Opinion on miRNA pipeline
0
0
Entering edit mode
12 weeks ago
ju_ra • 0

Dear colleagues,

I am currently in the process of evaluating miRNA Seq data and would like to present my pipeline for your review. Given the absence of a dedicated bioinformatician in my department, particularly for this specific use case, I am eager to gather feedback on the robustness of this workflow for potential peer review of the project.

Here is an overview of the key steps in my analysis:

1, Utilizing Cutadapt to eliminate any adaptors and the common sequence, which is ligated precisely at the 3' end of the read.

  1. Due to the utilization of a specific miRNA extraction and library kit, I opted to align the data to a miRNA genome obtained from RNA central, using the miRBase data for humans. This choice aims to minimize issues related to multi-mapping leveraging the specificity of the kits used.

  2. Experimenting with various aligners, I found that STAR produced the most satisfactory and acceptable results for me. Consequently, I decided to adhere to STAR, and because of my familiarity and experience with the tool. I fine-tuned the settings to optimize the mapping, achieving a 35-50% unique mapped reads rate, with most of the remaining reads classified as multi-mappers.

  3. For the subsequent analysis of Differentially Expressed Genes (DEG) using a DESeq2 pipeline, my intention is to focus solely on the uniquely mapped reads to mitigate any potential bias.
miRNA pipeline • 418 views
ADD COMMENT
0
Entering edit mode

Feel free to take bits from this pipeline - mirna_alignment.sh. It mimicks all of the steps in nf-core/smrnaseq and the advice given by Sean Davis in this biostars post

ADD REPLY
0
Entering edit mode

Thank you very much! I see you decided to go for Bowtie as aligner. Do you see any issue in using STAR (as a splice aware reader technically wouldn't be necessary)?

ADD REPLY
0
Entering edit mode

I have never used STAR for smRNA-Seq (no particular reason) so I can't comment, sorry.

I looked around and found that the ENCODE project uses STAR to profile miRNAs. Check out the pipeline page (https://www.encodeproject.org/pipelines/ENCPL337CSA/) it will bring you to a DNAnexus repository where you can find the steps they use. I would consider this a 'gold standard' to compare against.

ADD REPLY

Login before adding your answer.

Traffic: 1704 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6