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Old 12-15-2015, 10:35 AM   #1
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Location: San Diego

Join Date: Nov 2014
Posts: 6
Default Galaxy smallRNA Analysis help

I've been playing with Galaxy lately to do my data analysis for smallRNA (Illumina).
I think I'm getting what I want but I'm stuck at a certain point.
Here are the tools/workflow I have used:
1. FastQC (to check quality)
2. Clip (to trim off 3' adapter sequence)
3. Sickle (to discard any bad quality sequences)
4. MiRDeep2 mapper to collapse the reads from Sickle (seems to just make the sickle file compatible with MiRDeep2 Quantifier)
5. MiRDeep 2 Quantifier to map the collapsed reads to the reference fasta file of mature miRNA.
*Result is a list of miRNA in a tabular format
-Next I want to convert this file to a file compatible with edgeR for differential expression analysis. Any thoughts on how to do this? Any known tools?
-Also, there is one other tool: MiRDeep2 (identification of novel and known miRNAs). I can't for the life of me figure out if this tool is more useful or if I should be using this one instead of Quantifier however the output when I use this tool is not giving me the miRNA list I would expect. Any help would be appreciated. Thx!!
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Old 01-02-2016, 09:19 AM   #2
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does mirdeep provide you with a list of counts for each miRNA? if not, i guess it gives at least the genomic coordinates of your miRNAs, so you can make it as bedfile or gff file and use it to extract read counts with betools or other tools on your bam file. The bed file you need it anyway for edgeR
Then you just extract the coloumn with the counts in a new txt file and give to edgeR lie this
counts <- read.delim("counts.txt")
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Old 02-25-2016, 04:55 PM   #3
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Location: San Diego

Join Date: Nov 2014
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Thanks for your reply. I'm just getting back to trying to figure this out.
I get a list of counts (see attached image) for each miRNA when mapping to the mature fasta file downloaded from miRbase. These aren't necessarily assigned to regions in the genome however, just miRNA names. This is a tabular file. It seems like edgeR wants me to combine all my lists(samples and replicates) together into a matrix type tabular file. Do you know of a tool to do this easily? Will edgeR accept these counts if not assigned to a position in the genome and just a name? Any help is appreciated. Thx!
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data analysis, edger, galaxy, mirdeep2, smallrna

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