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I am a newbie in this world (just finished my master's on bioinformatics. As a practice, a researcher gave me 4 fastq files from a small RNAseq experiment, to see if I am able to reproduce their results but with an updated pipeline and tools (they used bowtie in 2014). I used fastqc for the quality check and I removed the adapters properly.

My first intention was to use Salmon for pseudo-align them, but after do some reading it seems not to be a good idea for such small RNAs.

Can anyone provide me with some light on this? Which alignment tool use and which parameters, and etc. Ideally a proper protocol/pipeline would be amazing.

Thank you in advance...

PD: I was thinking to use the HISAT2 with the full genome (or the pre-builded index from HISAT2 website).

Kind regards, and thank you in advance!

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I have heard that the Small RNA Workbench from UEA can be an easy solution for a full small RNA pipeline. It is java based, has a GUI, and is supposed to be easy to install and run. I am not an expert in small RNA analysis, but I think this pipeline will certainly given you an idea about the steps needed for a small RNA analysis, it has been put together by experts.

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  • $\begingroup$ Thanks a lot! I think that can be a good starting point. Also, probably I can get some help from the authors.... fingers crossed! $\endgroup$
    – FJ_Bioinf
    Nov 20, 2019 at 11:20
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piPipes is biased for piRNAs but should be a good starting point:

https://github.com/bowhan/piPipes/wiki/smallRNA-seq

Well documented and with many features.

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As far as I'm concerned, bowtie 1 is still the method of choice since it rather mature and fast.

And yes, pseudoaligner such as salmon are probably not the best choice For once, they need a reference "transcriptome", which is usually available for RNA-Seq but not so much for small-RNAs. Also the way they handle multiple mapping locations is probably not ideal for small-RNAs that may have excessive number mapping location.

I've also written a simple snakemake-pipeline which simply performs some standard pre-processing and mapping against a reference genome along with QC and coverage tracks. It's still work in progress, but I'd be curious about any feedback.

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Check nf-core for all the pipelines they have, including small-RNA seq. To run them, you have to use Nextflow.

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