Tag: Seurat

scrnaseq – Integrating scRNA-seq data using raw data

I believe when you say alignment, you mean aligning reads to a genome (sometimes to transcriptome) and count these to get count matrices. In the aforementioned paper, however, what is meant is “bringing different data sets to a level where they can be compared/integrated/…”. Basically scRNA-seq data are heavily prone…

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Combining multiple 10x scRNAseq datasets

Hi everyone, Wondering if someone can provide me with some guidance. I have previously sequenced 4 skin cancers using 10X chemistries and I would like to combine them into one dataset. My research question is to look at cancer stem cell populations, so I will need sensitivity. I have done…

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SCHNAPPs – Single Cell sHiNy APPlication(s)

Abstract : Single-cell RNA-sequencing (scRNAseq) experiments are becoming a standard tool for bench-scientists to explore the cellular diversity present in all tissues. Data produced by scRNAseq is technically complex and requires analytical workflows that are an active field of bioinformatics research, whereas a wealth of biological background knowledge is needed…

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Obs_name

Obs_name 0 Can someone please help explain what the Obs_name represents? I exported the anndata matrix (h5ad) and attempted to transform it to the Seurat object using something similar to the following: exprs <- t(adata$X) colnames(exprs) <- adata$obs_names$to_list() rownames(exprs) <- adata$var_names$to_list() # Create the Seurat object seurat <- CreateSeuratObject(exprs) #…

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Single cell RNAseq data analysis

Github repository  02-04 February 2022  SciLifeLab Solna, Tomtebodavägen 23b, Stockholm, Sweden This workshop will introduce the best practice bioinformatics methods for processing and analyses of single cell RNA-seq data via a series of online lectures and computer practicals. The total course duration is 45 hours, including the online lectures (15 hours)…

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Monocle3 differential expression failed when active.assay is not “RNA”

after run estimate_size_factors, data with active.assay = ‘integrated’ works too, but no deg in the result. > [email protected] = ‘integrated’ > cds_raw <- as.cell_data_set(seurat_object) Warning: Monocle 3 trajectories require cluster partitions, which Seurat does not calculate. Please run ‘cluster_cells’ on your cell_data_set object > cds <- cluster_cells(cds_raw) > pr_graph_test_res <-…

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Generation of Centered Log-RatioCentered log-ratio (CLR) Normalized Antibody-Derived TagAntibody-derived tag (ADT) Counts from Large Single-Cell Sequencing Datasets

Recent developments in single-cell analysis has provided the ability to assay >50 surface-level proteins by combining oligo-conjugated antibodies with sequencing technology. These methods, such as CITE-seq and REAP-seq, have added another modality  …more Recent developments in single-cell analysis has provided the ability to assay >50 surface-level proteins by combining oligo-conjugated…

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About ‘Estimated Number of cells’ in snRNA-seq

About ‘Estimated Number of cells’ in snRNA-seq 0 Hi all, I am analyzing single nucleus RNA-seq data using Seurat. And I have total four group and 24 samples (Brain region A Control & case and Brain region B Control & case; each n=6). I wonder what is the appropriate range…

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Telling apart control and treatment groups in Seurat Visualizations

Hi, I saw on one of the Seurat data visualization tutorials that if you have a dataset you generated from an experiment, you can split a dataset into the control and the treatment. For example, if you have the following dataset where the metadata is clearly split into groups, you…

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spatial 10X visum analysis

spatial 10X visum analysis 0 I have data of spatial 10X visium transcriptome. I are working on different conditions with two replicate each (A1, A2, B1,B2) . All samples were submitted in spaceranger pipeline along with “image” data . Now I have output from spaceranger ( feature matrix and so…

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conversion of ensembl to gene_symbol in seurat

I am working on spatial 10X visum data. Space ranger was used for the identification of filtered_feature_bc_matrix.h5 matrix and given as input in Seurat R package. For Quality filteration, I would like to have gene symbol but my features consist of ENSEMBL ids. How can I change the ENSEMBL to…

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RunPCA function – RDocumentation

RunPCA(object, …) # S3 method for default RunPCA( object, assay = NULL, npcs = 50, rev.pca = FALSE, weight.by.var = TRUE, verbose = TRUE, ndims.print = 1:5, nfeatures.print = 30, reduction.key = “PC_”, seed.use = 42, approx = TRUE, … ) # S3 method for Assay RunPCA( object, assay =…

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GitHub – satijalab/seurat at develop

