scATAC-seq (Sinac)

Question

[Synat]

Dear all,

I'm learning to analyse ATAC-seq data and got error once I reach the following first section human PBMC and got error when integrating with scRNAseq.

https://stuartlab.org/signac/articles/pbmc_vignette

pbmc_rna <- readRDS("pbmc_10k_v3.rds")
pbmc_rna <- UpdateSeuratObject(pbmc_rna)
transfer.anchors <- FindTransferAnchors(
  reference = pbmc_rna,
  query = pbmc,
  reduction = 'cca'
)

Error: No features to use in finding transfer anchors. To troubleshoot, try explicitly providing features to the features parameter and ensure that they are present in both reference and query assays.

Tried to fix and but still not worked. if anyone who is experienced with scATAC-seq, kindly help. Appreciate

Kind Regards,

Synat

Answer 1

[LChart]

You need to have features that are shared between objects. Since you have RNA the features will need to gene identifiers. You therefore need to quantify chromatin accessibility in a way that relates in some way to gene expression - this could be accessibility of the entire gene, or only the promoter, or you could annotate peaks with the gene into which they fall and create a new feature matrix by aggregating the counts in some way (median / mean /etc).

In the vignette this is done by:

gene.activities <- GeneActivity(pbmc)

# add the gene activity matrix to the Seurat object as a new assay and normalize it
pbmc[['RNA']] <- CreateAssayObject(counts = gene.activities)
pbmc <- NormalizeData(
  object = pbmc,
  assay = 'RNA',
  normalization.method = 'LogNormalize',
  scale.factor = median(pbmc$nCount_RNA)
)

DefaultAssay(pbmc) <- 'RNA'

I'd wager you skipped the DefaultAssay(pbmc) <- 'RNA' step. This sets the default assay to be the accessibility-based gene activity scores. The assay is named 'RNA' so that the next step (transfer.anchors) will match the 'RNA' name to the 'RNA' assay of the pbmc_rna object.

[Synat]

thanks you for your help.