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Using SCT assays #136

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mckoch234 opened this issue Jun 30, 2024 · 6 comments
Closed

Using SCT assays #136

mckoch234 opened this issue Jun 30, 2024 · 6 comments

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@mckoch234
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mckoch234 commented Jun 30, 2024 •
edited

I am getting this error. Error in x[[i, drop = TRUE]]:
! 'counts' not found in this Seurat object
Did you mean "nCount_SCT"?

How do I get it to recognize the counts? The seurat objects I'm working with have multiple assays but even when I change the assay it gives me that error.
@evanbiederstedt
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Hi @mckoch234 CC @saketkc

I'm guessing these are changes with Seurat V5: #133

I didn't change the codebase as I was hoping others would chip in.

We could probably make things V5 compatible. @saketkc or others in the Satija Lab might be able to lend a helping hand as well.

Please detail the issues you've having; an email might be necessary.

Best, Evan

@mckoch234
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mckoch234 commented Jul 9, 2024 via email

@mckoch234
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mckoch234 commented Jul 9, 2024 via email

@evanbiederstedt
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@mckoch234

Without having the data you are working with, I cannot really debug this given the information you've provided.

Here is the function:

conos/R/integrations.R

Lines 365 to 523 in 91110ba

#' Utility function to generate a pagoda2 app from a conos object
#'
#' @param conos Conos object
#' @param cdl list Optional list of raw matrices (so that gene merging doesn't have to be redone) (default=NULL)
#' @param metadata list Optional list of (named) metadata factors (default=NULL)
#' @param filename string Name of the *.bin file to seralize for the pagoda2 application if save=TRUE (default='conos_app.bin')
#' @param save boolean Save serialized *bin file specified in filename (default=TRUE)
#' @param n.cores integer Number of cores (default=1)
#' @param n.odgenes numeric Number of top overdispersed genes to use (dfault=3e3). From pagoda2::basicP2proc().
#' @param nPcs numeric Number of PCs to use (default=100). From pagoda2::basicP2proc().
#' @param k numeric Default number of neighbors to use in kNN graph (default=30). From pagoda2::basicP2proc().
#' @param perplexity numeric Perplexity to use in generating tSNE and largeVis embeddings (default=50). From pagoda2::basicP2proc().
#' @param log.scale boolean Whether to use log scale normalization (default=TRUE). From pagoda2::basicP2proc().
#' @param trim numeric Number of cells to trim in winsorization (default=10). From pagoda2::basicP2proc().
#' @param keep.genes optional set of genes to keep from being filtered out (even at low counts) (default=NULL). From pagoda2::basicP2proc().
#' @param min.cells.per.gene numeric Minimal number of cells required for gene to be kept (unless listed in keep.genes) (default=0). From pagoda2::basicP2proc().
#' @param min.transcripts.per.cell numeric Minimumal number of molecules/reads for a cell to be admitted (default=100). From pagoda2::basicP2proc().
#' @param get.largevis boolean Whether to caluclate largeVis embedding (default=TRUE). From pagoda2::basicP2proc().
#' @param get.tsne boolean Whether to calculate tSNE embedding (default=TRUE). From pagoda2::basicP2proc().
#' @param make.geneknn boolean Whether pre-calculate gene kNN (for gene search) (default=TRUE). From pagoda2::basicP2proc().
#' @param go.env GO environment for the organism of interest (default=NULL)
