OK, let's imagine you have lists (fasta files) of clean unique 18-30 nt reads with their frequences from several libraries.
How do you sort our the functionally important sRNA (particularly interested in siRNAs and miRNAs) from the junk (degraded mRNA fragments, contaminations, ribosomal, nuclear and tRNAs)?
How do you then classify the sRNAs into families?
Finally, how do you normalize the reads and analyse their difefrential expression?
Try srna workbench and tool kit
http://srna-workbench.cmp.uea.ac.uk/
Hello Kostya,
Since you are working with crop, and the genome is sequenced, I'd recommend you to use BWA-align to map your reads vs the genome. This aligner works very good with small sequences and it will give you the coordinates of your sequences in the genome. Later, and here's the complicated part, you should try and find the annotation for the genome, that is a list of all the genes for that specie with their coordinates. You can then compare them and, hopefully you will have a list of everything you have sequenced. Then with a small script grepping the specific words (miRNA,lncRNA,rRNA, tRNA, etc) you are interested on, you will get all the info you want.
About differential expression, the best thing you can do is go to the bioconductor website and look for DeSeq2, or EdgeR manual pages. Cuffdiff/Cufflinks is not recommended for small sequences.
PlantDario tool is quite powerful for this kind of analysis:
http://journal.frontiersin.org/article/10.3389/fpls.2014.00708/abstract
I agree with Jennifer. It depends on whether your plant genome is unveiled or not, and detailed information about annotations for the genome. Furthermore, you can align with the relatives of the species you are studying. The small RNA also share some similarity in some distance.
Check out http://www.nature.com/natureevents/science/events/47533-microRNA_Analysis_Using_Next_Generation_Sequencing
Thnak you for the course information David. Is this course focuses on animal miRNAs only?
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