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Alignment parameters
The parameters used for the alignment/mapping procedure can be important in the final counts produced, irrespective of the aligner used.
This can be a complex topic in itself and general users will be confused by the many options. The command line tool for smallrnaseq takes the simple approach of providing a default alignment parameter for general mapping to libraries, another for mapping miRNAs and one for reference genomes. All can be changed in the config file if needed. You can also set custom parameters per library in the aligner section.
For general mapping -v 1 --best is used. -v 1 reports read mappings with up to one mismatch, options --best orders the mappings from best to worse alignments.
In miRDeep2 when mapping to the mature miRNAs (miRBase sequences) for mature quantification the following parameters are used:
-v 1 -a --best --strata --norc
Here -a means report all valid alignments, options --best --strata orders the mappings from best to worse alignments according to the strata definition of bowtie. --norc means do not map reads to the reverse complement of the sequences.
For reference genome mapping miRDeep2 uses these parameters:
-n 0 -e 80 -l 18 -a -m 5 --best --strata
-m 2 -M 1 is the default for general alignment to libraries. If you use subread you can check the parameters by typing subread-align --help at the command line, or refer to the website.
In the aligner section set your parameters. In the example below bos_taurus is the name of the reference genome. We have also used custom settings for mirna and another library of tRNAs.
[aligner]
mirna_params = -n 1 -l 20
bos_taurus = -v 1 -k 50
bosTau8-tRNAs = -v 0 --best
If using the package in your python code, aligner parameters are set via the aligners module. This is done before calling mapping routines such as map_rnas.
for example:
from smallrnaseq import aligners
aligners.BOWTIE_PARAMS = '-v 0 --best'
aligners.SUBREAD_PARAMS = '-m 0 -M 1'
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Shi, J., Dong, M., Li, L., Liu, L., Luz-Madrigal, A., Tsonis, P. A., … Liang, C. (2015). mirPRo–a novel standalone program for differential expression and variation analysis of miRNAs. Scientific Reports, 5, 14617. http://doi.org/10.1038/srep14617
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Friedländer, M. R., Mackowiak, S. D., Li, N., Chen, W., & Rajewsky, N. (2012). miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic Acids Research, 40(1), 37–52. http://doi.org/10.1093/nar/gkr688