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 ...
user avatar
  • 873
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 ...
user avatar
  • 3,201
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 ...
user avatar
  • 1,688
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 ...
user avatar
  • 19.3k
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/...
user avatar
  • 1,688
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 ...
user avatar
  • 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 ...
user avatar
  • 3,472
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: ...
user avatar
  • 2,099
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 ...
user avatar
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 ...
user avatar
  • 1,119
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,...
user avatar
  • 873
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 ...
user avatar
  • 788
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/...
user avatar
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 ...
user avatar
  • 3,472
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 ...
user avatar
  • 1,468
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: ...
user avatar
  • 11.5k
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.
user avatar
  • 3,472
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 ...
user avatar
  • 3,472
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 ...
user avatar
  • 1,688
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.
user avatar
  • 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 ...
user avatar
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: ...
user avatar
  • 873
1 vote

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 ...
user avatar
1 vote

Aggregate sequencing/mapping/etc. metrics from cellranger across Illumina samples

I wrote a simple script to do it. Not sure whether that's what you want. ...
user avatar
  • 272

Only top scored, non community-wiki answers of a minimum length are eligible