SI text
Fungal ITS sequences generated from the fecal pellets were analysed in QIIME 1.7.0 using the
following script:
# Metagenomic analysis of ITS1f-ITS4 fungal data from 16 possum and 14 deer fecal
pellets, Craigieburn, New Zealand
# Three GS junior plates with 10 samples per plate
# Create folder within home directory for the project name, and a subfolder for
the individual sequencing run, e.g. ~/454_Ectomyco/Run1
# Place raw data files in this folder
# Create mapping file (map.txt) for samples with MID tag and primer data and
place in this folder
# Create folder ~/454_Ectomyco/Post_split
# Create folder ~/454_Ectomyco/Post_denoise
# STEP 1: Split libraries based on MID tags, truncate tags and primers based on
mapping file, filter sequences based on length and quality scores
split_libraries.py -m 454_Ectomyco/Run1/map.txt -f
454_Ectomyco/Run1/Run1_shotgun_pipeline/1.TCA.454Reads.fna -q
454_Ectomyco/Run1/Run1_shotgun_pipeline/1.TCA.454Reads.qual -o
454_Ectomyco/Run1/Mapping_output -d -M 2 -l 200 -L 900 -b 10 -w 50 -s 25 -H 15
split_libraries.py -m 454_Ectomyco/Run2/map.txt -f
454_Ectomyco/Run2/Run2_shotgun_pipeline/1.TCA.454Reads.fna -q
454_Ectomyco/Run2/Run2_shotgun_pipeline/1.TCA.454Reads.qual -o
454_Ectomyco/Run2/Mapping_output -d -M 2 -l 200 -L 900 -b 10 -w 50 -s 25 -H 15
split_libraries.py -m 454_Ectomyco/Run3/map.txt -f
454_Ectomyco/Run3/Run3_shotgun_pipeline/1.TCA.454Reads.fna -q
454_Ectomyco/Run3/Run3_shotgun_pipeline/1.TCA.454Reads.qual -o
454_Ectomyco/Run3/Mapping_output -d -M 2 -l 200 -L 900 -b 10 -w 50 -s 25 -H 15
# STEP 2: Concatenate the Mapping_output fna files to new file
~454_Ectomyco/Post_split/seqs.fna
# STEP 3: Pick OTUs at 97% similarity
pick_otus.py -i 454_Ectomyco/Post_split/seqs.fna -o
454_Ectomyco/Post_denoise/OTUs -t -s 0.97
# STEP 4: Select representative OTU sequences
pick_rep_set.py -i 454_Ectomyco/Post_denoise/OTUs/seqs_otus.txt -f
454_Ectomyco/Post_split/seqs.fna -o 454_Ectomyco/Post_denoise/Rep_OTUs_97 -m
most_abundant
# STEP 5: Assign taxonomy to representative OTU sequences using the UNITE 12_11
fungal ITS database
assign_taxonomy.py -i 454_Ectomyco/Post_denoise/Rep_OTUs_97 -t
454_Ectomyco/its_12_11_otus/taxonomy/97_otu_taxonomy.txt -r
454_Ectomyco/its_12_11_otus/rep_set/97_otus.fasta -m blast
# STEP 6: Create OTU table
make_otu_table.py -i 454_Ectomyco/Post_denoise/OTUs/seqs_otus.txt -t
454_Ectomyco/Post_denoise/blast_assigned_taxonomy/Rep_OTUs_97_tax_assignments.txt
-o 454_Ectomyco/Post_denoise/OTU_table
# STEP 7: Concatenate mapping files to new file
~454_Ectomyco/Post_denoise/concat_map.txt
# STEP 8: Plot taxonomic summary by sample
summarize_taxa_through_plots.py -o 454_Ectomyco/Post_denoise/Plot_samples -i
454_Ectomyco/Post_denoise/OTU_table -m 454_Ectomyco/Post_denoise/concat_map.txt c Sample -f -s