Download unbias reference databases
The unbias command attempts to correct for abundance bias
in amplicon sequencing by adjusting the counts in an OTU table
Unfortunately, the UNBIAS algorithm
does not work very well as a practical tool, achieving only a small improvement in OTU frequency estimates. Even after running UNBIAS, the correlation between cell frequency and OTU frequency is very low
, and I believe that the problem of abundance bias is not solvable at the present time. So, why provide a command that doesn't work? Mostly, to draw attention to the problem and emphasize the limitations of 16S amplicon sequencing as a quantitative method. Plus, doing something is better than nothing -- there is some improvement, even if it's not a full solution.
The unbias command requires three input files: an OTU table in QIIME classic format
and two tabbed text files with predictions for the SSU operon copy number and number of primer differences for each OTU. The tabbed text files are specified by the -copynrin and -diffsin options. The tabbed text files must have at least two fields. The first field is the OTU identifier, the second field is an integer giving the predicted copy number or number of primer differences for the OTU. These files can be generated using the sinaps command
using reference databases which can be downloaded here
. Predictions are needed for every OTU regardless of confidence, so the -noboot option of sinaps can be used for faster execution.
The -tabbedout option specifies a tabbed text file with one line for each OTU; fields are: OTU identifier, copy number, primer diffs, copy number correction factor, primer diffs correction factor, final correction factor.
Output is written in QIIME classic format to the file specified by the -output option.
usearch -sinaps otus.fa -db primerdiffs.udb -strand plus -tabbedout primerdiffs.txt \
-noboot -attr v4diffs
usearch -sinaps otus.fa -db copynr.udb -strand plus -tabbedout copynr.txt \
-noboot -attr copynr
usearch -unbias otutab.txt -diffsin primerdiffs.txt -copynrin copynr.txt \
-output otutab_unbias.txt -tabbedout unbias.txt