Questions tagged [scrnaseq]

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

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21 votes
3 answers
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 ...
13 votes
2 answers
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 ...
  • 1,733
13 votes
1 answer
227 views

scRNA-seq multi-dataset integration for small datasets

There have been a few methods proposed for integration (or batch correction) of scRNA-seq datasets, such as Seurat CCA, MNN Correct, Scanorama, and Harmony. The concern is generally about the maximum ...
  • 2,099
12 votes
2 answers
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 ...
  • 2,099
8 votes
2 answers
8k 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 ...
  • 1,733
7 votes
1 answer
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 ...
  • 1,733
7 votes
1 answer
137 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 ...
  • 2,584
7 votes
1 answer
199 views

Pseudo-temporal ordering in heterogeneous populations

What's the most widely accepted tool for doing pseudo-temporal ordering from scRNAseq data? Also is there away to separate differential expression that occurs based on "cell identity" or maybe more ...
7 votes
1 answer
72 views

Simulating 3' end tag-based scRNA-seq reads

Are there any tools that will simulate 3' end tag-based single-cell RNA-seq reads? For example, Drop-seq, 10X Chromium, ...
  • 561
6 votes
2 answers
5k 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”...
  • 1,733
6 votes
3 answers
513 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
2 answers
173 views

validating identified sub-populations of cells in scRNA-seq

In the analyses of single-cell RNA-seq data there are different unsupervised approaches to identify putative subpopulations (e.g. as available with Suerat or SCDE packages). Is there a good way of ...
  • 153
6 votes
2 answers
228 views

How I can test my hypothesises computationally

I have single cell RNA-seq data on about 2000 cells in 9 time point. I have clustered my cells in each time point by Seurat. I am seeing in some time points I have 3 clusters while in another time ...
  • 1,707
6 votes
1 answer
736 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 ...
  • 1,733
6 votes
1 answer
1k 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 ...
  • 1,733
6 votes
1 answer
225 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: ...
  • 1,733
6 votes
2 answers
303 views

Why are TPMs per 10k or 100k in many scRNA-seq studies?

I noticed that many scRNA-seq papers normalize TPMs to 10k or 100k as opposed to 1M (as the abbreviation defines them). It doesn't really matter since you are just moving the decimal point, so why ...
  • 2,099
6 votes
1 answer
209 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 ...
  • 19.3k
5 votes
1 answer
868 views

PCA vs tSNE in single cell RNA-seq

What makes tSNE being the preferred dimensional reduction for visualization in single cell RNA-seq over PCA? I am aware that tSNE works better at showing local structures and fails to capture global ...
  • 1,022
5 votes
4 answers
527 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, ...
5 votes
3 answers
8k 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 ...
  • 51
5 votes
3 answers
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 ...
  • 1,733
5 votes
1 answer
892 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 ...
  • 873
5 votes
1 answer
83 views

What is and how to detect a dephased read

In the methods of this paper, the authors say: To simplify analysis, we first removed any dephased reads in our library (last 6 bases of read did not match the expected sequence). I have read this ...
  • 1,733
5 votes
3 answers
2k 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, ...
  • 200
5 votes
2 answers
287 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 ...
5 votes
1 answer
901 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 ...
  • 873
5 votes
4 answers
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 ...
  • 2,584
5 votes
1 answer
834 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 ...
  • 1,733
5 votes
1 answer
408 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?
  • 1,733
4 votes
1 answer
3k 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
1 answer
161 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, ...
  • 1,733
4 votes
2 answers
64 views

Arrange ggplot Figure for scRNA-seq data

I have generated a ggplot for 8 single-cell libraries, with the purpose of visualizing the tSNE facet plot by sample, colored by cell type -- with percentages. The best I could get to is this - ...
  • 51
4 votes
1 answer
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 ...
4 votes
3 answers
988 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 (...
  • 157
4 votes
1 answer
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....
  • 2,584
4 votes
1 answer
1k views

Understanding PCHeatmap outputs

I am currently trying to understand the purpose of these PCHeatmaps - part of the seurat package in R: All the online documentation I have searched for has only ...
  • 806
4 votes
1 answer
221 views

Problem: "Pair-end" reads scRNA seq data (Drop-seq)

In case of Drop-seq, we have paired end data. Read 1: Cell code + UMI (unique molecule identifier) Read 2: The transcript information But I have a problem/doubt with the sample I am working on. ...
's user avatar
4 votes
1 answer
1k 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 ...
  • 1,733
4 votes
2 answers
272 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 ...
  • 818
4 votes
0 answers
701 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 ...
  • 41
3 votes
2 answers
2k views

Using Seurat to compare mutant vs.wt

I am interested in using Seurat to compare wild type vs Mutant. I don't know how to use the package. How can I test whether mutant mice, that have deleted gene, cluster together?
  • 423
3 votes
2 answers
130 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 ...
  • 1,733
3 votes
2 answers
550 views

PCA plot shows big difference but not many differentially expressed genes are found

I got a PCA plot of bulk RNA-seq experiment that looks the following way: It was generated by the following code: ...
3 votes
2 answers
986 views

Changing a wide range of colours to a limited gradient

I returned a FeaturePlot from Seurat to ggplot. My plot has a weird range of colours as below I produced this plot by this code ...
  • 1,707
3 votes
3 answers
3k views

How do I pull singe cell RNA sequencing data from GEO database?

I am new to R and computational biology. I am trying to look through a published data set to check for gene expression for my own project. I am having trouble finding materials to teach me how to ...
3 votes
2 answers
304 views

Differential gene expression bias due to effect of an individual sample

I am analysing a human single cell RNA seq experiment, where we have 4 groups, four samples each. Data has been analysed using Seurat, with the canonical workflow. I have tried DE using various ...
  • 31
3 votes
1 answer
58 views

public multi-modal single-cell data

There is a scRNAseq Bioconductor package with a few different example scRNA-seq datasets. Are there any R packages that offer multiple modalities of single-cell data? For example, hashtags or ADTs or ...
  • 2,099
3 votes
1 answer
2k views

How to set the position of groups in a Seurat object on a FeatureHeatmap plot

I am analysing singe cell sequence data and I have followed this tutorial, https://satijalab.org/seurat/pbmc3k_tutorial.html to perform QC and various differential analyses using the Seurat package on ...
  • 516
3 votes
2 answers
4k 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 ...
  • 1,733

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