MiRNA analysis just using BLAST against

I have sequencing data (Illumina). Library prep was focused on short ncRNAs. I would like to identify human miRNAs. Do you think it is feasible to simplyBLASTagainst a current version of mirBase and filter the output for mature, human miRNAs without usingbowtie/tophatagainsthg19first?


You don't need to useTopHatbut it is better to usebowtieinstead ofBLAST. First of all you need to get rid of the adapter sequences (along with other processing steps prior to alignment).

Now there are two aspects here:

  • miRNA quantification
  • miRNA discovery

First one is relatively straightforward whereas the second one requires you to perform additional tests such as prediction of stem loops etc. There are published software that can handle both the aspects (mirDeep,miRScanetc). See this review for details on different miRNA gene finders. I have used miRdeep and it usesbowtie-1to align.

BLASTwould work but you have to set parameters which are suitable for small sequences (increase E-value cutoff, reduce word size etc). Moreover,BLASTdoes not have a cutoff option for the number of mismatches and the length of alignment (it only has a post alignment filter for percentage identity).

I also personally preferbowtiebecause it has something called asn-alignment mode. In this mode the entire read is divided intoseedandnon-seedregions. You can specify the length of the seed region and the mismatch cutoff. Since for miRNAs the seed region (bases 2-8) is critical for its function, I generally set theseedmismatch cutoff to zero while allowing one or two mismatches in thenon-seedregion (note that inbowtietheseedalways starts at 1).

In general, I would advise that you should go formiRdeepinstead ofBLAST. However,miRdeepdoesn't straight away perform an alignment against the mature sequences. It maps the location of mature sequences in the pre-miRNA sequences (stem loops) and then aligns the reads to the pre-miRNA sequences. If the location of reads has a significant overlap with the mature region (you can adjust the window) then the read is considered as a valid miRNA.

Watch the video: Bioinformatics Practicals on MicroRNA Target and Pathway Prediction Part 1 (December 2021).