My samples are patient-derived xenografts that contain both human epithelial cancer cells and mouse fibroblast/blood cells. I run cellranger count using the human and mouse combined reference genome, which was downloaded from 10xgenomics (https://cf.10xgenomics.com/supp/cell-exp/refdata-gex-GRCh38-and-mm10-2020-A.tar.gz). After Leiden clustering, the human and mouse cells can be separated by the marker genes found with scanpy.tl.rank_genes_groups. Then the data can be separated into human cells and mouse cells. The mouse genes in human cells were removed and the raw data of human cells can be re-clustered.
I am wondering if is this the correct practice for the analysis.
Is it possible to filter out the human cell from the fastq data and run cellranger count using only human reference genome?
Thanks.
2 answers
One thing I like doing is mapping raw FASTQs to combined human+mouse reference to figure out which cell barcodes are human and which cell barcodes are mouse (e.g. if >75% of reads/UMIs belonging to a given barcode are of human origin, it's probably human and not mouse).
Then, I map the original raw FASTQ to human-only reference and throw away the cell barcodes that were of non-human origin from the previous step.
For scRNA-seq, I like this approach of deciding at the cell barcode-level what's human and what's mouse.
To split FASTQ based on cell barcodes, I tried running subset-bam:
https://github.com/10XGenomics/subset-bam
and then run bamtofastq:
https://github.com/10XGenomics/bamtofastq
and then map the fastq files to human-only and mouse-only references.
Log in to answer this question.
-
Cellranger: Demux pooled (hashing antibodies) GEX and VDJ 10x sequence fastq data
written by Flipstone •
-
cellranger count matrix
written by shamza •
-
cellranger count: an extremely low rate of correct barcodes was observed
written by Alex •
-
mouse+human cells in 10x scRNA-seq
written by Sy80 •
-
Any reason for scRNA data of mouse lung samples to fail to capture epithelial cells?
written by 윤여가(대학원 바이오정보학) •
-
where to get PBMC (68k) labels?
written by mk
-
cellranger count error "Your reference does not contain the expected files, or they are not readabl…
written by leranwangcs •
-
Filtering out mouse cells based on mouse/human genes ratio
written by fifty_fifty •
-
Cellranger count argument for 10xgenomic RNA+surface protein
written by Stemcellguy •
-
How to filter out mouse cells to from a mixture library (mouse and human cells) by cellranger?
written by mzhoufulai •
One option is to use Sargasso (https://github.com/biomedicalinformaticsgroup/Sargasso) in order to split the raw FASTQ reads based on their respective species. I have used this program before, but I am unsure of its usage in scRNA-seq.