small RNA-seq pipelines

Question: small RNA-seq pipelines

5.7 years ago by

Saad Khan • 400

United States

Saad Khan • 400 wrote:

Hi,

I have to analyze a small RNA-seq data. This is my first time working with small RNA. I wanted to do differential expression and micro-rna comparison along with GO annotation. Something that mirtools does. But mirtools takes fasta files and I have paired end fastq files. I was wondering which publicly available pipelines (based on ease of usage and installation) can anybody recommend to do a comprehensive small RNA-seq analysis.

pipelinles small rna-seq reliable • 7.6k views

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modified 5.7 years ago by Mike Axtell • 240 • written 5.7 years ago by Saad Khan • 400

5.7 years ago by

Brian Bushnell ♦ 17k

Walnut Creek, USA

Brian Bushnell ♦ 17k wrote:

If you want something standalone, easy to use, and easy to install, I suggest the BBMap package - it's already compiled and will run on any platform that has Java. It does not do annotation, but it does do adapter-trimming with bbduk.sh (which is important for micro-RNA) and alignment with bbmap.sh, or alignment-free RNA quantification against a transcriptome with seal.sh. You could also use reformat.sh to convert your fastq files to fasta.

ADD COMMENT • link modified 12 months ago by RamRS ♦ 30k • written 5.7 years ago by Brian Bushnell ♦ 17k

How is the alignment of small rna reads going to be different from general RNA-seq reads? What bbmap options would be specific to small rna read mapping?

ADD REPLY • link written 5.7 years ago by Saad Khan • 400

Mainly BBMap is just easy to install and use, which is what you asked for. And it supports direct output of coverage and RPKM, which is often useful for RNA-seq.

ADD REPLY • link written 5.7 years ago by Brian Bushnell ♦ 17k

What I am really asking is what parameters should I change or specifically use for aligning small RNA reads when using bbmap?

ADD REPLY • link written 5.7 years ago by Saad Khan • 400

Oh, the defaults are usually fine. You could set "maxindel=20" if you want to speed it up a little since the defaults are geared toward long RNAs, and small RNAs don't contain long introns. Just be sure to adapter-trim the reads first, because small RNAs are expected to have adapter sequence, which interferes with mapping.

ADD REPLY • link written 5.7 years ago by Brian Bushnell ♦ 17k

So once I have mapped the reads I'll have to depend on other tools to do further analysis. Is there a way I can leverage other tools like srnabench once I have the mapped sam file or do any functionalities exist in BBtools to do so.

ADD REPLY • link written 5.7 years ago by Saad Khan • 400

BBTools can output coverage across the genome, or generate RPKMs if you are mapping to a transcriptome. And it can produce various metrics on coverage, like the fraction of bases across a transcript that were covered, but it does not do most RNA-seq-specific things like differential expression analysis. Many RNA analyses use mapped sam files, but I don't personally do small-RNA analysis so I'm not sure which downstream tools are best.

ADD REPLY • link written 5.7 years ago by Brian Bushnell ♦ 17k

5.7 years ago by

Mike Axtell • 240

Penn State University

Mike Axtell • 240 wrote:

You could try Shortstack ..https://github.com/MikeAxtell/ShortStack , http://www.ncbi.nlm.nih.gov/pubmed/23610128

ADD COMMENT • link written 5.7 years ago by Mike Axtell • 240

Does not support paired end reads!!

ADD REPLY • link written 5.7 years ago by Saad Khan • 400

5.7 years ago by

gaurav.singh • 10

United States

gaurav.singh • 10 wrote:

http://mirnablog.com/web-based-tools-for-microrna-analysis-from-deep-sequencing-data/

ADD COMMENT • link written 5.7 years ago by gaurav.singh • 10

I am looking for standalone applications not webservers

ADD REPLY • link written 5.7 years ago by Saad Khan • 400

5.7 years ago by

David Langenberger ♦ 9.5k

Deutschland

David Langenberger ♦ 9.5k wrote:

You could try sRNAbench.

