy

# convert OTU abundance table into a tab separated text file, readable with Excel

biom convert -i OTU_18S/otu_table_mc2_w_tax.biom -o OTU_18S/otu_table_mc2_w_tax.tsv --to-tsv --header-key taxonomy

http://nbviewer.jupyter.org/github/biocore/qiime/blob/1.9.1/examples/ipynb/Fungal-ITS-analysis.ipynb