Bioinformatics Asked by Yach on May 4, 2021
I am trying to follow a tutorial from Sanger institute (from May 2019) on analysis of single cell RNA Seq data. They use calculateQCMetric function to calculate the quality metrics, but I am getting an error message that calculateQCMetric is a deprecated (isSpike function is also deprecated). I want to calculate the quality metrics. Can anyone provide code for getting QCMetric step? I attached the previous defunct code below:
umi <- calculateQCMetrics(
umi,
feature_controls = list(
ERCC = isSpike(umi, "ERCC"),
MT = isSpike(umi, "MT")
)
And I use StupidWolf’s code from this question isSpike function in SingleCellExperiment package is deprecated? to instead isSpike function. I also attached below:
Spikein_names = grep("^ERCC-",rownames(molecules),value=TRUE)
SpikeIn = molecules[Spikein_names,]
g = genes(EnsDb.Hsapiens.v86)
MTgene_names = g[seqnames(g)=="MT"]$gene_id
MTgenes = molecules[rownames(molecules) %in% MTgene_names,]
Have a vector of genes to keep:
keep = setdiff(rownames(molecules),c(SpikeIn,MTgenes))
Then make the single cell object:
sce <- SingleCellExperiment(
assays=list(counts=as.matrix(molecules[keep,])),colData=annotation)
Use altExp to slot in the stuff:
altExp(sce, "spike-in") <- SummarizedExperiment(SpikeIn)
altExp(sce, "MTgenes") <- SummarizedExperiment(MTgenes)
I try the below code, which did not work.
> perCellQCMetrics(sce,
subsets = list(Spikein_names,MTgene_names),
flatten = TRUE,
exprs_values = "counts",
use_altexps = TRUE)
Thanks in advance.
It would be helpful if you can provide the error message. Guessing from your codes, it's possible that the alternative experiments weren't set correctly. My suggestion:
altExp(sce, "spike-in") <- SummarizedExperiment(assays=list(counts=as.matrix(SpikeIn)))
altExp(sce, "MTgenes") <- SummarizedExperiment(assays=list(counts=as.matrix(MTgenes)))
If you're using altExp, the subsets argument in perCellQCMetrics is unnecessary in this case.
perCellQCMetrics(sce,
exprs_values="counts",
use_altexps=TRUE)
Answered by mattcwh on May 4, 2021
Ok I have figured it out and I am sharing this in case anyone can get help. I used
sce <-addPerCellQC(sce)
which calculated the QC for all the altexps also and adds them to the colData.
Answered by Abhay on May 4, 2021
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