Counts per million to differential expression

Question: Counts per million to differential expression

17 months ago by

Hi All,

Strange question so a bit of background:

I have used a targeted RNAseq panel so it is essentially a huge PCR reaction with specific primers to around 600 genes. Which are subsequently subjected to next gene sequencing.

The output from the manufacturers website is essentially a table of gene identifiers (from refseq) with the number of counts associated to each gene.

So to normalise FPKM/RPKM is not valid as the fragments amplified with the specific primers do not relate to the size of the gene. I have therefor simply normalised by count per million (cpm) so am left with a list of 600 genes with the CPM from each sample.

My question is how to proceed from here using Galaxy to get differential expression analysis. I have 3 donors and 3 conditions. I would ideally like to get volcano plots with annotated genes. Perhaps the plots require a third party program which someone could suggest?

Thank you,

Reuben

rna-seq cpm • 730 views

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modified 17 months ago by Jennifer Hillman Jackson ♦ 25k • written 17 months ago by reubenmcgregor88 • 0

17 months ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

Hello Reuben,

In Galaxy, there are several tools available for count-based differential expression analysis (DEXSeq, DeSeq2, others in the Main Tool Shed - but probably not Cuffdiff). However, those wrapped for Galaxy are expecting specific inputs (usually from other Galaxy-wrapped tools, BAM mapped datasets, and/or the original fastq reads). The tool group NGS: RNA Analysis at Galaxy Main (http://usegalaxy.org) includes the tool options available publically on that server. You might be able to reformat your data so that the target tool you choose accepts the input you currently have to produce meaningful results.

Other public Galaxy servers have even more domain-specific tools (including those for RNA-seq). So, perhaps review those along with the currently wrapped tools in the Tool Shed to get the scope of what is available and use the other servers and/or install the tools from the Tool Shed into a local or cloud Galaxy for your project. These options are explained in more detail here: https://galaxyproject.org/choices/

Nearly any tool can be wrapped for Galaxy if is not already. If that interests you, please see: https://galaxyproject.org/develop/

This is a tough question to address directly with the given input format and experimental design. Although, others may still reply and add in their ideas. You also may want to ask this question at https://www.biostars.org/ (or another public bioinformatics forum) if you are comfortable working on the command line.

Hopefully, this gives some options! Jen, Galaxy team

ADD COMMENT • link written 17 months ago by Jennifer Hillman Jackson ♦ 25k

Thanks Jen, I will explore the options.

Not comfortable on command line, yet. Perhaps something I should look to do.

ADD REPLY • link written 17 months ago by reubenmcgregor88 • 0

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