RunPrestoAll / Please only specify either assay or reduction / No DE genes identified

[jdime]

Hello,

I'm trying to use RunPrestoAll(), but I'm getting warnings that end up in no DE being detected.

I'm creating a Seurat object SmallRO following the vignette and doing SCTransform, although I'm calling the "RNA" assay for the DE calculation (see below). https://satijalab.org/seurat/v3.2/pbmc3k_tutorial.html With ~1k PBMC's from 10X public datasets.

First, I tried to run RunPrestoAll with all default parameters but got error: error in evaluating the argument 'x' in selecting a method for function 't': Cannot find '0.01' in this Seurat object

> so.RunPrestoAll <- RunPrestoAll(object = SmallRO)
Calculating cluster 0
Calculating cluster 1
...etc
Calculating cluster 12
Warning: No DE genes identified
Warning: The following tests were not performed: 
Warning: When testing 0 versus all:
	error in evaluating the argument 'x' in selecting a method for function 't': Cannot find '0.01' in this Seurat object

Then, I indicated the assay assay = "RNA" but I got Please only specify either assay or reduction. warnings and the result is empty.

> so.RunPrestoAll <- RunPrestoAll(object = SmallRO, assay = "RNA")
Calculating cluster 0
Calculating cluster 1
... etc
Calculating cluster 12
Warning: No DE genes identified
Warning: The following tests were not performed: 
Warning: When testing 0 versus all:
	Please only specify either assay or reduction.
... etc
Warning: When testing 12 versus all:
	Please only specify either assay or reduction.
> so.RunPrestoAll
data frame with 0 columns and 0 rows

Then, I added reduction = NULL. The warning is gone but the results are still empty.

> so.RunPrestoAll <- RunPrestoAll(object = SmallRO, assay = "RNA", reduction = NULL)
Calculating cluster 0
Calculating cluster 1
... etc
Calculating cluster 12
Warning: No DE genes identified
> so.RunPrestoAll
data frame with 0 columns and 0 rows

Just to note, if I use FindAllMarkers() with the same object I get DE.

so.FindAllMarkers <- FindAllMarkers(object = SmallRO, assay = "RNA", only.pos = F)
> head(so.FindAllMarkers)
            p_val   avg_logFC pct.1 pct.2    p_val_adj cluster gene
TRAC 1.606406e-52  10.1013930 0.952 0.322 2.100215e-48       0 TRAC
IL32 3.388294e-52 -41.6934132 0.959 0.332 4.429856e-48       0 IL32
IL7R 2.395640e-51  12.4201524 0.816 0.217 3.132060e-47       0 IL7R

My questions are:

1. May the conflict of RunPrestoAll(..., assay = "RNA") when not specifying reduction be a bug? Is reduction related to PCA, etc? I couldn't find reduction in the RunPrestoAll() Usage or Arguments. What reduction would I be using if I set reduction=NULL?

2. What may be wrong in my commands, since I can run FindAllMarkers() but not RunPrestoAll() with basically the same parameters?

This is my session info:

R version 4.0.2 (2020-06-22)
Platform: x86_64-apple-darwin17.0 (64-bit)
Running under: macOS High Sierra 10.13.6

Matrix products: default
BLAS:   /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRlapack.dylib

locale:
[1] en_CA.UTF-8/en_CA.UTF-8/en_CA.UTF-8/C/en_CA.UTF-8/en_CA.UTF-8

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
[1] Seurat_3.2.1         SeuratWrappers_0.3.0

Thanks.

[qindan2008]

I met the same problem, when run RunPrestoAll: Warning message: “No DE genes identified” Warning message: “The following tests were not performed: ” Warning message: “When testing Fibroblast versus all: object 'p_val' not found” Warning message: “When testing Endothelial versus all: object 'p_val' not found” But when run FindAllMarkers, all is ok.