pick_closed_reference_otus.py – Closed-reference OTU picking/Shotgun UniFrac workflow.

Description:

This script picks OTUs using a closed reference and constructs an OTU table. Taxonomy is assigned using a pre-defined taxonomy map of reference sequence OTU to taxonomy. If full-length genomes are provided as the reference sequences, this script applies the Shotgun UniFrac method.

Note: If most or all of your sequences are failing to hit the reference, your sequences may be in the reverse orientation with respect to your reference database. To address this, you should add the following line to your parameters file (creating one, if necessary) and pass this file as -p:

pick_otus:enable_rev_strand_match True

Be aware that this doubles the amount of memory used.

Usage: pick_closed_reference_otus.py [options]

Input Arguments:

Note

[REQUIRED]

-i, --input_fp

The input sequences

-o, --output_dir

The output directory

[OPTIONAL]

-r, --reference_fp

The reference sequences [default: /Users/caporaso/.virtualenvs/qiime/lib/python2.7/site-packages/qiime_default_reference/gg_13_8_otus/rep_set/97_otus.fasta]. NOTE: If you do not pass -r to this script, you will be using QIIME’s default reference sequences. In this case, QIIME will copy the corresponding reference tree to the output directory. This is the tree that should be used to perform phylogenetic diversity analyses (e.g., with core_diversity_analyses.py).

-p, --parameter_fp

Path to the parameter file, which specifies changes to the default behavior. See http://www.qiime.org/documentation/file_formats.html#qiime-parameters . [if omitted, default values will be used]

-t, --taxonomy_fp

The taxonomy map [default: /Users/caporaso/.virtualenvs/qiime/lib/python2.7/site-packages/qiime_default_reference/gg_13_8_otus/taxonomy/97_otu_taxonomy.txt]

-s, --assign_taxonomy

Assign taxonomy to each sequence using assign_taxonomy.py (this will override –taxonomy_fp, if provided) [default: False]

-f, --force

Force overwrite of existing output directory (note: existing files in output_dir will not be removed) [default: None]

-w, --print_only

Print the commands but don’t call them – useful for debugging [default: False]

-a, --parallel

Run in parallel where available [default: False]

-O, --jobs_to_start

Number of jobs to start. NOTE: you must also pass -a to run in parallel, this defines the number of jobs to be started if and only if -a is passed [default: 1]

--suppress_taxonomy_assignment

Skip the taxonomy assignment step, resulting in an OTU table without taxonomy (this will override –taxonomy_fp and –assign_taxonomy, if provided) [default: False]

Output:

Pick OTUs, assign taxonomy, and create an OTU table against a reference set of OTUs. ALWAYS SPECIFY ABSOLUTE FILE PATHS (absolute path represented here as $PWD, but will generally look something like /home/ubuntu/my_analysis/).

pick_closed_reference_otus.py -i $PWD/seqs.fna -r $PWD/refseqs.fna -o $PWD/otus_w_tax/ -t $PWD/taxa.txt

Pick OTUs and create an OTU table against a reference set of OTUs without adding taxonomy assignments. ALWAYS SPECIFY ABSOLUTE FILE PATHS (absolute path represented here as $PWD, but will generally look something like /home/ubuntu/my_analysis/).

pick_closed_reference_otus.py -i $PWD/seqs.fna -r $PWD/refseqs.fna -o $PWD/otus/

Pick OTUs, assign taxonomy, and create an OTU table against a reference set of OTUs using usearch_ref. ALWAYS SPECIFY ABSOLUTE FILE PATHS (absolute path represented here as $PWD, but will generally look something like /home/ubuntu/my_analysis/).

pick_closed_reference_otus.py -i $PWD/seqs.fna -r $PWD/refseqs.fna -o $PWD/otus_usearch/ -p $PWD/usearch_params.txt -t $PWD/taxa.txt

Pick OTUs using usearch_ref, assign taxonomy, and create an OTU table against a reference set of OTUs using usearch_ref. ALWAYS SPECIFY ABSOLUTE FILE PATHS (absolute path represented here as $PWD, but will generally look something like /home/ubuntu/my_analysis/).

pick_closed_reference_otus.py -i $PWD/seqs.fna -r $PWD/refseqs.fna -o $PWD/otus_usearch_ref/ -p $PWD/usearch5.2_params.txt -t $PWD/taxa.txt

Pick OTUs, assign taxonomy, and create an OTU table against a reference set of OTUs using sortmerna. ALWAYS SPECIFY ABSOLUTE FILE PATHS (absolute path represented here as $PWD, but will generally look something like /home/ubuntu/my_analysis/).

pick_closed_reference_otus.py -i $PWD/seqs.fna -r $PWD/refseqs.fna -o $PWD/otus_sortmerna/ -p $PWD/sortmerna_params.txt -t $PWD/taxa.txt