GitHub – satijalab/seurat at develop You can’t perform that action at this time. You signed in with another tab or window. Reload to refresh your session. You signed out in another tab or window. Reload to refresh your session. Read more here: Source link

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NA values for mitochondrial gene percentage

NA values for mitochondrial gene percentage 0 I am running Seurat on publicly available dataset of ~400k cells. More than 80% of the cells are returned as NA when I use percentageFeatureSet(object, pattern = “^MT-“). How should I interpret these result? Does this mean the 80% of cells are of…

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Labeling Clusters in ggplot

Labeling Clusters in ggplot 1 I have a data matrix I extracted from seurat and I want to plot the tSNE plot by using ggplot. I don’t know how to label the clusters on the plot with 0..15. Help is appreciated. ggplot single cell • 1.3k views This is what…

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How do I access inflection points in Seurat object?

How do I access inflection points in Seurat object? 0 I ran the following code below to calculate inflection points for the UMI counts for my single cell data using Seurat. seurat_obj <- CalculateBarcodeInflections(seurat_obj,barcode.column = “nCount_RNA”,group.column = “orig.ident”,threshold.low = NULL,threshold.high = NULL) I want to obtain the inflection points so…

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Correct usage of FindConservedMarkers() in Seurat

Correct usage of FindConservedMarkers() in Seurat 0 Dear all, I have a Seurat object of a certain cell type with a UMAP of 7 clusters. I also have information about the sample’s origin (primary tumor/metastatic) in my metadata. Looking at the UMAP I can clearly see that clusters 1 and…

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How to use “SingleR” on the marker genes from `FindAllMarkers` for each cluster?

How to use “SingleR” on the marker genes from `FindAllMarkers` for each cluster? 0 Hi, I tried to use SingleR to identify cell types for clusters. I have the table of results from FindAllMakers of Seurat package. I know that I can use: SingleR(GetAssayData(seurat.object, assay = assay, slot = “data”),…

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Seurat Integration Input Question

Seurat Integration Input Question 0 I have two datasets I am trying to run a Seurat integration on. I am following the vignette on the Seurat website. One of my dataset has a bunch of Nan row names (the genes columns), and if I drop them before the integration I…

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Which trajectory method is better !?

Which trajectory method is better !? 2 Hello I was engaged with a basic problem. I have dataset consist ~2000 cells and composed 8-9 clusters using Seurat package, then I transfer Seurat object to the Monocle. I tried monocle2 and monocle3. The problem is, how to make the trajectory ?…

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PC p values in JackStrawPlot in Seurat are not in order

PC p values in JackStrawPlot in Seurat are not in order 0 Hi, I got the Seurat object, then I did: pbmc <- JackStraw(pbmc, num.replicate = 100) pbmc <- ScoreJackStraw(pbmc, dims = 1:20) And when I did: JackStrawPlot(pbmc, dims = 1:15) The plot looks like this: As you can see,…

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Can SingleR be reused to identify cell types after subsetting the original Seurat Object

Can SingleR be reused to identify cell types after subsetting the original Seurat Object 0 Hi, I got a Seurat object and applied SingleR to identify cell types. Then subset the epithelial cell then redid the clustering on this cell type only. So now I got some new clusters. My…

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Are Variable Features ranked in seurat?

Are Variable Features ranked in seurat? 0 Dear all, does anybody know if variable features obtained by FindVariableFeatures(object, selection.method = “vst”, nfeatures = 2000) in Seurat are ranked? If I look at the top variable genes in var_gene var_genes <- object[[“integrated”]]@var.features I see e.g. : “Ccl19” “Cxcl13” “Cxcl2” “Mt1” “Ube2c”…

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Ways to analyze treatment changes in scRNAseq

Ways to analyze treatment changes in scRNAseq 0 Hi, I am working with single cell RNA seq to determine gene expression changes in individual cells using two conditions: untreated and treated. I have merged the untreated and treated files into a single seurat object to generate featureplots of genes that…

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What is the difference between filtering cells before creating Seurat object and after that?

What is the difference between filtering cells before creating Seurat object and after that? 0 I tried filter cells that have unique feature counts over 5000 or less than 350 and > 2% mitochondrial counts before creating Seurat object, and I also tried to do this step after creating Seurat…

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How do I load a GEO dataset into Seurat?

