10 votes

determining doublets in single-cell RNA-seq

Expected rates of doublets / duplets / multiplets Fluidigm C1 doublet rate: around 1-5% depending on chip type used. More information: Fluidigm white paper: Redesign of C1 Medium-Cell 96 IFCs ...
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9 votes
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What is the index fastq file (sample_I*.fastq.gz) generated when demultiplexing Illumina paired-end runs?

tldr - The I*.fastq.gz file contains the read index sequences. long explanation Illumina uses a program called bcl2fastq to demultiplex sequencing runs. This ...
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  • 3,071
5 votes
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Raw vs Filtered in the output of cellranger count

I'm unsure whether this is the answer you are looking for, but when looking into 10X cellranger documentation for the Matrices Output: Unfiltered gene-barcode matrices: Contains every barcode ...
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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 ...
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  • 3,211
5 votes

Merging two dataframes in R

For merging dataframes, I find it easiest to use the tidyverse / dplyr functions inner/full/left/right_join. See the "Data Transformation Cheatsheet" on this page. For the merge that @...
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4 votes
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10x Genomics Chromium single-cell RNA-seq data analysis options?

Data preparation Cell Ranger uses the Illumina sequencing output (.bcl) files Make fastq files: cellranger mkfastq ==> ...
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  • 2,574
4 votes
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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 ...
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  • 1,662
4 votes

determining doublets in single-cell RNA-seq

We cannot assume that doublets will produce more UMIs I would caution the assumption that all doublets will have twice the UMI levels of isolated single-cells. Many "doublets" could contain ...
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4 votes
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Derive a GTF containing protein coding genes from a GTF file with Exons and CDS

In your case, I would definitely suggest following @Emily_Ensembl's advice and using the Arabidopsis GTF from Ensembl. But for future reference, if an Ensembl GTF wasn't available, you could build ...
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  • 3,211
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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  • 1,662
4 votes

Single-cell sequencing dataset has too many barcodes

The BAM file you downloaded has all of the detected 10X barcodes instead of those that represent cells. In a given 10X experiment the number of input barcodes vastly outnumbers the input cell count. ...
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  • 727
3 votes
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Why do I have >10,000 cells in the 10X matrix produced by cellranger?

The raw data from cell ranger contains all of the barcodes detected in the experiment. These raw barcodes are filtered by cell ranger to identify the barcodes that likely represent cells rather than ...
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  • 727
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 ...
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  • 131
3 votes

How to normalise scRNASeq data for differential expression analysis

I would suggest using a likelihood ratio test for differential expression using logistic regression with batch as a latent variable. In Seurat you can do: ...
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  • 674
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 ...
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  • 3,432
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: ...
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3 votes
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Why are there more barcodes than GEMs in 10X chromium data?

Your initial guess is almost certainly correct. I don't know about the linked read libraries, but in the 10X single cell sequencing protocol, separating real barcodes from noise barcodes is an ...
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  • 3,211
2 votes
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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 ...
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  • 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,...
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  • 873
2 votes

What is the index fastq file (sample_I*.fastq.gz) generated when demultiplexing Illumina paired-end runs?

It took me a while to figure out that the "index" is the same thing as the "barcode" that says which sample each sequence is from on a multiplexed run. If your data is not demultiplexed (single <...
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  • 121
2 votes
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How to search a specific sequence in BAM files for 10X experiment

The bam file has bam tags which say what reads belong to what cells. https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/output/bam
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  • 1,662
2 votes
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10X Illumina demultiplexing sample sheet issue

So, we figured it out. If you delete everything until the [Data] tag in the sample sheet, leaving the [Data] tag, it starts ...
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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 ...
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  • 768
2 votes

Reading multiple raw files in Seurat

Seurat has a vignette specifically for combining multiple 10x libraries.
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2 votes
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Making Seurat object from a processed file

The function you need is CreateSeuratObject() and not Read10X() as you start from TPMs.
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  • 3,432
2 votes
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color by clusters and sampled in Seurat

Here is a solution that makes use of LabelClusters() from Seurat: ...
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  • 3,432
2 votes
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What does this Seurat argument mean

Checkout the first Seurat tutorial. To filter cells with >20% mitochondrial counts: ...
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  • 3,462
2 votes
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TCR-seq or scRNA-seq

The 3' gene expression protocol will capture TCR and BCR mRNAs but this may not be very helpful to you. As you already mentioned only the 3' end will be sequenced, which are the constant regions. With ...
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  • 768
2 votes
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Some definitions about RNA-seq

I am pretty sure that is read length. Both reads in a pair (2x) can be 50, 75, ... 250 bases long. And I am even more sure you will find this information on the webpage of the provider and if not, you ...
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  • 5,287
1 vote
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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: ...
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