7
votes
Accepted
Using Seurat to compare mutant vs.wt
Single-cell analysis to compare samples is a long a difficult process. There is very good documentation for 10x Genomics cellranger, the DropSeq Pipeline and the Seurat R package. These tools all have ...
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5
votes
Script to allow gene set enrichment analysis of 10x genomics data in R
Seruat will give you a list of genes which it thinks are upregulated in a particular cluster. Look at the functions that talk about marker genes - these functions basically do a DE analysis of the ...
- 3,271
4
votes
Accepted
What is cellranger doing in comparison to other methods?
cellranger mkfastq is not necessary anymore. It used to be that the cellranger software wanted the reads to be interleaved, and you could use cellranger to do that for you if you couldn't do it ...
- 1,812
4
votes
What process and input data is required for a cellranger reference transcriptome?
Your problem is caused by using the transcriptome fasta file rather than the genome fasta file. You've already given it transcriptome information with ...
- 19.5k
4
votes
BAM to gene expression matrix (UMI counts per gene per cell),10X
You should be able to parse out what you need using the tags in the .bam. 10xGenomics' website says what tags they add.
https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/...
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3
votes
BAM to gene expression matrix (UMI counts per gene per cell),10X
How to re-analyze 10X BAM files?
This is a great question and honestly, I don't think there was an easy way to do this at the time the question was asked. The reason is that if you want to re-do the ...
- 131
3
votes
Low custom tdtomato gene content
A few suggestions:
Manually inspecting the BAM file with a genome browser like IGV, as @GWW suggested and checking the qualities of the alignments in your BAM/SAM file (SAM is the human readable ...
- 3,672
3
votes
What is cellranger doing in comparison to other methods?
The best approach would be to contact 10x Genomics support: support@10xgenomics.com . They usually respond within a few hours.
Otherwise, you can check the methods from the first 10x Genomics paper:
...
- 2,139
2
votes
Using Seurat to compare mutant vs.wt
There is no single ended answer to your question as everything will depend on your data. For example: Is this unsupervised data where you have to browse to 100 cell types for each sample or did you ...
- 574
2
votes
Accepted
Can I get the graph generated by cellranger
Cell Ranger
You can't download the tSNE coordinates for cells directly from the Analysis tab of the fancy, polished .html document that Cell Ranger produces. If you have access to the machine on ...
- 1,129
2
votes
Script to allow gene set enrichment analysis of 10x genomics data in R
There is no purpose-built R package to perform gene set enrichment analysis on single-cell data but there does not need to be. You should be able to tools developed for bulk-RNA-Seq or microarray data,...
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2
votes
10X cellranger count, [error] The chemistry was unable to be automatically determined
The issue was that I supplied wrong indexes during fastq files generation, however, they were generated successfully, and only the next step failed with not that ...
- 2,538
2
votes
Low custom tdtomato gene content
The solution to your problem probably is adding the full mRNA sequence of your transgene to the reference (as also suggested by acrux).
I had a very similar problem recently when tdTomato expression ...
- 858
2
votes
Clustering using cellranger
The direct answer is yes. If you have never worked with hdf5 you can start here.
But, cellranger outputs several hdf5 files. You can extract the count matrix from the "/outs/...
- 574
2
votes
Accepted
Cellranger results have too many cells
The Barcode Rank plot shows very high background in your sample.
The cell estimation from Cell Ranger will look for a sudden drop in the number of UMIs and use this ...
- 858
2
votes
Running cellranger on scRNASeq data with feature barcoding (10x + antibody capture)
Check your FASTQ headers - if they've been demultiplexed, the index barcodes can be found in the header. The barcodes are usually separated by a + in the comment ...
- 1,745
2
votes
In cellranger, can I change the minimum number of reads for autodetecting chemistry?
I don't think you can reduce the number of reads required for autodetection, and it would be a bad idea anyway, because the guess could go wrong.
From 10X Q&A:
TotalSeq B is specifically designed ...
- 252
1
vote
Low fraction reads in cell metric in snRNA-seq data
Yes, "low fraction reads in cells" is more common with snRNA-seq from frozen samples, I wouldn't worry about it by itself (you're just throwing away some sequencing data). You should still ...
- 3,858
1
vote
In CellRanger output, what are the reads with an xf:i:17 tag?
From the documentation at https://kb.10xgenomics.com/hc/en-us/articles/115003646912-How-is-sequencing-saturation-calculated-
All of these samflag 0x400 reads have an xf tag value of 17, which consist ...
- 11
1
vote
How to solve correlation problems between different samples in scRNA-seq?
The picture below shows an heatmap of the R² by doing a linear
regression between 2 samples' count matrices.
In addition to doing a heatmap, I'd do a conventional UMAP/t-SNE plot and see how much ...
- 3,858
1
vote
Problem with Seurat reference mapping
Yes! Multiome seems to have cells with much higher mitochondrial gene content. I don't understand the biology behind this. But in using a cut off of 5% (i.e. remove cells with >5% MT genes) I lose ...
1
vote
Accepted
10X scRNAseq: Sample mix-up
If the samples were from different individual organisms you could try looking for mitochondrial variants that differentiate between them. You should have reasonable coverage of the entire ...
- 1,568
1
vote
Accepted
Cellranger gives error
Two of those command line parameters should be accessible locations (see here). Could you please add the output of the following commands:
...
- 13k
1
vote
cellranger mkfastq with full path to --id flag
You should be able to use the --output-dir argument to specify a folder of your choice and use --id=demux_10x as before.
- 3,672
1
vote
Cell Ranger) Error during performing a test run
Answer from PPK, converted from comments:
The memory warning is only letting you know that the upper limit you set on memory (112 gb) was higher than the available memory. Do you share the server with ...
1
vote
Accepted
Using cellranger mkgtf w/ NCBI GFF3 files
Regarding your comment:
This GFF file does not have the same key nomenclature as seen in
ENSEMBL or USCS
The link you have provided corresponds to an entry in ...
- 3,672
1
vote
Using cellranger for non-10x data
Possible? Sure. Your biggest problem is that 10x is expecting barcodes from its whitelist. I used to alter the python scripts to accept a 'new' kind of chemistry where the barcodes and umi lengths ...
- 1,812
1
vote
processing fastq files using cellranger on linux
this has been resolved. I downloaded the windows file and this caused trouble but when downloading the file within the Linux, it seems to be working.
- 29
1
vote
Low custom tdtomato gene content
We had the same issue. Turned out, our tdTomato transgene was followed up by WPRE and BGH polyA signals. These sequences are quite large, and their presence precludes sequencing into the tdTomato ...
- 11
1
vote
No counts for added gene in cellranger (scRNA-seq)
The first column of your GTF must correspond exactly to the name of the sequence in your FASTA file. Typically this is the chromosome so a GTF line:
...
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