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The chimera.uchime command reads a fasta file and reference file and outputs potentially chimeric sequences. The original uchime program was written by Robert C. Edgar and donated to the public domain, http://drive5.com/uchime. If you would like to follow along, please download UchimeExample.zip.
- 1 Default Settings
- 2 Options
- 3 Revisions
The fasta parameter is required, and you must provide a name file or reference file.
mothur > chimera.uchime(fasta=stool.trim.unique.good.align, reference=silva.gold.align)
The output to the screen should look like:
uchime by Robert C. Edgar http://drive5.com/uchime This code is donated to the public domain. Checking sequences from stool.trim.unique.good.align ... 00:41 23Mb 100.0% Reading stool.trim.unique.good.align 00:41 23Mb 15.4k sequences 00:52 31Mb 100.0% Reading silva.gold.align 00:52 31Mb 5181 sequences Processing sequence: 100, 6 chimeras found. Processing sequence: 200, 9 chimeras found. Processing sequence: 300, 11 chimeras found. Processing sequence: 400, 16 chimeras found. Processing sequence: 500, 23 chimeras found. Processing sequence: 600, 28 chimeras found. ...
Output in stool.trim.unique.good.denova.uchime.chimeras:
0.1101 F11Fcsw_92 S000546342 7000004128191216 98.7 83.8 98.2 82.5 98.2 1 0 0 36 2 2 0.45 N 0.1335 F11Fcsw_455 7000004131499028 S000428418 82.3 74.7 75.3 74.7 78.3 14 1 12 20 5 16 9.40 N 0.0489 F11Fcsw_966 S000366775 S000428418 83.3 81.7 75.8 78.0 79.2 22 9 19 3 0 2 1.97 N 0.0000 F11Fcsw_1136 * * * * * * * * * * * * * * N 0.1347 F11Fcsw_1915 S000436394 S000398603 82.5 75.9 75.0 67.9 76.1 15 1 5 32 15 16 8.70 N 0.0140 F11Fcsw_2390 7000004131499064 S000539267 100.0 99.5 98.6 98.1 99.1 3 1 0 1 0 0 0.47 N ...
The chimeras file format is explained here. Note: You may also set reference=self, in this case mothur will run unique.seqs and check for chimeras using more abundant sequences as the reference.
You can provide a name file to check for chimeras using more abundant sequences as the reference.
mothur > chimera.uchime(fasta=stool.trim.unique.good.align, name=stool.trim.good.names)
If you are using reference=self and provide a groupfile, mothur will use the more abundant sequences from the same sample to check the query sequence.
mothur > chimera.uchime(fasta=stool.trim.unique.good.align, name=stool.trim.good.names, group=stool.good.groups)
The count file is similar to the name file in that it is used to represent the number of duplicate sequences for a given representative sequence. The count file can also contain group information.
mothur > make.table(name=stool.trim.good.names, group=stool.good.groups) mothur > chimera.uchime(fasta=stool.trim.unique.good.align, count=stool.trim.good.count_table)
The processors parameter allows you to specify how many processors you would like to use. Default processors=Autodetect number of available processors and use all available.
The dereplicate parameter can be used when checking for chimeras by group. If the dereplicate parameter is false, then if one group finds the sequence to be chimeric, then all groups find it to be chimeric, default=f. If you set dereplicate=t, and then run remove.seqs with dups=f you can remove only the redundant chimeric sequences.
Let's look at an example:
>seq1 attgacat.... >seq4 ttgacaga.... seq1 seq1,seq2,seq3 seq4 seq4,seq5,seq6 seq1 group1 seq2 group2 seq3 group3 seq4 group1 seq5 group2 seq6 group3
If dereplicate=f and dups=t, (default settings in mothur), and seq2 is found to be chimeric by group2. The results would be:
>seq4 ttgacaga.... seq4 seq4,seq5,seq6 seq4 group1 seq5 group2 seq6 group3
If dereplicate=t and dups=t, and seq2 is found to be chimeric by group2. The results would be:
>seq4 ttgacaga.... seq4 seq4,seq5,seq6 seq4 group1 seq5 group2 seq6 group3
If dereplicate=t and dups=f, and seq2 is found to be chimeric by group2. The results would be:
>seq1 attgacat.... >seq4 ttgacaga.... seq1 seq1,seq3 seq4 seq4,seq5,seq6 seq1 group1 seq3 group3 seq4 group1 seq5 group2 seq6 group3
The abskew parameter can only be used with template=self. Minimum abundance skew. Default 1.9. Abundance skew is: min [ abund(parent1), abund(parent2) ] / abund(query).
