Question: Aligning miRNA reads to miRbase
1
gravatar for glady
11 months ago by
glady250
glady250 wrote:

Hello.,

Recently I have got some miRNA reads from the small RNA seq data. I have filtered them for quality and removed the adapters with FASTX. I want the align the reads to the miRbase by using SHRiMP.

  1. Which SHRiMP parameters should I consider while aligning the reads to miRBase?
  2. Which miRbase file mature/precursor file should I use as reference?
  3. In there a need to create an index for the reference miRBase file before aligning the miRNA reads to it?

Thank you.

rna-seq • 804 views
ADD COMMENTlink modified 11 months ago by karthic100 • written 11 months ago by glady250

I recommend you to use both, mature and hairpin because you may found new miRNA variants or "isoforms". And also, recommend you to use bowtie (no bowtie2 or blast) to align them, it is easy to handle and filter multihits.

ADD REPLYlink modified 11 months ago • written 11 months ago by Buffo1.7k

Thank you for your reply. But how can I use both the mature & hairpin files as a reference. Since, for preparing the index, I can only pass on one file.

For example, I prepared the reference file in SHRiMP by using this cmd: media/SHRiMP_2_2_3/utils/project-db.py --seed 00111111001111111100,\ 00111111110011111100,00111111111100111100,00111111111111001100,\ 00111111111111110000 --h-flag --shrimp-mode ls /project/SHRiMP_ref/miRBase_22/mature_ref_hsa.fa

ADD REPLYlink written 11 months ago by glady250

$cat hairpin.fa mature.fa > both.fa

ADD REPLYlink modified 11 months ago • written 11 months ago by Buffo1.7k

Thank you for the suggestions. How can we get the read count from the final SAM output file?

ADD REPLYlink written 11 months ago by glady250
0
gravatar for karthic
11 months ago by
karthic100
karthic100 wrote:

I guess performing a blast with task as short blast and setting as ungapped will do. It depends upon what you are looking for, it is common to go for the mature miRNAs from the mirbase. You can even download mirnas for the selected species of your interest. If you are going to do blast, index file is not required to create, but if you are going to align using bowtie or bwa, then you need to create an index file.

Thanks,

KK

ADD COMMENTlink written 11 months ago by karthic100

Which file should we consider to create an index? miRbase mature or hairpin file? In the above comment, I used an miRbase mature file of homo sapiens.

ADD REPLYlink written 11 months ago by glady250

I have used mature miRNAs.

ADD REPLYlink written 11 months ago by karthic100

What are the parameters you used for mapping? Because I'm getting very low mapping percentage - 2.77%. the command which I used is;

/SHRiMP_2_2_3/bin/gmapper-ls -L /SHRiMP_ref/mature_ref_hsa-ls --ignore-qvs -Q /sample.fastq --mode mirna >sample.sam 2>sample.log

ADD REPLYlink written 11 months ago by glady250
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