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How to correct counts between 10X and Smart-seq2 dataset? #43

@Paperplane1031

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@Paperplane1031

Thank you for bringing us such a fantastic atlas in your paper.
I have seen that you perform 10X and Smart-seq2 sequencing for most of the same tissue. After using DecontX for removing ambient RNA, and with batch correction for donor and technology, you perform downstram analysis like detect DEGs and compare the gene expression level between different tissues like you have said in the supplementary material:

Data pre-processing and cell type annotations
Gene count tables were combined with the metadata variables using the Scanpy (41) Python
package version 1.7.2. We removed cells that did not have at least 200 detected genes. For FACS
we removed cells with less than 5000 counts and for droplet cells with less than 2500 UMIs.
Ambient RNA and barcode hopping (42) are known problems in 10x sequencing. To remove cells
generated by barcode hopping, we removed all cells sharing both the cell and transcript barcode
but not the same sample barcode in each sequencing run. In order to filter out reads from ambient
RNA we ran DecontX (43) separately for each organ, using default parameters and with batch
correction for donor and technology. After the DecontX filtering step, we re-filtered the dataset by
excluding the mitochondrial encoded genes when removing cells that did not contain the minimum
number of genes and/or minimum of counts/UMIs. The filtered gene-count matrix was then used
for the analysis. All downstream analyses were performed after correcting counts for potential
ambient mRNA contamination and dissociation artifacts, which would otherwise result in the
detection of differentially expressed genes (DEGs) that are specific to a tissue rather than to the
cell type of interest.

DecontX is used for contaminant RNA removal, and if you set the batch vector, removal will be perform in each batch. So I wonder how you use DecontX for batch correction for donor and technology? Or maybe you correct it using some other package? As I seen It seems that there is no batch correction function in DecontX. And for most of the batch correction package, most people will advise not to use the corrected count for DEGs analysis or some other downstream analysis. Could you help to answer my questions? I am quite new to scRNA-seq analysis especially for the 10X and Smart-seq2 integration analysis. Thanks a lot!

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