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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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 ...
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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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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5
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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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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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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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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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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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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4
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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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
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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3
votes
Accepted
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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3
votes
What is cellranger doing in comparison to other methods?
The best approach would be to contact 10x Genomics support: [email protected] . 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
Accepted
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
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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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 ...
- 1,139
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
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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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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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 ...
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2
votes
Accepted
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 ...
- 2,538
2
votes
Can a customized GRCh38 .gtf file be used with any of the GRCh38 released patches?
You can see the exact assembly by looking in the README file in Ensembl's genome download page. ftp://ftp.ensembl.org/pub/release-89/fasta/homo_sapiens/dna/README
As you can see the current assembly ...
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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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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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2
votes
Adding metadata to Seurat object
Answer from @user438383, converted from comment:
Did you try the AddMetaData function? This post may also be useful.
...
2
votes
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Adding metadata to Seurat object
If you have single-dimension per-cell metadata, and it's arranged identically to the cell order in the Seurat object, I find it easier to use the double bracket notation to add metadata to a Seurat ...
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