Ballgown Package of R for the Differential Expressed Genes (DEGs)

Question

[HaroonPakistan]

Hi, I am running a Ballgown package of R to find DEGs in my samples. I have three samples (b73,f1, and mo17). I have run all the initial commands successfully. I have given the very right paths and I am sure about that. But for a specific command like in below, I am facing difficulty. I have a seprate file by the name of CSV file which is usually edited by the author byself because it contains the information of samples and their characteristics.

Commmand;

results_transcripts <- stattest(bg_filt, feature="transcript", covariate="sex", adjustvars = c("population"), getFC=TRUE, meas="FPKM")

Output:

*Error in stattest(bg_filt, feature = "transcript", covariate = "sex", :

There must be at least two replicates per group. Make sure covariate is categorical; if continuous, consider the timecourse option, or specify your own models with mod and mod0.*

Request to you all; Please help if you can! Thanks!

[cpad0112]

Please refer to https://support.bioconductor.org/p/105354/. If you do not have replicates per group (which is not suggested in any comparative experiment), quantify the expression units by featurecounts, then use edgeR for differential analysis. But with one replicate per group, it is difficult to draw any meaningful observations.

[HaroonPakistan]

Thanks, I follow it.

[HaroonPakistan]

Ok. So for that, which file I should use? I mean merged transcript file or ctab files?

[cpad0112]

alignment (bam, outside R) -> read count (featurecounts - outside R) -> edgeR (within R)

Answer 1

[ATpoint]

Ballgown was intended for transcript level differential analysis, and you currently seem to have transcript-level data, do you? Yet you mention differential gene analysis.

Not discussing that 1 vs 1 comparisons are meaningless from a stats standpoint but you should be using a gene level workflow, e.g. aggregating the transcript level abundances you have to gene level with tximport from Bioconductor and then do gene level analysis with any of the tools mentioned in the tximport vignette.