How do I load a GEO dataset into Seurat? 1 I’m very new to single cell clustering, but have been able to get results from Seurat using sample datasets from 10x Genomics and also some datasets that were in H5 format. I’m looking for scRNA-seq datasets that are specific to…

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WNN in Seurat

Dear all, I am trying to follow the WNN vignette here satijalab.org/seurat/articles/weighted_nearest_neighbor_analysis.html After the steps below, I would like to annotate my clusters, hence I need to know the markers which best represent each cluster. pbmc <- FindMultiModalNeighbors(pbmc, reduction.list = list(“pca”, “lsi”), dims.list = list(1:50, 2:50)) pbmc <- RunUMAP(pbmc, nn.name

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Trouble running Cyclum for my scRNA-seq analysis

I have been analyzing some mouse T cell scRNA-seq data for a few months now using mostly the Seurat pipeline run with default parameters, and I have noticed that regressing out the ‘S.Score’ and ‘G2M.Score’ obtained from default Seurat::CellCycleScoring seems to be insufficient to remove (seemingly large) variation originating from…

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Searching expression for one particular gene in Seurat object issue (Seurat, R)

Searching expression for one particular gene in Seurat object issue (Seurat, R) 0 I don’t know if it’s normal but when I filter my seurat object by a particular gene expression, I have the following results: My gene expression quantiles look like this: `0% 25% 50% 75% 100% : 0.0…

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subsetting out cells from seurat object based on expression of 1 gene

subsetting out cells from seurat object based on expression of 1 gene 1 I have a seurat object, with raw counts stored in the RNA assay at object@assays[[“RNA”]]@counts Lets say that the count matrix is simple and looks like this, where letters are genes and numbers are cells: [1] [2]…

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SCENIC-openloom not working

SCENIC-openloom not working 0 I have been trying to open a loom file which was produced by converting seurat object to loom.Using the following command: loom <- open_loom(loomPath) It is showing the following error : Error in H5File.open(filename, mode, file_create_pl, file_access_pl) : It is not possible to open the file….

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How to count gene expression level in R?

How to count gene expression level in R? 1 Hi! Could anyone suggest some reasonable quick way (library or tutorial) to get table with gene expression level with data listed below? .tsv with barcodes .tsv with genes and .mtx matrix I am new in this and I will appreciate any…

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which matrix should NMF use in single cell RNA seq data to find diferential gene program?

which matrix should NMF use in single cell RNA seq data to find diferential gene program? 2 Hi there, I’m new to scRNA-seq(use the seurat pipeline to analysis) and nmf. Recently, I’m going to do nmf in the scRNA-seq to find the diferent programs(like markers for some cells). But I…

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Cell cycle genes for D. melanogaster and C. elegans (for scRNA-seq analysis)

Cell cycle genes for D. melanogaster and C. elegans (for scRNA-seq analysis) 0 Hello all, It’s pretty common to do cell cycle correction when analyzing scRNA-seq data, especially for some tasks (e.g. trajectory inference). In Seurat, there’s a built-in list of gene names (cc.genes or cc.genes.updated.2019) that define S and…

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Differential splicing/transcript usage/isoform analysis of iCell8 (SMART-seq v2) full transcriptome single cell data in conjunction with Seurat

Differential splicing/transcript usage/isoform analysis of iCell8 (SMART-seq v2) full transcriptome single cell data in conjunction with Seurat 0 Dear all, I am currently analyzing single-cell data gathered using the iCell8 platform (Takara) that captures full transcripts using the SMART-seq v2 chemistry. The official pipeline by Takara (CogentAP) uses STAR for…

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Honeycomb Biotechnologies hiring Bioinformatics Scientist, Single-Cell Genomics in Waltham, Massachusetts, United States

Company Description:  Honeycomb Biotechnologies is an early-stage company focused on making scalable solutions for storage and single cell genomic analysis of precious clinical samples. We enable translation of the rich biological information encoded in clinical specimens into high-resolution digital information, which can be queried and analyzed with sequencing. The resulting…

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Change location of ‘fragments’ in multiome Seurat file

Change location of ‘fragments’ in multiome Seurat file 1 Hello, I have a multiome dataset of RNA +ATAC seq that was given to me as an analysed .RData file by a collaborator. This is already integrated and fully analysed. When I attempt to visualise something in the multiome Seurat object…

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How to export count data as .csv file in Seurat

How to export count data as .csv file in Seurat 1 Hi, I’m trying to export the log normalized count data from Seurat in to a .csv file and get the following error: library (Seurat) fib.data <- subset(onlyharmonfib, subset = nFeature_RNA > 200 & nFeature_RNA < 2500 & percent.mito <5)…

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Best compression for single cell RNA-seq object

