usage: micca otu [-h] -i FILE [-o DIR] [-r FILE]
[-m {denovo_greedy,denovo_swarm,closed_ref,open_ref}] [-d ID]
[-n MINCOV] [-t THREADS] [-c] [-g {dgc,agc}] [-s MINSIZE]
[-a {both,plus}] [--swarm-differences SWARM_DIFFERENCES]
[--swarm-fastidious]
micca otu assigns similar sequences (marker genes such as 16S rRNA and
the fungal ITS region) to operational taxonomic units (OTUs).
Trimming the sequences to a fixed position before clustering is
*strongly recommended* when they cover partial amplicons or if quality
deteriorates towards the end (common when you have long amplicons and
single-end sequencing).
Removing ambiguous nucleotides 'N' (with the option --maxns 0 in micca
filter) is mandatory if you use the de novo swarm clustering method.
micca otu provides the following protocols:
* de novo greedy clustering (denovo_greedy): sequences are clustered
without relying on an external reference database, using an
approach similar to the UPARSE pipeline (doi: 10.1038/nmeth.2604):
i) predict sequence abundances of each sequence by dereplication; ii)
greedy clustering; iii) remove chimeric sequences (de novo, optional)
from the representatives; iv) map sequences to the representatives.
* de novo swarm (denovo_swarm): sequences are clustered without relying
on an external reference database, using swarm (doi:
10.7717/peerj.593, doi: 10.7717/peerj.1420,
https://github.com/torognes/swarm): i) predict sequence abundances of
each sequence by dereplication; ii) swarm clustering; iii) remove
chimeric sequences (de novo, optional) from the representatives.
* closed-reference clustering (closed_ref): sequences are clustered
against an external reference database and reads that could not be
matched are discarded.
* open-reference clustering (open_ref): sequences are clustered
against an external reference database and reads that could not be
matched are clustered with the 'de novo greedy' protocol.
Outputs:
* otutable.txt: OTU x sample, TAB-separated OTU table file,
containing the number of times an OTU is found in each sample.
* otus.fasta: FASTA file containing the representative sequences (OTUs);
* otuids.txt: OTU ids to original sequence ids (tab-delimited text file)
* hits.txt: three-columns, TAB-separated file:
1. matching sequence
2. representative (seed)
3. identity (if available, else '*'), defined as:
matching columns
-------------------------------- ;
alignment length - terminal gaps
* otuschim.fasta (only for 'denovo_greedy', 'denovo_swarm' and
'open_ref' when --rmchim is specified): FASTA file containing the
chimeric otus;
optional arguments:
-h, --help show this help message and exit
arguments:
-i FILE, --input FILE
input fasta file (required).
-o DIR, --output DIR output directory (default .).
-r FILE, --ref FILE reference sequences in fasta format, required for
'closed_ref' and 'open_ref' clustering methods.
-m {denovo_greedy,denovo_swarm,closed_ref,open_ref}, --method {denovo_greedy,denovo_swarm,closed_ref,open_ref}
clustering method (default denovo_greedy)
-d ID, --id ID sequence identity threshold (for 'denovo_greedy',
'closed_ref' and 'open_ref', 0.0 to 1.0, default
0.97).
-n MINCOV, --mincov MINCOV
reject sequence if the fraction of alignment to the
reference sequence is lower than MINCOV. This
parameter prevents low-coverage alignments at the end
of the sequences (for 'closed_ref' and 'open_ref'
clustering methods, default 0.75).
-t THREADS, --threads THREADS
number of threads to use (1 to 256, default 1).
-c, --rmchim remove chimeric sequences (for 'denovo_greedy',
'denovo_swarm' and 'open_ref' OTU picking methods).
-g {dgc,agc}, --greedy {dgc,agc}
greedy clustering strategy, distance (DGC) or
abundance-based (AGC) (for 'denovo_greedy' and
'open_ref' clustering methods) (default dgc).
-s MINSIZE, --minsize MINSIZE
discard sequences with an abundance value smaller than
MINSIZE after dereplication (>=1, default 2).
Recommended value is 2 (i.e. discard singletons) for
'denovo_greedy' and 'open_ref', 1 (do not discard
anything) for 'denovo_swarm'.
-a {both,plus}, --strand {both,plus}
search both strands or the plus strand only (for
'closed_ref' and 'open_ref' clustering methods,
default both).
Swarm specific options:
--swarm-differences SWARM_DIFFERENCES
maximum number of differences allowed between two
amplicons. Commonly used d values are 1 (linear
complexity algorithm), 2 or 3, rarely higher. (>=0,
default 1).
--swarm-fastidious when working with SWARM_DIFFERENCES=1, perform a
second clustering pass to reduce the number of small
OTUs (recommended option).
Examples
De novo clustering with a 97% similarity threshold and remove
chimeric OTUs:
micca otu -i input.fasta --method denovo_greedy --id 0.97 -c
Open-reference OTU picking protocol with a 97% similarity
threshold, without removing chimeras in the de novo protocol step
and using 8 threads:
micca otu -i input.fasta --method open_ref --threads 8 --id 0.97 \
--ref greengenes_2013_05/rep_set/97_otus.fasta
De novo swarm clustering with the protocol suggested by the authors
using 4 threads (see https://github.com/torognes/swarm and
https://github.com/torognes/swarm/wiki):
micca otu -i input.fasta --method denovo_swarm --threads 4 \
--swarm-fastidious --rmchim --minsize 1