satijalab / pasta Goto Github PK

I'm currently working on applying PASTA, and I'm having trouble creating a fragments TSV file using Sinto. I've looked through the documentation and searched online, but I'm still not clear on how to do it.

Here's what I've tried so far:

I've installed Sinto and its dependencies using pip.
I've run the blocks function in Sinto to create a BED file of the genomic blocks or fragments from PolyApipe Bam output.
However, I'm not sure how to convert the BED file to a TSV file that I can use in my analysis or if this is even the right file

I would really appreciate any guidance or resources that you could provide to help me create a fragments TSV file and what was used as an input to create it.

Thank you for your time and assistance.

Hi! Thanks for the very useful tool for single-cell APA analysis!

I have a few questions about the analysis.

First, how much does the selection of background cells matter when you have the mixture of disease cells and control cells with different cell types? For example, in my case, I am comparing leukemia cells and healthy cells where the data is biased to have more leukemia cells than healthy. In this case would you recommend to use control cells as background, or to subsample, or just use entire cells as background?

And I wonder if you can provide the script for the global shortening/lengthening analysis in figure3/4 in the paper. That would be super helpful.

Thanks!!

Best,
Soobeom

I try the "PASTA vignette", and get a error as fowllowing:

######################## code

pbmc <- CalcPolyAResiduals(pbmc, assay = "polyA", features = features.last.exon, gene.names = "Gene_Symbol", verbose = TRUE)

######################## info

Calculating background distribution
Using all cells in order to estimate background distribution
Removing 321 sites without a gene annotation
Running Dirichlet Multionmial Regression
Regularizing Dirichlet Multionmial Variance
Error in validObject(object = value) :
invalid class "polyAsiteAssay" object: features in 'scale.data' must be in the same order as in 'data'
In addition: Warning messages:
1: The slot argument of AverageExpression() is deprecated as of Seurat 5.0.0.
ℹ Please use the layer argument instead.
ℹ The deprecated feature was likely used in the Seurat package.
Please report the issue at https://github.com/satijalab/seurat/issues.
This warning is displayed once every 8 hours.
Call lifecycle::last_lifecycle_warnings() to see where this warning was generated.
2: In asMethod(object) :
sparse->dense coercion: allocating vector of size 3.1 GiB
3: In .M2v(x) : sparse->dense coercion: allocating vector of size 3.1 GiB
4: In asMethod(object) :
sparse->dense coercion: allocating vector of size 3.1 GiB
5: In asMethod(object) :
sparse->dense coercion: allocating vector of size 3.1 GiB

#######################################

Could you tell me it is the error of Seurat 5.0.0 or PASTA? Thanks in advanced!