Best compression for single cell RNA-seq object 0 I generated a scRNA-seq object (counts, PCA, UMAP embeddings, DEGs etc.) in Scanpy or Seurat. What is the best data structure to store this in to reduce the size of the object? I’m considering H5AD (scanpy/anndata), RDS or H5Seurat (Seurat), or Loom…

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WNN (Seurat v4) vs. totalVI (scvi-tools) for CITE-seq data

WNN (Seurat v4) vs. totalVI (scvi-tools) for CITE-seq data 0 I want to build a UMAP from CITE-seq data with a joint embedding of the scRNA-seq and protein ab data. What’s the ‘best’ method in terms of representing the most accurate embedding? In the totalVI paper, they say totalVI >…

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Strategies to learn about a gene of interest from single-cell RNA-seq data

Strategies to learn about a gene of interest from single-cell RNA-seq data 0 Using a large public single-cell RNA-seq dataset from brain where cells are already segregated by brain region, cell type, marker gene cluster, etc. I am looking to do exploratory analyses to learn whatever I can about a…

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How to transform the deg gene list from seurat to a gene list input to clusterProfiler compareCluster ?

Sorry for lateness, I wanted to do something similar. This is what I did for reference: Using a Seurat generated gene list for input into ClusterProfiler to see the GO or KEGG terms per cluster. I’ll keep the meat and potatoes of the Seurat vignette in this tutorial: library(dplyr) library(Seurat)…

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VisCello Shiny app code for hosting snRNA-seq data

I am trying to set up VisCello shiny app for hosting some of our single cell data, analyzed in Seurat: github.com/qinzhu/VisCello I am using the following code though running into a problem, the app launches however I dont see the umaps and when I click over to differential expression the…

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spots not filling the whole tissue image

Issue with Seurat SpatialPlot: spots not filling the whole tissue image 0 In Seurat, SpatialPlot generates a plot with an enlarged/expanded image of tissue section as compared to the original spot image. This seems to happen on the relatively small image with a number of spots around 500. I ‘d…

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Seurat re-clustering a cell subset but cell identity numbers are not completely showing up

Seurat re-clustering a cell subset but cell identity numbers are not completely showing up 0 Hi, After running T-SNE plot on dataset, I tried to extract a specific cell type then do reclustering. But I found that the some of the cell identity IDs are skipped in the reclustered T-SNE…

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UMAP of TRA/B

Hello, I have output from a single cell sequencing run that has both the VDJ and gene expression data. For the same cells, we also used a hybrid capture approach to sequence the TCR sequences. I have compared the TCR sequences across the two approaches and I have found a…

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How to display TCR data in tsne space via seurat object

How to display TCR data in tsne space via seurat object 1 Hi Guys, I am trying to work out how I can display by VDJ usage within my tsne plot for some 10x data. I added everything to the Seurat object and tried to do a feature plot to…

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How to load Seurat Object into WGCNA Tutorial Format

As far as I can find, there is only one tutorial about loading Seurat objects into WGCNA (ucdavis-bioinformatics-training.github.io/2019-single-cell-RNA-sequencing-Workshop-UCD_UCSF/scrnaseq_analysis/scRNA_Workshop-PART6.html). I am really new to programming so it’s probably just my inexperience, but I am not sure how to load my Seurat object into a format that works with WGCNA’s tutorials (horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/)….

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Violion plot with statistics

Violion plot with statistics 0 Hi I am using Seurat for scRNA-Seq analysis. Along the analysis, i used Seurat’s VlnPlot function as the following: VlnPlot(Myeloid.object, features=”Mki67″, split.by = ‘Sample’, pt.size = 0) The feature is then represented in a different color for each Sample, and is divided by cluster. I…

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Normalisation for single nuclei RNA-seq

Normalisation for single nuclei RNA-seq 0 For single cell RNA-seq the typical workflow includes a normalisation step to account for variable sequencing depth. In Scanpy/Seurat, CPM (Counts per million) is a simple and common choice. We don’t normally normalize for gene length (like we would with full length transcript bulk…

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Splitting a Seurat Object HTO classification

Splitting a Seurat Object HTO classification 1 Hello, I am trying to split a Seurat object based on HTO classification. I have 3 different HTO based on different stimulation conditions. I want to pull out the unstimulated condition and make it its own Seurat object. I have tried Sample_1_unstim<- SplitObject(Sample_1@meta.data$HTO_classification,…

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Best approach to finding correlation between two genes from MAGIC imputed single-cell RNA-seq data

Best approach to finding correlation between two genes from MAGIC imputed single-cell RNA-seq data 0 Hi, I have single-cell RNA-seq data from two conditions that has been run through the Seurat pipeline, had macrophage cells extracted and MAGIC imputation performed to restore the data to its underlying expression structure so…

