FindIntegrationAnchors() returns "no applicable method for 'Assays' applied to an object of class "NULL""

[lucas-vicuna]

Hello,
I have a list of seurat objects that I read into a list. Then I merge them, split them by experimental condition, perform standard steps but when running the function FindIntegrationAnchors(), I get:

Error in UseMethod(generic = "Assays", object = object) :
no applicable method for 'Assays' applied to an object of class "NULL"

However, I see that the class of the merged object is "RNA", so I don't get why I get class NULL afterwards.

Please help, I have been stacked in this issue for several days.

Below you can see the script

Thank you very much !

library(Seurat)
library(dplyr)
library(ggplot2)
library(SeuratObject)
library(future.apply)

workDir <- "/project/lbarreiro/USERS/lucas/czi/data/scrna/seurat/human_iav_tcruzi"
setwd(workDir)

# Read first 4 files:

file_paths <- list.files(workDir, pattern = "^seuratObj4_[1-4]\.rds$", full.names = TRUE)

# Initialize an empty list to store Seurat objects

seurat_list <- future_lapply(file_paths, readRDS)

# Merge the Seurat objects concurrently

merged_obj <- Reduce(merge, seurat_list) # Takes ~20 min

# class(merged_obj) # Seurat

slotNames(merged_obj)

#[1] "assays"       "meta.data"    "active.assay" "active.ident" "graphs"      
#[6] "neighbors"    "reductions"   "images"       "project.name" "misc"        
#[11] "version"      "commands"     "tools" 

# > Assays(merged_obj)
# [1] "RNA"


# split the dataset into a list of four seurat objects (C1, C2, C3, C4)

ifnb.list <- SplitObject(merged_obj, split.by = "expCond1") # very fast

# class(ifnb.list) # list

# normalize and identify variable features for each dataset independently

ifnb.list <- lapply(X = ifnb.list, FUN = function(x) {
x <- NormalizeData(x)
x <- FindVariableFeatures(x, selection.method = "vst", nfeatures = 2000)
})

# class(ifnb.list) # list

# select features that are repeatedly variable across datasets for integration run PCA on each
# dataset using these features

features <- SelectIntegrationFeatures(object.list = ifnb.list)
# class(features) # "character"

ifnb.list <- lapply(X = ifnb.list, FUN = function(x) {
x <- ScaleData(x, features = features, verbose = FALSE)
x <- RunPCA(x, features = features, verbose = FALSE)
})
# class(ifnb.list) # list

# Active assays:

assayNames(ifnb.list)

immune.anchors <- FindIntegrationAnchors(object.list = ifnb.list, anchor.features = features, reduction = "rpca", assay = "RNA", normalization.method = "LogNormalize")

Error in UseMethod(generic = "Assays", object = object) :
no applicable method for 'Assays' applied to an object of class "NULL"

[kevinrue]

Having a similar issue with the following:

> obj <- IntegrateLayers(
+   object = obj,
+   method = RPCAIntegration,
+   normalization.method = "SCT",
+   verbose = F
+ )
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=00s  
  |                                                  | 0 % ~calculating  Error in UseMethod(generic = "Assays", object = object) : 
  no applicable method for 'Assays' applied to an object of class "NULL"

Based on tutorial https://satijalab.org/seurat/articles/seurat5_integration#perform-streamlined-one-line-integrative-analysis

I don't want to hijack this issue. I'm not expecting a reply to my own issue here. I mainly post here to be notified of replies to the issue reported above, but I thought that maybe the maintainers might be interested in knowing that the issue might affect more than one use case.

Version of Seurat packages

 P Seurat               * 5.0.3      2024-03-18 [?] CRAN (R 4.3.1)
 P SeuratData           * 0.2.2.9001 2024-04-19 [?] Github (satijalab/seurat-data@4dc08e0)
   SeuratObject         * 5.0.1      2023-11-17 [1] CRAN (R 4.3.1)
 P SeuratWrappers       * 0.3.5      2024-04-19 [?] Github (satijalab/seurat-wrappers@8d46d6c)

[zskylarli]

Hi - to the original issue, do you run into the same issue when running the vignette with the ifnb dataset, and/or would you be able to create a reproducible example? Could you check the class() of the individual assays before running FindIntegrationAnchors by running class(ifnb.list[1] etc.?