But paired-end sequencing ist also not very common when doing small RNA-Seq. How long are your reads?

ADD COMMENT • link written 5.7 years ago by David Langenberger ♦ 9.5k

BTW: sRNAbench is also available as standalone: http://arn.ugr.es/sRNAbench/

ADD REPLY • link written 5.7 years ago by David Langenberger ♦ 9.5k

I believe sRNAbench does not handles paired end reads atleast according to their documentation

ADD REPLY • link written 5.7 years ago by Saad Khan • 400

But I actually do not see any sense in using paired-end sequencing for molecules of ~18-30 nt in length. Why should someone do that? To waste money?

ADD REPLY • link modified 5.7 years ago • written 5.7 years ago by David Langenberger ♦ 9.5k

I would have to ask my collaborator that :( . BTW the length of the sequences in the fastq files is 50nt I am sure it does includes some adapter sequences that I would have to remove.

ADD REPLY • link written 5.7 years ago by Saad Khan • 400

That is actually a very important tipp: Know your data, before you do any analysis!

ADD REPLY • link written 5.7 years ago by David Langenberger ♦ 9.5k

Well it was just a one-off analysis I had to do for my collaborator to add on some results to a paper. So I don't know much about why they used paired end (Which I have read on Illumina website don't help much in case of small RNA or miRNA).

ADD REPLY • link written 5.7 years ago by Saad Khan • 400

BTW do you happen to know of any sirna pipeline that works with paired end data?

ADD REPLY • link written 5.7 years ago by Saad Khan • 400

No, since paired-end makes no sense in my eyes.

But if your paired-end data is strand-specific (what I really hope you checked), then you can just create a single-end file out of it. mate1 is in the correct direction and mate2 is directed in reverse direction. So, you can just take mate1.fastq and add the reverse complement of mate2.fastq. Assure to do that after clipping the adapters!

I'm not sure, but probably there are some tools available which do the re-direction of mate2 for you. But I never needed it, so I don't know. But you can use the resulting file single-end file with all small RNA-Seq tools available.

ADD REPLY • link written 5.7 years ago by David Langenberger ♦ 9.5k

What if they are not strand specific. What can I do with such data then??

ADD REPLY • link written 5.7 years ago by Saad Khan • 400

Not that much, I would say

ADD REPLY • link written 5.7 years ago by David Langenberger ♦ 9.5k

What should I do then. I was wondering just aligning after removing the reads and using edger etc for differential expression.

ADD REPLY • link written 5.7 years ago by Saad Khan • 400

Just adapter-trim, then throw away read 2 and use read 1.

ADD REPLY • link written 5.7 years ago by Brian Bushnell ♦ 17k

How about using the 2nd read pair as a replicate??

ADD REPLY • link written 5.7 years ago by Saad Khan • 400

It's not a replicate, it's the same; so no.

ADD REPLY • link modified 5.7 years ago • written 5.7 years ago by Brian Bushnell ♦ 17k

Ask the lab, which preparation protocol they used!

ADD REPLY • link written 5.7 years ago by David Langenberger ♦ 9.5k

Actually I just found out it is strand specific data. Do you know who can direct me to a package/tool that does the redirection of mate2?

ADD REPLY • link written 5.7 years ago by Saad Khan • 400

Hi David,

I have finally done the mapping etc for small rna. Since sRNAbench does not handle data which has no replicates (like in my case) what would be a good procedure to use in order to give the required results. Since both edgeR and DEseq handle replicate free data for RNA-seq do you think these techniques can be extended to small rna as well.

ADD REPLY • link written 5.6 years ago by Saad Khan • 400

5.7 years ago by

Vivek Todur • 50

Bangalore

Vivek Todur • 50 wrote:

The UEA small RNA Workbench is another option, Its cross platform GUI (Graphical User Interface) application, You can run either on Linux or Windows seamlessly. It has different modules for different analysis such as Mirprof for Known miRNA Analysis and Mircat for Novel miRNA Analysis.

ADD COMMENT • link written 5.7 years ago by Vivek Todur • 50

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