#' @param cell.subset string Cells to subset with the conos embedding conos$embedding. If NULL, uses all cells via rownames(conos$embedding) (default=NULL)
#' @param max.cells numeric Limit to the cells that are included in the conos. If Inf, there is no limit (default=Inf)
#' @param additional.embeddings list Additional embeddings to add to conos for the pagoda2 app (default=NULL)
#' @param test.pathway.overdispersion boolean Find all IDs using GO category against either org.Hs.eg.db ('hs') or org.Mm.eg.db ('mm') (default=FALSE
#' @param organism string Organism of interest, either 'hs' (Homo sapiens) or 'mm' (Mus musculus, i.e. mouse) (default=NULL). Only used if test.pathway.overdispersion is TRUE. If NULL and test.pathway.overdispersion=TRUE, then 'hs' is used.
#' @param return.details boolean If TRUE, return list of p2 application, pagoda2 object, list of raw matrices, and cell names. If FALSE, simply return pagoda2 app object. (default=FALSE)
#' @return pagoda2 app object
#'
#' @export
p2app4conos <- function(conos, cdl=NULL, metadata=NULL, filename='conos_app.bin',
save=TRUE, n.cores=1, n.odgenes=3e3, nPcs=100, k=30, perplexity=50, log.scale=TRUE, trim=10,
keep.genes=NULL, min.cells.per.gene=0, min.transcripts.per.cell=100, get.largevis=TRUE,
get.tsne=TRUE, make.geneknn=TRUE, go.env=NULL, cell.subset=NULL, max.cells=Inf,
additional.embeddings=NULL, test.pathway.overdispersion=FALSE, organism=NULL, return.details=FALSE) {
if (!requireNamespace("pagoda2", quietly = TRUE)) {
stop("You have to install the pagoda2 package to use p2app4conos(). Please refer to <https://github.com/kharchenkolab/pagoda2>.")
}
if (!requireNamespace("GO.db", quietly = TRUE)) {
stop("Package \"GO.db\" is needed for this function to work. Please install it from Bioconductor.", call. = FALSE)
}
if (!requireNamespace("org.Hs.eg.db", quietly = TRUE)) {
stop("Package \"org.Hs.eg.db\" is needed for this function to work. Please install it from Bioconductor.", call. = FALSE)
}
if (!requireNamespace("org.Mm.eg.db", quietly = TRUE)) {
stop("Package \"org.Mm.eg.db\" is needed for this function to work. Please install it from Bioconductor.", call. = FALSE)
}
if (!requireNamespace("AnnotationDbi", quietly = TRUE)) {
stop("Package \"AnnotationDbi\" is needed for this function to work. Please install it from Bioconductor.", call. = FALSE)
}
if (is.null(conos$embedding)){
stop("The input Conos object must contain an embedding. Please generate with the method embedGraph().")
}
if (is.null(cdl)) {
#cdl <- lapply(conos$samples,function(p) t(p$misc$rawCounts))
cdl <- lapply(rawMatricesWithCommonGenes(conos),t)
}
samf <- lapply(conos$samples,function(x) rownames(x$counts))
samf <- as.factor(setNames(rep(names(samf),unlist(lapply(samf,length))),unlist(samf)))
if (!is.null(cell.subset)) {
cell.subset <- intersect(cell.subset,rownames(conos$embedding))
} else {
cell.subset <- rownames(conos$embedding)
}
samf <- droplevels(samf[names(samf) %in% cell.subset])
cdl <- lapply(cdl, function(x) x[,colnames(x) %in% cell.subset,drop=FALSE])
# limit to the cells that are included in the conos
vc <- unlist(lapply(cdl,colnames))
if (length(vc)>max.cells) { # subsample
cat("subsampling",length(vc),"cells down to",max.cells,'...')
vc <- sample(vc,max.cells)
cat('done\n')
}
cdl <- lapply(cdl,function(d) d[,colnames(d) %in% intersect(vc,names(samf))])
cm <- do.call(cbind,cdl)
cp2 <- pagoda2::basicP2proc(cm, n.cores=n.cores, n.odgenes=n.odgenes, nPcs=nPcs, k=k, perplexity=perplexity, log.scale=log.scale, min.cells.per.gene=min.cells.per.gene, min.transcripts.per.cell=min.transcripts.per.cell, get.largevis=get.largevis, get.tsne=get.tsne, make.geneknn=make.geneknn)
if (test.pathway.overdispersion) {
if (is.null(organism) || organism =='hs') {
ids <- unlist(lapply(mget(colnames(cp2$counts),org.Hs.eg.db::org.Hs.egALIAS2EG,ifnotfound=NA),function(x) x[1]))
