Finding Isomirs In Alingnment Files. Next Steps After Mapping To The Pre-Mirnanome ?

Question

[Agatha]

Hi,

I am currently trying to find isoMiRS in Illumina RNA-seq libraries and I am currently stuck at the alignment step. I have aligned the trimmed qualified reads to the human pre-miRNAs from miRBase using Bowtie with 3 mismatches. Now I am trying to analyze the BAM files.

How can I make sure that my reads are aligned with a 3 nt extension at the end?

Is there any specific pipeline for this task?

Also, which tools can I use for getting an output similar to the one from the image attached?

Image description :

"Example of small RNA reads aligning to a known miRNA precursor (miR-1-1). The mature miR-1-1 sequence, as annotated in miRBase, is underlined. The minimum free energy secondary structure is denoted by dot-bracket notation, represent complementary bases and represent non-complementary bases. Numbers in the right column represent the abundance of digital read counts corresponding to each sequence."

Thank you!

http://www.freeimagehosting.net/bugfu

[Nicolas Rosewick]

I think miRdeep2 output such picture

[Agatha]

@NicoBxl- thank you I will have a look

Answer 1

[Doctoroots]

Ive just preformed a very similar analysis, basically i wanted to look at the isomiR population in my sequenced data so i preformed the following steps:

1. create a reference file of the mature miR sequnces with a flanking region on both 5 and 3 prime ends (i chose flank=3 bases)

2. collapse the sequence reads file using fastx-collapser

3. align the collapsed fasta file to the miRs-with-flank reference created in step 1

4. parse the resulting sam file (including only uniquely aligned reads) so that each collapsed sequence represents an isomiR for the miR it aligned to.

this way you can get the abundance of each isomiR (from the read name) and mark variations (edit distance) in both the complete and the seed sequence.

i hope this helps, good luck

[Agatha]

@doctoroots - sounds good, thanks a lot. I will try :-)

[Dg]

I known mirExplorer can finished this work, you can download this tool from http://biocenter.sysu.edu.cn/mir, if any question can sent email to [e-mail]

Answer 2

[Dg]

I known mirExplorer can finished this work, you can download this tool from http://biocenter.sysu.edu.cn/mir, if any question can sent email to [e-mail]