BoatCC123 commented 6 months ago

用的seurat.V5对象,进行时出现报错，请问该如何解决呢？麻烦了

chuiqin commented 6 months ago

首先，检查一下是否有celltype这一列存在，以及随机挑选一个Assay验证一下是否存在，可以这样操作：

unique(Neutrophi_rename.final2$celltype)

Neutrophi_rename.final2[["AUCell"]]@scale.data[1:2,1:2]

如果存在，随机挑一个Assay检查一下这些基因集是否真的在这些细胞类型中存在差异；

Idents(Neutrophi_rename.final2) <- Neutrophi_rename.final2$celltype
a <- Seurat::FindAllMarkers(object = Neutrophi_rename.final2, assay = "AUCell",
                         slot = "scale.data", 
                         test.use = "wilcox", min.pct = -Inf, logfc.threshold = 0,
                         min.cells.group = 0, min.diff.pct = -Inf, verbose = F,
                         min.cells.feature = 0)

以上方法都失败的话，我们要先用示例数据测试一下irGSEA流程是否顺畅。如果示例数据跑irGSEA流程不顺畅，说明安装存在问题：

# load PBMC dataset by R package SeuratData
library(Seurat)
library(SeuratData)
# download 3k PBMCs from 10X Genomics
InstallData("pbmc3k")
data("pbmc3k.final")
pbmc3k.final <- SeuratObject::UpdateSeuratObject(pbmc3k.final)

# Seurat object
pbmc3k.final <- irGSEA.score(object = pbmc3k.final, assay = "RNA",
slot = "data", msigdb = T, species = "Homo sapiens",
category = "H", geneid = "symbol",
method = c("AUCell", "UCell", "singscore", "ssgsea"), kcdf = 'Gaussian')

# Integrated analysis
result.dge <- irGSEA.integrate(object = pbmc3k.final,
group.by = "seurat_annotations", metadata = NULL, col.name = NULL,
method = c("AUCell","UCell","singscore","ssgsea"))

BoatCC123 commented 6 months ago

好的，非常感谢您，我会尽快尝试。有问题再跟您交流，辛苦了！ ------------------ Original ------------------ From: @.>; Date: Sat, Mar 2, 2024 11:50 PM To: @.>; Cc: @.>; @.>; Subject: Re: [chuiqin/irGSEA] irGSEA.integrate (Issue #33)

首先，检查一下是否有celltype这一列存在，以及随机挑选一个Assay验证一下是否存在，可以这样操作： unique(Neutrophi_rename.final2$celltype) @.***[1:2,1:2]
如果存在，随机挑一个Assay检查一下这些基因集是否真的在这些细胞类型中存在差异； Idents(Neutrophi_rename.final2) <- Neutrophi_rename.final2$celltype a <- Seurat::FindAllMarkers(object = Neutrophi_rename.final2, assay = "AUCell", slot = "scale.data", test.use = "wilcox", min.pct = -Inf, logfc.threshold = 0, min.cells.group = 0, min.diff.pct = -Inf, verbose = F, min.cells.feature = 0)
以上方法都失败的话，我们要先用示例数据测试一下irGSEA流程是否顺畅。如果示例数据跑irGSEA流程不顺畅，说明安装存在问题：

load PBMC dataset by R package SeuratData library(Seurat) library(SeuratData) # download 3k PBMCs from 10X Genomics InstallData("pbmc3k") data("pbmc3k.final") pbmc3k.final <- SeuratObject::UpdateSeuratObject(pbmc3k.final) # Seurat object pbmc3k.final <- irGSEA.score(object = pbmc3k.final, assay = "RNA", slot = "data", msigdb = T, species = "Homo sapiens", category = "H", geneid = "symbol", method = c("AUCell", "UCell", "singscore", "ssgsea"), kcdf = 'Gaussian') # Integrated analysis result.dge <- irGSEA.integrate(object = pbmc3k.final, group.by = "seurat_annotations", metadata = NULL, col.name = NULL, method = c("AUCell","UCell","singscore","ssgsea"))

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chuiqin commented 6 months ago

还可以排除一下Seurat的版本。错误的发生是因为Seurat从5.02开始，增加了一个fc.slot参数。如果你要计算scale.data的差异，不管是FindAllMarkers还是FindMarkers函数，没有指定fc.slot，计算的时候都会出错。恰好irGSEA的打分矩阵都存放在scale.data里面。我在新版本的irGSEA（version 3.2.4）里面已经修复了这个错误，只要重新安装irGSEA即可解决这个问题。

chuiqin / irGSEA

irGSEA.integrate #33