Skip to main content
4 votes

Identify differentially covered genes only between two samples

Not really an answer but an extended comment... and most likely something you don't like to hear I guess by technical replicates, it means taking the same biological sample and making 2 methylation ...
StupidWolf's user avatar
  • 1,688
3 votes
Accepted

Help with Limma-model

It looks good to me. One think I could suggest is to see the distribution of your data/model (with limma/voom). After you get the list of your genes with: ...
flavinsky's user avatar
  • 224
3 votes

TPM or rlog(CPM) for comparing expression?

For comparing the counts of different samples from DESeq2, Michael Love recommends using the variance-stabilized transform. It'd be great if you could provide some specific code examples in your ...
gringer's user avatar
  • 14.3k
3 votes

Smallest group size for differential expression in limma (bulk RNA-Seq)

While I share Llopis' concern about estimating variance from 2 samples, the statement you quoted is about avoiding false positives from genes that are only expressed in a few samples. It's fairly ...
heathobrien's user avatar
  • 1,816
3 votes
Accepted

Smallest group size for differential expression in limma (bulk RNA-Seq)

The problem with most of the methods is that use the gene's variance for each group, which can't be calculated (reliable) when the sample is <= 2. Also, statistically, it would have extremely low ...
llrs's user avatar
  • 4,713
3 votes

How to get log2 fold change of RNA-Seq data for time series experiment?

If you just want the change with time then use time as a continuous variable. The log2FC will then be the change per hour (you can use the default Wald test to ...
Devon Ryan's user avatar
  • 19.6k
2 votes
Accepted

Significant gene set testing - limma

The mroast function has an argument to specify which contrast do you want to test, quoting from the help page: contrast contrast for which the test is required....
llrs's user avatar
  • 4,713
2 votes

Can I use a regular liner regression model when I'm working with DNA methylation data?

You can use either, but lmFit has the benefit of returning an object that can be used with eBayes() so you can pool information ...
Devon Ryan's user avatar
  • 19.6k
2 votes

TPM or rlog(CPM) for comparing expression?

To add to what @gringer, when you do use TPM, the normalization done is for both library size and gene length. When you use rlog, the normalization is done via median normalization (https://www.ncbi....
StupidWolf's user avatar
  • 1,688
2 votes
Accepted

Any way to filter out highly correlated genes with limma linear model?

The problem is that you are transposing the vector of GA values with t(ano$GA). Why would you do that? It produces a row matrix that is inappropriate for input to <...
Gordon Smyth's user avatar
2 votes
Accepted

Any way to quantify the variation of genes that expressed in Affymetrix expression data?

You can use the following code to calculate the coefficient of variation: ...
llrs's user avatar
  • 4,713
2 votes
Accepted

R limma alternatives in Python

August 2021 To date, it seems that the response to this question is "No". However, there is a GitHub repository called edgePy, aiming to "become an ...
Gian Arauz's user avatar
2 votes

Identify differentially covered genes only between two samples

You may run edgeR for methylation analysis without replicates (https://f1000research.com/articles/6-2055). But I also recommend you to have a look at the R DSS package. It has a smoothing step which ...
thomas duge de bernonville's user avatar
2 votes
Accepted

How to use the contrast function in ebayes for making different comparisons

A design matrix is a matrix of values of the grouping variable. ANOVA needs such a matrix to know which samples belong to which group. Since limma performs an ANOVA ...
Nitesh Shriwash's user avatar
2 votes

To find genes that don't change in RNA-seq, Deseq2 has altHypothesis="lessAbs". Is there a way to make limma do the same thing for proteomics?

There is no direct support for this by best knowledge. Similar questions have been asked previously in the Bioconductor support forum, for example: https://support.bioconductor.org/p/64300/#64793 They ...
ATpoint's user avatar
  • 1,326
2 votes
Accepted

How do I fix this limma line?

My guess is you need to index matchedgeneTPM before you run lmFit(), not after: ...
user438383's user avatar
  • 1,689
2 votes
Accepted

How do I correctly format my limma/eBayes code?

You need to look at how to create a model matrix : https://www.rdocumentation.org/packages/stats/versions/3.6.2/topics/model.matrix In your code, you did not specify the data from which the Sex ...
Basti's user avatar
  • 266
1 vote

Problems in conducting batch effect correction

The ComBat-seq method requires raw counts which can be modelled as a negative binomial. The other methods are typically used with normalized counts transformed to log2 scale. Obviously for any method ...
Boggs Diamond's user avatar
1 vote
Accepted

Assumptions of batch effect removal

You're basically subtracting a constant per-gene per-level. The relevant portion of the code is: ...
Devon Ryan's user avatar
  • 19.6k
1 vote
Accepted

Limma decideTests function: what kind of multiple hypothesis testing correction does parameter "method" involve?

More context from the docs, looking at the arguments to decideTests(): method: character string specifying how genes and contrasts are to be combined in the ...
Maximilian Press's user avatar
1 vote

Help with DIA-Mass Spectrometry data analysis with several conditions (limma?)

Regarding the big FC values, I just realise the Limma lmfit function requires as input a log2 matrix... so I just transformed my data and now makes more sense. Still I have the doubt regarding the ...
FJ_nw's user avatar
  • 31
1 vote
Accepted

Toptable error, wont recognize condition

The error is telling you that there isn't any Infected column on the model.matrix. Check the column names of mm. Also not sure that having two ...
llrs's user avatar
  • 4,713
1 vote

Determining what RNAseq data is filtered on volcano plot

edgeR functions do not do any filtering, i.e., they do not remove any genes from the results or from the plots. The only filtering was that which you did explicitly yourself by: ...
Gordon Smyth's user avatar
1 vote

the variation between treatments is less than the variation between replicates in RNA-seq data

If you got DEGs with statistically significant p-values after multiple testing following a valid pipeline, I'd believe that, even if looking at the first few PCs doesn't look promising.
swbarnes2's user avatar
  • 1,921
1 vote
Accepted

Voom transformation of RNA seq raw counts data

Try to use DElist() function before you transform, and also make rownames first. ...
benn's user avatar
  • 3,571

Only top scored, non community-wiki answers of a minimum length are eligible