rids <- names(ids)
names(rids) <- ids
# list all the ids per GO category
go.env <- list2env(eapply(org.Hs.eg.db::org.Hs.egGO2ALLEGS, function(x) as.character(na.omit(rids[x]))))
} else if (organism =='mm') {
# translate gene names to ids
ids <- unlist(lapply(mget(colnames(cp2$counts),org.Mm.eg.db::org.Mm.egALIAS2EG,ifnotfound=NA),function(x) x[1]))
# reverse map
rids <- names(ids)
names(rids) <- ids
# list all the ids per GO category
go.env <- list2env(eapply(org.Mm.eg.db::org.Mm.egGO2ALLEGS, function(x) as.character(na.omit(rids[x]))))
} else {
stop("Unknown organism. Currently only 'hs' (human) and 'mm' (mouse) supported.")
}
}
if (!is.null(go.env)) {
#cp2$getHierarchicalDiffExpressionAspects(type='PCA',clusterName='community',z.threshold=3)
# here we test for pathway overdispersion, but recursive diff expression, as shown above will work just as well
cp2$testPathwayOverdispersion(go.env,verbose=TRUE,correlation.distance.threshold=0.95,recalculate.pca=FALSE,top.aspects=15)
termDescriptions <- AnnotationDbi::Term(GO.db::GOTERM[names(go.env)]) # saves a good minute or so compared to individual lookups
geneSets <- lapply(sn(names(go.env)),function(x) {
list(properties=list(locked=TRUE,genesetname=x,shortdescription=as.character(termDescriptions[x])),genes=c(go.env[[x]]))
})
} else {
hdea <- cp2$getHierarchicalDiffExpressionAspects(type='PCA',z.threshold=3)
geneSets <- pagoda2::hierDiffToGenesets(hdea)
}
# add all kinds of embeddings
# various joint embeddding versions
#cp2$embeddings$Conos <- list("All"=t(conO$embedding),"cochlea"=t(conC$embedding),"DRG"=t(conD$embedding),"Merged"=t(conO$embedding),"Refined"=t(con2$embedding))
cn <- rownames(cp2$counts) # cell names
cp2$embeddings$Conos <- list("All"=conos$embedding[cn,])
# hack: add placeholder spaces, so that old p2 version picks up the new embeddings
cp2$reductions$Conos <- list()
cp2$embeddings$sample <- lapply(conos$samples,function(x) { em <- x$embeddings$PCA[[1]]; em[rownames(em) %in% cn,] }) # embeddings of the individual samples
cp2$embeddings$sample <- cp2$embeddings$sample[!unlist(lapply(cp2$embeddings$sample,is.null))]
if (length(cp2$embeddings$sample)>1) {
cp2$reductions$sample <- list()
} else {
cp2$embeddings$sample <- NULL
}
if (!is.null(additional.embeddings)) {
cp2$embeddings$Other <- lapply(additional.embeddings, function(em) { em[rownames(em) %in% cn,] })
cp2$reductions$Other <- list()
}
# additional metadata with different factors .. you probably want to include something like sample or tissue (or patient type)
metadata <- c(metadata,list(sample=samf))
metadata <- lapply(metadata, function(d) d[cn])
meta <- lapply(sn(names(metadata)),function(n) pagoda2::p2.metadata.from.factor(droplevels(as.factor(metadata[[n]])),displayname=n))
p2app <- pagoda2::make.p2.app(cp2, dendrogramCellGroups = as.factor(conos$clusters[[1]]$groups[cn]), additionalMetadata = meta, geneSets = geneSets,innerOrder='odPCA')
# Optional showing of app
#show.app(p2app, name='newPagoda',browse=FALSE)
# Save serialised web object, RDS app and session image
if(save){
p2app$serializeToStaticFast(binary.filename = filename,verbose=TRUE)
}
if(return.details) {
return(list(app=p2app,p2=cp2,cdl=cdl,cn=cn))
} else {
invisible(p2app)
}
}

If you run the sections line-by-line, you might be able to track down the bug. That would be helpful. Otherwise, please email the data to me so I could help out.

It's not something seen before.

Best, Evan

@mckoch234
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mckoch234 commented Jul 9, 2024 via email

@mckoch234
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mckoch234 commented Jul 9, 2024 via email

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@evanbiederstedt @mckoch234