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Old 02-02-2016, 01:46 AM   #1
danova
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Location: France

Join Date: Sep 2010
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Default bbduk for mirna mapping

Hi,
I have run a miseq run for microrna detection, filtered the data (removed adaptors and ncRNAs (others than microRNAs) and im now going to identify reads mapping to microRNAs in Human.

As for the strategy not sure if i have to map to the human genome or i can directly map to the mature.fa database from mirbase. Any suggestion ? The reason i have choosen not to map to the human genome is that not really interested in denovo microRNAS but on the contrary im not sure if i have first to remove reads that can map to other parts of the genome.

So far i have decided to align to mature mirbase database.

Im using the following command:

bbduk.sh ref=mature.fa.gz in=1.fa.gz stats=statsmature.txt out=mirna.fa hdist=0


1/ What is the best approach to identify full match microRNAs (no mismatch) with bbduk.
2/ How to identify isomirs ? Do i need to cluster reads first with vsearch with a 99% identity and then map to mirbase with 100% full match.

Thanks for your comments
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Old 02-03-2016, 07:01 PM   #2
Brian Bushnell
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Default

By default BBDuk will allow one mismatch of the middle base in a kmer (even with "hdist=0"). You need to add the flag "mm=f" to require exact matches. mm stands for "maskmiddle".

If you want to require every kmer in a sequence to also be present in the reference, you can also add "mkf=1" (which stands for min kmer fraction). That will make the process completely intolerant of any errors or remaining adapter sequence, though.

Be sure to specify a kmer length. If you are looking for RNAs as short as 17bp, then set "k=17"; the default is much longer.

So, the full command might look like:

Code:
bbduk.sh ref=mature.fa.gz in=1.fa.gz stats=statsmature.txt out=mirna.fa hdist=0 mm=f mkf=1 k=17
...but you should determine for yourself what value you want for K.
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