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Estimating sequencing depth or mean reads per cell

Estimating sequencing depth or mean reads per cell 0 I have been working with multiple single cell Rna datasets.Inorder to compare the sequencing dpeth of multiple sample,I am trying to find the mean reads per cell.I used the following code: counts_per_cell <- Matrix::colSums(sample1) Mean(counts_per_cell) The value is coming out to…

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CROP-seq data analysis

CROP-seq data analysis 1 Hi, I am a new bie to single cell sequencing analysis. I have to analyze CROP-seq data, I am going through the following paper, www.nature.com/articles/nmeth.4177. I have to use cell ranger ( instead of DROP-seq software) as the first step to process single cell data.I wanted…

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Sequencing file conversion

Sequencing file conversion 0 Hi, friends, I downloaded a set of scATAC-seq BAM files from an article database, and the author said that a BAM file is information about a cell. However, after a few days’ analysis of the script given by the author, I found that a CSV file…

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%% error in Rstudio

%% error in Rstudio 1 dc.markers %>% group_by(cluster) %>% top_n(2, wt = avg_logFC) the above code is giving error even after using dplyr and matrix libraries in seurat analysis in rstudio error : Error: Problem with filter() input ..1. i Input ..1 is top_n_rank(2, avg_logFC). x object ‘avg_logFC’ not found…

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Seurat to Trajectory Analysis

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Signac CallPeaks from multiple fragment files

Signac CallPeaks from multiple fragment files 0 I am attempting to run Macs2 CallPeaks on some multiome data and running into a problem when attempting to run CallPeaks command on multiple fragment file paths in Seurat object. peaks<-CallPeaks(DataCombined, macs2.path = “/anaconda3/bin/macs2”) FileNotFoundError: [Errno 2] No such file or directory: ‘/Users/Desktop/multiome/sc291/atac_fragments.tsv.gz…

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Annotating cell types via integrating a query dataset with a reference dataset and then cluster

  Mostly because it’s typically unnecessary given that reference-based classification should yield a similar result without being subjected to potential biases introduced during the integration process. SingleR (and presumably Seurat, I don’t know as I don’t use it) uses a reference dataset and asks “Which reference sample’s expression profile is…

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Manual annotation of cell types in single cell RNA-seq

Manual annotation of cell types in single cell RNA-seq 1 I have recently started working with scRNA-seq data. I am following the tutorials by the creators of Seurat. In the final section titled “Assigning cell type identity to clusters”, the authors mention that Fortunately in the case of this dataset,…

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seurat `@assays$RNA@counts` vs `@assays$RNA@data`?

seurat `<obj>@assays$RNA@counts` vs `<obj>@assays$RNA@data`? 1 I have two matrices called <object>@assays$RNA@counts and <object>@assays$RNA@data that are both real non-negative. What is the difference between these? seurat • 46 views • link 11 minutes ago by mk &utrif; 230 Source link

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remove effect of latent variables from log fold change

The findMarkers function of seurat allows users to specify latent variables to be adjusted for when finding differentially expressed genes. I am testing for differences in gene expression between 2 groups – disease vs normal. For the statistical test, I am using LR, described below: LR: disease state is modelled…

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Corelate TCR data to clusters/GEX/CITEseq data

Corelate TCR data to clusters/GEX/CITEseq data 1 Hello everyone, I just added my TCR VDJ data as metadata to my Seurat object (as described in the tutorial here). So, I basically ended up with two different collumns of metadata where my barcodes are assigned to the clonotypes and the cdr3…

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Integrated Dimension Reduction Plot for CD4/CD8 sorted Feedback

Integrated Dimension Reduction Plot for CD4/CD8 sorted Feedback 1 Hello, I have recently followed adopted the Harvard Chan Bioinformatics Core guidelines for SC QC/Normalization/Clustering (hbctraining.github.io/scRNA-seq_online/schedule/links-to-lessons.html). I have integrated CD4+/CD8+ T cells from two time points. I recently received feedback that my integrated dimension reduction plot clustering looked problematic. Specifically, the…

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Gene read count-level batch correction in scRNA-seq?

Gene read count-level batch correction in scRNA-seq? 0 Hi, I’m working on the integration of several scRNA-seq datasets. After trying Seurat v3 and Harmony, I realized they outputs dimension reduction matrix rather than correct read counts, therefore not suitable for some downstream analysis on gene-expression level. I wonder if there…

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