Seurat issues I run the following commands: install. You could try renaming the You signed in with another tab or window. I began this question on #8635 but am still having issues. Nevertheless, the issue still persisted with SCT v2 satijalab / seurat Public. It seems that it's partially answered by referring to point 4 of the FAQ, but I'm still unclear about how the scaled. I have been using the function FindMarkers with You signed in with another tab or window. It worked fine in Seurat V4, but now produces the following error: > sc_obj An object of class Seurat 23341 features across 17601 samples within Depending on what your downstream analysis is, it might be possible to select features without creating a new Seurat object. Issue with running AddModuleScore in seurat v5 #7536. h5ad (anndata), and I now want to load it in R by converting it to a Seurat object, Thanks for using Seurat! It appears that this issue has gone stale. 1 (2023-06-16) Platfo A toolkit for quality control, analysis, and exploration of single cell RNA sequencing data. txt. The author mentioned this . It seems this is a R related issue not an issue with Seurat, but I will help you since this is your first issue ever 😃 First of all please look here to read more on reading data types Hello, I am trying Seurat for the latest visium HD data and trying to use SpatialFeaturePlot function. cca, rpca and harmony. Hi there. Closed Hi, I have a Seurat object in which I did a SCTransform integration and now I'm trying to define marker genes for the identified clusters from a hierarchical clustering approach. Sign up for a free GitHub account I was previously generating violin plots with 'split. They seem to be I'm curious if others are facing similar challenges with Seurat V5. In an effort to keep our Issues board from getting more unruly than it already is, we’re going to begin closing out issues that haven’t had any activity since the Hi there, In issues #3692 and #1227, it was suggested the issue can be resolved by setting min. Spots as shown in the partial image below. The addition is just overriding the I have installed the Seurat 5 beta and it's all working well for me bar the FastNMM part. Many thanks! Hey everyone, I have a question regarding the VlnPlot function. Sign up for a free GitHub account to open Hi, Thank you guys making such a great tool for the single cell community. You are right. genes <- I want to generate count matrix and perform the same DGE analysis as we do for bulk RNA seq data for single cell RNA seq data in Seurat. This comes from multiple samples that Hi Seurat Team, I am trying to create a chromatin assay from a feature matrix from fragment files of my data outputted by CellRanger ARC. As we say in the documentation (and as you note above), the major difference is that in Seurat v5, we perform the integration in low-dimensional space. When I reclustered a subset of cells I set DefaultAssay(object) <- "integrated" After integrating the 2 Seurat objects "TCE. You Is it ever ok to use Seurat for clustering bulk samples? I am looking at FPKM data from ~750 bulk RNA-seq samples. The issue is this Running: obj <- Hi everyone! Now I am struggling with a couple of issues related to Seurat v5. I have seen previous issues related to cell cycle scoring #6625, #5329, #5518 but did not seem to report the same problem. How can I create TSNE plot just based on list of genes. The reason it returns an empty vector is that there are no variable features that have been compute for this assay. Prior to integration, For now we will close the issue as this is expected behavior, Discussed in #9442 Originally posted by bbmorris1 October 31, 2024 I have scRNAseq from a tumor cell line +/- drug treatment (sample 1 = no drug, sample 2 = drug Recently, we used your powerful Seurat software and I met some questions. How do I load That's correct: corrected counts here reflect counts that have been adjusted for sequencing depth. combined <- IntegrateLayers( object = mic. Additionally, I’m looking for Hello Andrea, If the plotted colours are the default colours of ggplot2, you can get these colours using the hue_pal() function of the scales packages in R. My dataset has a lot of metadata which includes tissue and time point. However, I need to subset two clusters of interest from the total object and Im doubting on how to do it. In an effort to keep our Issues board from getting more unruly than it already is, we’re going to begin closing out issues that haven’t had any activity since the invalid class “Seurat” object: 10: All cells in images must be present in the Seurat object invalid class “Seurat” object: 11: A What I take from this is that it seems that Seurat built You signed in with another tab or window. Seurat aims to enable users to identify and interpret sources of heterogeneity from single-cell Fix issue in SingleCellExperiment conversion where the mainExp would not be set properly; Fix for default dispersion info displayed in VariableFeaturePlot() I have a large expression matrix (~ 350,000 x 20,000). Navigation Menu Find and fix vulnerabilities Actions. I'm trying to recluster the clusters I found earlier. You switched accounts on another tab or window. However, I don't understand what join layers is doing? If you have I performed a standard analysis of data coming from different subjects with seurat, then I wrote a function to subset my dataset, with this command: subset_seurat_object <- seurat_object[, seurat_object@meta. 0 into a Seurat object using Load10X_Spatial from Seurat 5. I have two seurat objects, one with about 40k cells and another with around 20k cells. I would like to randomly downsample the larger object to have the same You signed in with another tab or window. To compute the score, I Hello, I understand that when Seurat regresses out the unwanted sources of variation, it stores the scaled data in scale. ) Hi David Collins, Thank you for providing the information. However, now using a similar pipeline, we consistently run into a problem when we use the RunPCA Hi, I'm on a Mac and am trying to install Seurat, which I have used before and never had these issues. Finally I found out that having a large number of cells is one of the reason for not plotting the graph, when you have more than 30k cells. I am not sure whether we should use the sequencing-based analysis method (e. data(), a dot plot would show that some gene have negative average expression in some sample, with examples shown in the I am trying to load Seurat in RStudio to analyse sc-RNAseq data. . 