Questions tagged [10x-genomics]

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12
votes
2answers
3k views

determining doublets in single-cell RNA-seq

Doublets are a known problem with scRNA-seq experiments, where 2 or more cells are sometimes captured instead. To determine their presence, there are studies that mix multiple species (such as human ...
9
votes
3answers
6k views

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

What is the index fastq file that comes with some Illumina sequencing datasets? (The samplename_I*.fastq.gz file.) For example, I recently received some 10X ...
6
votes
1answer
203 views

Can a customized GRCh38 .gtf file be used with any of the GRCh38 released patches?

I got a customized GRCh38.79 .gtf file (modified to have no MT genes) and I need to create a reference genome out of it (for 10xGenomics CellRanger pipeline). I suspect that the .79 part is the ...
5
votes
2answers
1k views

BAM to gene expression matrix (UMI counts per gene per cell),10X

I am trying to reproduce some results of a scRNASeq experiment. However I am new to the server-side aspect of such analyses and am very confused at the moment. The data provided by the authors of the ...
5
votes
3answers
7k views

Script to allow gene set enrichment analysis of 10x genomics data in R

I have 10x single cell RNA seq data. Which R package is best suited for analysis of the 10x data matrix. What is the script to prepare the data for downstream GSEA analysis. I have already processed ...
5
votes
1answer
649 views

Derive a GTF containing protein coding genes from a GTF file with Exons and CDS

Why I need a compatible file I’m trying to run velocyto with the R package to analyse RNA velocity (cell trajectories) with single cell RNASeq data. I have performed single cell analysis from 10x ...
5
votes
1answer
773 views

How to normalise scRNASeq data for differential expression analysis

I wish to perform differential expression analysis for cluster-specific gene expression in single-cell data (with a tool such as MAST or SCDE). I have data for 3 biological replicates. I performed ...
4
votes
2answers
2k views

What is cellranger doing in comparison to other methods?

I've recently started working with the 10X-Genomics platform with Illumina (MiSeq and HiSeq) for single-cell RNA-Seq. I've been recommended the "cellranger" (version 2.1.0) which I understand handles ...
4
votes
1answer
2k views

Raw vs Filtered in the output of cellranger count

After running cellranger count I got two relevant for further analysis folders: filtered_gene_bc_matrices and ...
4
votes
1answer
2k views

10x Genomics Chromium single-cell RNA-seq data analysis options?

Provide an overview of 10x data analysis packages. 10x provides Cell Ranger which prepares a count matrix from the bcl sequencer output files and other files (see bottom of page https://support....
4
votes
2answers
186 views

High percentage of poly A sequences in 10X chromium R2 read

I'm currently analyzing two samples of eosinophil cells isolated from mouse lung and the samples are of very different quality. According to the Cell Ranger summary 56% of the reads can be mapped to ...
3
votes
2answers
2k views

10X Illumina demultiplexing sample sheet issue

Also posted on biostars. I am trying to use cellranger or bcl2fastq to convert the .bcl ...
3
votes
2answers
2k views

Handling sample identity in aggregated 10x libraries?

cellranger aggr can combine multiple libraries (samples), and appends each barcode with an integer (e.g. AGACCATTGAGACTTA-1). The sample identity is not recorded in ...
3
votes
0answers
521 views

Low Fraction of usable antibody reads in CiteSeq

we performed a combined gene expression and CiteSeq experiment with the 10x VDJ kit and 20 conjugated antibodies and sequenced on hiseq. I used cellranger to process the sequencing output. The ...
2
votes
4answers
2k views

Low custom tdtomato gene content

I have a set of scRNA-seq samples expressing TdTomato, which has high content in microscope. I followed the 10x cellranger pipelines to finsh the work, my procedures are as follows: added TdTomato on ...
2
votes
1answer
763 views

Why do I have >10,000 cells in the 10X matrix produced by cellranger?

In my scRNA seq experiment, single-cell libraries were generated using the GemCode Single-Cell Instrument and Single Cell 3′ Library & Gel Bead Kit v2 and Chip Kit (10x Genomics) according to the ...
2
votes
1answer
1k views

Why are there more barcodes than GEMs in 10X chromium data?

Question: Why are there more barcodes than GEMs in 10X chromium data? Introduction I have several 10X genomics chromium libraries made with the de novo assembly/genome protocol. The 10X chromium ...
2
votes
2answers
244 views

Can I get the graph generated by cellranger

I’ve run the cellranger analysis pipeline on single cell RNASeq datasets. I can import the matrix and graph-based clusters into R. Doing this I can optimise the dimension reduction and plot cells with ...
1
vote
1answer
252 views

color by clusters and sampled in Seurat

I have a Seurat object I want to have both cluster numbers and coloured cells by sample names like this figure (from a Nature paper) I have tried group.by argument ...
1
vote
1answer
1k views