The chimealns parameter allows you to indicate you would like a file containing multiple alignments of query sequences to parents in human readable format. Alignments show columns with differences that support or contradict a chimeric model.
The minh parameter - mininum score to report chimera. Default 0.3. Values from 0.1 to 5 might be reasonable. Lower values increase sensitivity but may report more false positives. If you decrease xn you may need to increase minh, and vice versa.
The mindiv parameter - minimum divergence ratio, default 0.5. Div ratio is 100%% - %%identity between query sequence and the closest candidate for being a parent. If you don't care about very close chimeras, then you could increase mindiv to, say, 1.0 or 2.0, and also decrease minh, say to 0.1, to increase sensitivity. How well this works will depend on your data. Best is to tune parameters on a good benchmark.
The xn parameter - weight of a no vote. Default 8.0. Decreasing this weight to around 3 or 4 may give better performance on denoised data.
The dn parameter - pseudo-count prior on number of no votes. Default 1.4. Probably no good reason to change this unless you can retune to a good benchmark for your data. Reasonable values are probably in the range from 0.2 to 2.
The xa parameter - weight of an abstain vote. Default 1. So far, results do not seem to be very sensitive to this parameter, but if you have a good training set might be worth trying. Reasonable values might range from 0.1 to 2.
The chunks parameter is the number of chunks to extract from the query sequence when searching for parents. Default 4.
The minchunk parameter is the minimum length of a chunk. Default 64.
The idsmoothwindow parameter is the length of id smoothing window. Default 32.
The minsmoothid parameter - minimum factional identity over smoothed window of candidate parent. Default 0.95.
The maxp parameter - maximum number of candidate parents to consider. Default 2. In tests so far, increasing maxp gives only a very small improvement in sensivity but tends to increase the error rate quite a bit.
The skipgaps parameter controls how gapped columns affect counting of diffs. If skipgaps is set to T, columns containing gaps do not found as diffs. Default=T.
The skipgaps2 parameter controls how gapped columns affect counting of diffs. If skipgaps2 is set to T, if column is immediately adjacent to a column containing a gap, it is not counted as a diff. Default=T.
The minlen parameter is the minimum unaligned sequence length. Defaults 10. Applies to both query and reference sequences.
The maxlen parameter is the maximum unaligned sequence length. Defaults 10000. Applies to both query and reference sequences.
The ucl parameter - use local-X alignments. Default is global-X or false. On tests so far, global-X is always better; this option is retained because it just might work well on some future type of data.
The queryfract parameter - minimum fraction of the query sequence that must be covered by a local-X alignment. Default 0.5. Applies only when ucl is true.
- 1.22.0 Added group option
- 1.22.0 mothur now calls the uchime executable.
- 1.23.0 Paralellized for Windows.
- 1.23.0 Added column headings to .chimera file.
- 1.23.0 fixed bug with uchime executable location. - http://www.mothur.org/forum/viewtopic.php?f=4&t=1379
- 1.28.0 Added count parameter
- 1.28.0 Bug Fix - spaces in uchime path causing crash
- 1.29.0 Added dereplicate and strand parameters.
- 1.30.0 With count file and dereplicate=t will create a *.pick.count_table file.
- 1.30.0 - Bug Fix: dereplicate=t, remove.seqs(dups=f) was not removing all redundant chimeras.
- 1.32.1 Bug Fix: count table and dereplicate=t caused total=0 error message. - http://www.mothur.org/forum/viewtopic.php?f=4&t=2620
- 1.33.0 Improved work balance load between processors when processing by group.
- 1.36.0 Adds method tag to output files. - http://www.mothur.org/forum/viewtopic.php?f=5&t=3636&p=10748#p10748
- 1.38.0 Error generated when filenames are too long for uchime program.
- 1.39.0 Fixes Windows multiple processors with groups crash.
- 1.40.0 Rewrite of threaded code. Default processors=Autodetect number of available processors and use all available.