Questions tagged [scrnaseq]

Use this tag for questions related to single-cell RNA-seq.

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1
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2answers
384 views

SC-RNA seq percent.mito

Is that possible that the percent.mito level is zero? I have done vlnplot and it shows that its percent.mito level is zero. Does that mean my mitochondrial gene didn't detect? It is violin plot. And ...
2
votes
1answer
73 views

RCurl issue when installing SCnorm

I am working on the Rivanna cluster that is of SLURM-type. Because of that I do not have write permissions and need to install ...
2
votes
2answers
3k views

Interpretation of the violin plots from sc-RNA-seq

I'm confused about the meaning of the black dots and the red shape in the violin plots from the seurat tutorial:
-1
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1answer
1k views

Changing the colour of each cell in tSNE plot

I have plotted a tSNE plot of my 1643 cells from 9 time points by seurat like below as 9 clusters. But, you know I should not expected each cluster of cells contains only cells from one distinct time ...
2
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1answer
2k views

How I can reproduce this heat map

I have single cell RNA-seq on 8 time points. I have clustered each time point to 2-3 clusters by seurat. I also have a list of differentially expressed genes between all my 1562 cells in 8 time points....
3
votes
2answers
154 views

Hemoglobin subunits genes in scRNA-seq

In one scRNA-seq sample I encountered the genes: Hbb-bs, Hba-a1 and Hba-a2. These genes appear on top of the list of the highest expressed genes but the 75% percentile of cells have very low counts. ...
-3
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1answer
594 views

Installing a R package from git

I am trying to install STEMNET R package but a permanent error stops me, what can I do please? ...
1
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0answers
99 views

Variability in genes across platforms

I have a matrix of gene expression for single cell (cells in column and genes in row) in 9 time points with about to 200 cells for each time point. I have also a matrix of gene expression in bulk RNA-...
5
votes
1answer
747 views

No counts for added gene in cellranger (scRNA-seq)

I have a set of scRNA-seq samples enriched with FACS for cells expressing a specific gene reporter (TdTomato). In particular the gene I want to report has positive counts in the resulting matrix for ...
1
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0answers
80 views

Ordering the cells by the expression of specific genes

I have 209 cells. I want to show the expression of two genes by ordering my cells. Imagine something like a plot on which y axis shows the read counts range from 0 to 10000 and x axis shows the number ...
2
votes
2answers
2k views

Which data is being used for violin plot?

Sorry I just got totally confused conceptually. If this is my raw count data Likely, Seurat divides each value by sum of the column afterward times by 10000. which gives so ...
4
votes
1answer
2k views

Is it possible to identify cells that are expressing two or more genes in Seurat?

I'm interested in looking into cells that are positive for two (or in some cases more) genes. I know I have some double positive just by looking at the FeaturePlot of those genes, but now I'm trying ...
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 ...
7
votes
1answer
105 views

Network comparison of single cells (from sequencing data)

What methods can we use to compare networks (PPI, gene regulatory etc) created from single-cell gene expression (or proteomics) data? There are methods that construct networks by comparing two ...
5
votes
4answers
434 views

Specific cell type identification in Single Cell Sequencing

In order to define which cell is of which type we need to identify a set of rules, for instance neurons should express one of the following: Thy1, ...
3
votes
1answer
3k views

RunUMAP in Seurat not working: module 'umap' has no attribute 'UMAP'

I am trying to run UMAP in the following way: ...
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 ...
5
votes
4answers
2k views

Pathway level analysis of single-cell gene expression

I'm looking for single-cell specific methods to construct (using gene expression data) new features that express pathway "level" or "activity", and then use these for clustering cells. One example ...
2
votes
1answer
761 views

What process and input data is required for a cellranger reference transcriptome?

I'm analysing single-cell RNA-Seq data using the 10X Genomics cellranger platform. While they provide reference data for Mouse and Humans, other species require a ...
0
votes
1answer
206 views

Demultiplexing and preprocessing for custom single-nucleus Drop-seq data

I am trying to reproduce the preprocessing of paired-end sequencing reads in Lake et al. (ref. 1). First, paired-end reads were filtered out if read 1 had more than four non-T bases in the last ten ...
13
votes
2answers
2k views

How to decide number of neighbors and resolution for Louvain clustering

I am using Louvain clustering (1,2) to cluster cells in scRNAseq data, as implemented by scanpy. One of the parameter required for this kind of clustering is the number of neighbors used to construct ...
5
votes
3answers
1k views

Regressing out unwanted sources of variation in single cell RNA-seq data

I have a dataset of single cell count data and I want to regress out the variation caused by the number of UMI's and the percentage of mitochondrial genes. I know that count data is discrete data, ...
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 ...
6
votes
1answer
2k views

What's a template switching site?

Reading Islam et al. (2011): From each transcript, a single read is obtained, corresponding to a template-switching site located preferentially at the 59 end of the mRNA. By reading this page I ...
2
votes
1answer
2k views

What is a poly(T) primer anchor sequence?

I am reading Tang et al. (2009). In the method description it is stated that: A single cell is manually picked under a microscope and lysed. Then mRNAs are reverse-transcribed into cDNAs using a ...
1
vote
1answer
51 views

Is it possible to use spike-ins with droplet technologies?

By reading Bacher & Kendziorski (2016), I found the following statement: recent droplet technologies are not yet able to accommodate spike-ins What's the reason why current droplet ...
5
votes
1answer
387 views

Which tools for differential expression analysis in scRNA-Seq?