Reading multiple raw files in Seurat

I have multiple single cell samples to analyze and I'm following the instructions in Satija Lab's website. I want to merge all the count files from all the samples at once, and associate the metadata ...
1
vote
1answer
154 views

How to search a specific sequence in BAM files for 10X experiment

I have to search a specific sequence in a set of cells (> 1k cells) from a single-cell experiment done with 10X genomics. As input file I have a single bam file, and 24 fastqs, therefore each file ...
0
votes
2answers
61 views

Merging two dataframes in R

I have a file with cluster number of a scRNA-seq and corresponding annotated cell type like ...
0
votes
1answer
156 views

Single-cell sequencing dataset has too many barcodes

I am analyzing a single-cell sequencing dataset from the website 10xgenomics, with 2000 cells. It is a BAM file and I am trying to obtain the individual cells per sample. I used the command ...
0
votes
1answer
62 views

TCR-seq or scRNA-seq

I need idea, intuition, suggestion please I have 10X scRNA-seq of PBMC (multiome's 3' poly-A capture), can I capture ...
0
votes
2answers
76 views

Help with Seurat QC ambiguity

I have four PBMC samples from 10X scRNA-seq ...
0
votes
1answer
40 views

What does this Seurat argument mean

I have extensively read about percent mito in Seurat but I got more and more confused Let's say we want to keep cells with ...
0
votes
1answer
62 views

Avoiding the warning 'The following features were omitted as they were not found in the scale.data slot for the SCT assay' in Seurat

I have a Seurat object I want to plot a list of genes but I got a warning saying most of genes removed I have tried with both my genes of interest and all variable genes but the results is very ...
0
votes
1answer
110 views

Seurat Dimplot with different clustering IDs

In Seurat metadata I have assigned cells to some cell types with different resolutions I have added the cluster identities to the object via Idents: ...
0
votes
2answers
98 views

10X scRNAseq: Sample mix-up

The student who was working on scRNA seq of KO and WT lines has made a mistake and he mixed both lines and generate the final sequencing data. Now, we are having gene expression data but don't know ...
0
votes
2answers
80 views

cellranger mkfastq with full path to --id flag

I have always used cellranger mkfastq to demultiplex 10x genomics runs manually, though recently the commands to do so have been incorporated into a script that ...
0
votes
1answer
2k views

10X cellranger count, [error] The chemistry was unable to be automatically determined

While running cellranger count using the following .slurm file: ...
0
votes
0answers
36 views

Error in colSums(cts[[i]]) : 'x' must be an array of at least two dimensions

I'm getting an error when creating the counts from a single cell experiment.I'm identifying shared barcodes as well as lowUMI barcodes with the follow code: ...
0
votes
0answers
47 views

Making a box plot of the proportion of cells in each cell types in two groups

I have number of cells in three cell types T, B, M in a Seurat object For two groups of patients, cancers and controls like ...
0
votes
2answers
255 views

Adding metadata to Seurat object

I have a Seurat object of 8 patients. I want to add metadata to that so that I have origin of each cell. At the moment UMAP just shows a bunch of cells while I want to color clusters by sample This is ...
0
votes
2answers
157 views

10X single cell sequencing issue

Can you please help me in a technical issue? We sent some samples to single cell sequencing The company says Many of the reads were not assigned to cell-associated barcodes. This could be caused by ...
0
votes
0answers
39 views

which marker identifications method is recommended when single-nuclei RNA seq datasets are integrated across regions

I'm analyzing Sn-RNA seq datasets which include different brain regions and spinal cord in patients and normal controls. I would have different comparison within and between individuals. I've got a ...
-1
votes
1answer
105 views

Seurat heatmap for two conditions

I have a Seurat object of four cancers and four controls ...
-1
votes
1answer
58 views

Filtering cells from values in metadata

I metadata slot of Seurat object I have mapping score of each cell to a reference PBMC data like ...
-1
votes
1answer
152 views

Cellranger gives error

I am trying to run cellranger but I get fastq permission denied error ...
-1
votes
1answer
41 views

Problem with Seurat reference mapping

I have 10X scRNA-seq multiome's 3' poly-A capture (scRNA-seq+ATAC-seq) from PBMC Using Seuratreference mapping, I mapped my scRNA-seq part on reference PBMC (for ...
-1
votes
1answer
76 views

sctransform - mitochondrial expression filtering / transformation

I have a Seurat object which has a high expression of mitochondrial genes ...
-2
votes
2answers
81 views

Removing cells zero for a gene from a scRNA-seq data

I have a big single-cell RNA seq data ...
-3
votes
1answer
92 views

Getting conventional gene symbol for Seurat [duplicate]

I have a Seurat object made by human single cells I am mapping some genes on that but no sign of expression When I GOOGLE for those genes I see the genes have different names How I know Seurat uses ...
-5
votes
1answer
80 views

Making Seurat object from a processed file [closed]

I have downloaded log2(TPM/10+1) values of 11,548 genes and 9609 cells from ...