Information regarding Differentiation Gene Expression analysis

Question

[khaiber.shinvari]

Hello Greetings everyone I am new to Bioinformatics, I download GEO data set and now want to analysis it through R, kindly guide me step by step I am familiar with R just want to know that step by step what should I do to analyze this data and get the results.

Thanks

Answer 1

[Charles Warden]

In general, I think you should expect multiple rounds of analysis (and discussion, if the roles of data generation and analysis are separate, but require close communication).

In terms of getting an idea of the variation in results, you can take a look at "Target_Recovery_Status.xlsx" from this page.

For each program, I think there is usually some pretty good documentation / tutorials:

DESeq2: http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html

edgeR: https://bioconductor.org/packages/release/bioc/html/edgeR.html (I think there is a similar tutorial as DESeq2, but I am not finding it at the moment)

limma-voom: https://www.bioconductor.org/packages/devel/workflows/vignettes/RNAseq123/inst/doc/limmaWorkflow.html

You also need some upstream steps of alignment and pre-processing. I show some variation of that in the Excel file, but there are also other options that you may want to consider (if something seems off about your preliminary results). There is also code, but that is probably not the best way to learn from scratch.

Here are some links for the other steps that I am talking about:

Genome Alignment:

STAR: https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf

HISAT2: https://ccb.jhu.edu/software/hisat2/index.shtml

TopHat2: https://ccb.jhu.edu/software/tophat/index.shtml

Genome Quantification (post-alignment):

htseq-count: https://htseq.readthedocs.io/en/release_0.11.1/count.html

featureCounts: http://bioinf.wehi.edu.au/featureCounts/

Transcriptome Quantification (without alignment):

Salmon: https://combine-lab.github.io/salmon/

kallisto: https://pachterlab.github.io/kallisto/about

You can also do a transcriptome alignment (using Bowtie1 or Bowtie2, for example), with quantification using eXpress or RSEM.

[khaiber.shinvari]

Thank you so much if you could give me your email, I would contact you in case of a problem with the analyzing. Thanks

[Charles Warden]

Please keep questions public in the Biostars forum.

I will certainly help when I can, but that allows you to possibly get help from others (for example, I am on Biostars a lot more some weeks than others).

I hope everything is otherwise going well with you!