I am starting to run analysis for differential expression in scRNA-Seq. Which tools are available for this kind of analysis? Can tools for bulk RNA-Seq like DESeq be used for scRNA-Seq?
6
votes
1answer
172 views

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

I have a number of single-cell projects processed with cellranger from 10x Genomics. The pipeline produces a number of handy metrics that are summarized for each Illumina sample in web page. These ...
5
votes
3answers
3k views

Is it possible to merge scRNAseq data from experiments with different design?

I have 4 different single-cell RNAseq experiments, each one representing a different sample of cell type population. I'd like to merge them to a single dataset. However, different cell types are ...
4
votes
1answer
134 views

Why is ribosomal RNA difficult to remove even with Poly(A) selection?

In this answer (actually in a comment), it is stated that: As you've noticed from your own analysis, the ribosomal genes have quite variable expression across cells. They're expressed everywhere, ...
2
votes
2answers
2k views

ComBat for batch effects removal on scRNA-seq data

Is it possible to use sva's ComBat for batch effects removal on scRNA-seq data? Is there any difference between RNA-seq and scRNA-seq data which doesn't allow to use ComBat on single-cell data? (I am ...
3
votes
2answers
116 views

Detect transcript isoform abundance for a specific gene in scRNA-seq

I want to detect the count of isoform transcripts for a specific gene in scRNA-seq data. Data is coming from cells of Mus Musculus. For transcript isoforms I mean the different alternatives provided ...
0
votes
1answer
44 views

Looper: can't specify the individual path for one pipeline run

I have multiple samples that I need to run one pipeline on. I am using Looper: https://github.com/pepkit/looper I have a real trouble specifying a separate location for each pipeline output files. ...
8
votes
2answers
7k views

Are mitochondrial genes to exclude in scRNA-seq such as ribosomal genes?

In this answer, it is stated that ribosomal genes should be excluded prior to normalization in scRNA-seq as contaminants. Do mitochondrial genes have to be excluded as well? I plotted the top 50 ...
1
vote
1answer
37 views

Looper: missing argument error

While trying to run looper I am getting the following error: Error (missing attribute): 'sandro_rna_seq' requires sample attribute '/scratch/nv4e/sandro_looper/pipeline/jyvo_experiment-metadata....
1
vote
1answer
20 views

Looper: Duplicate sample name error

I am using Looper. While running looper run project_config.yaml I am getting duplicate sample name error. I have two ...
0
votes
1answer
87 views

What is the detection limit of protocol in scRNA-seq

In Shapiro et al., when discussing about loss of molecules as source of error in single-cell sequencing, it is written that: Another source of error is losses, which can be severe. The detection ...
5
votes
2answers
248 views

Assign cell types to groups of cells based on their gene expression profiles

I have large filtered, normalized dataset of scRNA-seq data of C.Elegans species. Rows are genes (10 000), columns are cells (66 ...
6
votes
3answers
496 views

Improve scRNA-seq dataset for further analysis

I got a dataset from C.Elegans scRNA-seq paper: GSM2599701_Gene.count.matrix.celegans.cell.Rdata in ...
6
votes
1answer
640 views

Drawbacks of upper quartile normalization for scRNA-seq data

I would like to use Upper Quartile normalization for scRNA-seq data defined as: The upperquartile (UQ) was proposed by (Bullard et al. 2010). Here each column is divided by the 75% quantile of the ...
1
vote
1answer
59 views

Error in .checkedCall : subset indices out of range [closed]

After normalization the way it is described here: https://bioconductor.org/packages/3.7/bioc/vignettes/scran/inst/doc/scran.html Using quickCluster() function ...
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 ...
6
votes
1answer
989 views

Order of batch effects removal, data imputation and library size normalization in scRNA-seq data

I am preprocessing scRNA-seq data. What is the best practice in use to run both ComBat for batch effects removal, data imputation (to mitigate dropout) and library size normalization? I thought that ...
3
votes
2answers
3k views

What are RIKEN genes?

I am using data from the Tabula Muris Consortium. These are gene counts from scRNA-seq of mouse cells. There are some specific genes with name ending with suffix 'Rik' (e.g. 0610005C13Rik or ...
1
vote
2answers
4k views

How to filter ribosomal RNA from scRNA-seq data

I want to filter out ribosomal RNA from scRNA-seq data (downloaded from here). Is there a list of known ribosomal RNA? The only solution I found is SortMeRNA, however it works with raw sequencing ...
4
votes
1answer
904 views

Inspection of gene expression in scRNA-seq data

I am running the data preprocessing pipeline for scRNA-seq data presented here. 3.8.6.1 Gene expression In addition to removing cells with poor quality, it is usually a good idea to exclude genes ...
6
votes
2answers
4k views

What are doublets in single cell RNA-seq data?

I am reading The Tabula Muris Consortium et al. (pp). In some organs, cells with more than 2 million reads were also excluded as a conservative measure to avoid doublets. How exactly is a “doublet”...
6
votes
1answer
212 views

Scaling by linear regression against the number of reads

I am trying to build the preprocessing pipeline presented in The Tabula Muris Consortium et al. (pp). It is a pipeline to preprocess single-cell sequencing data. There is one step that is not clear: ...
4
votes
3answers
881 views

How to compare transcriptomic profiles of two cell types (single cell RNA-seq)?

I found this interesting Single RNA-seq data set in GEO, but I am not sure how to analyze it appropriately. They have deposited transcriptomic profiles of human and mouse pancreatic islets (...
19
votes
3answers
2k views

What is the actual cause of excessive zeroes in single cell RNA-seq data? Is it PCR?

First, sorry if I am missing something basic - I am a programmer recently turned bioinformatician so I still don't know a lot of stuff. This is a cross post with a Biostars question hope that's not ...