RNAseq Viz

Question

[icodenow]

Hi All,

I'm really excited to have my first RNAseq expt. results back. However, I'm trying to find the best pipeline to work with in R that best matches my coding experience (little to none). I came across Searchlight2 which seems perfect as it is automated and will produce many figures that will allow me to visualize and interpret my data but, I'm already stuck on what to do in terms of installing Python, etc. Is there any way to get one-on-one help untilI I can get on my feet in R? Or, is there another current pipeline that folks are using that I can start learning from with a lower bar. I already have the .tsv file with the following headers in this order:

Feature_ID
entrezgene
external_gene_name
gene_biotype
external_gene_source
transcript_count
description logFC
CI.L
CI.R
AveExpr t
P.Value adj.P.Val
B
linearFC

I have 2 groups each with 6 replicates

Thanks, icodenow

Answer 1

[Kevin Blighe]

Based on those column names, it seems that somebody [or some service] has already processed the data for you. Your job would now be to interpret the results. You may seek advice from the person [or service] who did this initial processing for you.

For what it's worth: I can already infer that the program used by this person [or service] was an R package called limma. They also seemed to have used biomaRt for gene annotation.

[icodenow]

Hi and thanks so much for your reply!

Yes, our core did all of the processing but the figures are very general and hard to see/work with to prep for presentations/manuscript. Per your suggestions though, I will seek their advice too.

I was playing around last night with this and figured out I could just use DESeq and ggplot to get me started remaking figures and such. I even played around with EnhancedVolcano which is super cool. I guess my question should have been, how do I take my .tsv file (first convert to .csv so that DESeq can use it) that the core gave me and remake figures that suit my needs, similar to EnhancedVolcano and Searchlight2. I plan to play around more with the Searchlight2 code to see if I can get past installing the packages I need for Python using R. I installed Python 3.10 .. now need work on installing SciPy and NumPy.

I hope to be able to document my steps and annotate for other users at a similar starting point as I.

Thanks!!

[Kevin Blighe]

Yes, I co-developed EnhancedVolcano many years ago with Sharmila. For DESeq2, you will require raw counts - the core facility should give this to you upon request. If they want to charge extra for it, I can tell you right now that it should only take maximum 5 minutes to generate and send to you via e-mail.

Then, to get familiar with DESeq2, you should start with this example: http://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#count-matrix-input

I cannot comment on Python packages.

[icodenow]

Ahh I see this is really helpful, thanks tons.. I do have an all.gene_counts.tsv file and an all.transcript_counts.tsv. So. I'll get working with those after going through the DESeq2 example.