9042 and I'm getting very different results using AddModuleScore than I got with previous versions of Seurat. gz. Hi Seurat Team @yuhanH @timoast @satijalab Below are the procedures I summarized for subclustering a SCTransform-normalized integrated object, but I'm not sure they're correct or not. CPU and memory were quite ok and this data was not big. 4 on my laptop (macbook M1) ever since i updated to version 5. I converted tissue_positions. rds. 1. However, I can no longer do this! When using 'split. I need to revert back to seurat v4 as several of my codes written using this version do not work for I'm wondering if there's a way to add this to the Seurat object? Thanks. After defining such subclusters, i would like to bring back the clusterinfo of the new subclusters to the parent Seurat object, in order to find I had the same issue. You switched accounts on another tab Hi, I am trying to add median lines to VlnPlot, like this: I know geom_violin(draw_quantiles = 0. I am trying to use 'DietSeurat()' but it seems that I am loosing graphs and dim reductions. How can I set up to directly obtain appropriate parameters Hello, I am wondering if SCTransform is compatible with the new IntegrateLayers function in v5? A vignette would be awesome if it is! Thanks! Hi, I attempted to use the future framework for parallelization during the FindIntegrationAnchors step in Seurat while working in R Studio on my Mac, but it caused R VariableFeatures() is an accessor function, and is still present in Seurat v4. [] I use Seurat 3. After scale. We are unable to reproduce the issue in From the looks of Seurat::Read10X_Coordinates we assume that tissue_position_list. Using the latest release of Seurat 3. I have been using Seurat method to integrate these together so that I estimate RNA If I understand correctly, you are trying to set the idents to a character string and then subset based on a specific metadata column. As suggested for FPKM data, I manually input log transformed data to the @data slot [cd138_bm@data <- Dear Seurat Devs, Is there any known maximum to the size of a Seurat object? I am trying to merge a large amount of Seurat objects in a loop. 2, using this code: Hello, Thanks a lot for reporting this. 5 with R version 3. plot=TRUE' I am informed the function is only supported for Hi Seurat team, How can I extract the full counts matrix from a seurat object (with rows as features/genes and columns as cell barcodes). If your issues persist or if you are having different problems, please feel free to open a new issue. 16K" datasets, which both have a response column with values "R" and "NR", I visualized it by group. Your website indicated that, "count,TPM,FPKM" are allowed as the input of Seurat, but the input expression matrix should not be log-transformed. data' (as in the first example below), vs. My merged data is around 8G and working with 37RAM computer. cell=1 and calculate the cell cycle gene scores on SCTransform-ed object. I seem to have fixed it by uninstalling both Seurat and SeuratObject remove. R toolkit for single cell genomics. Hello, I've downloaded GSE139555_all_integrated. All of my Seurat objects seem consistent with each other, and the matrices don't seem to have any From my point of view, I would only use merge alone if I am dealing with technical replicates. You can assign variable features using the You signed in with another tab or window. Hi -- thanks for your help. the v4 session is like: ''' R version 4. I tested with different data, but always this issue come. For example, de. When applying the same code to identical datasets on different devices, we I've been trying to replicate my previous scripts and data pipeline from earlier versions of Seurat. data slot is Contribute to satijalab/seurat-object development by creating an account on GitHub. Instant dev environments Issues. You switched accounts I am attempting to load Visium HD data analyzed using spaceranger 3. Search syntax tips. I know that Loupe Browser 6 has a new feature where it can export a CSV file of the projection' You signed in with another tab or window. Notifications You must be signed in to change notification settings; Fork 927; Star 2. You switched accounts I have some CD8 and TCRseq data that has been processed, clustered and analyzed in Seurat. 5) can achieve this when using ggplot function, but here I think I may have to I am trying to run the FindSpatiallyVariableFeatures function in CosMX data with the following command: cancer <- FindSpatiallyVariableFeatures(cancer,features = Dear Seurat Team, # cols - vector with colors SpatialDimPlot(spatialData, cols= cols) The cols parameter changes the colors. I am trying to use Seurat 4. 3. Plan Hi @DRSEI first of all I am not a Seurat dev,. 1, calling SCTransform and then SketchData does not work Running the Sketch pipeline (using NormalizeData) as in the tutorial works fine. I would like to extract only these genes from Seurat object and create TSNE Hi @mhkowalski and @samuel-marsh,. e. Seurat is an R package designed for QC, analysis, and exploration of single-cell RNA-seq data. I am currently analysing an 8HTO dataset. , 10 × Xenium or Nanostring how to Exports a seurat object as CSV files. However, when I check the meta data, most of the time My dataset contains different cell types. We strongly urge users to not rely on calling slots directly using @, as this In the past, I had the same problem with Scanpy with the same gene list and Seurat object. by() and split. Sign up for a free GitHub account to open an issue and Hi Seurat team, When I use SpatialDimPlot to draw and set clip=FALSE, the spots in the graph I get overlap. But nothing happens. I also understand that the data in To begin, I know that when you subset data, you should select recorrect_umi=FALSE . 9. I’ve attached my code below for review and would appreciate any insights. Navigation Menu Toggle navigation. Specifically, I am encountering a problem where R crashes and restarts during the RunPCA step in the standard Here I am doing the normalization and find variables in my merged_seurat which is the result of step 2 (and therefore the normalization and findvariables etc is being done by Hi Seurat Team, Thanks for the wonderful package. Would you be able to I am working with several published datasets (i. 2. After I subset out a cell type, do I need to re-normalize and re-scale the data, or do I just need to run PCA/UMAP and plot the cells on a new graph? You signed in with another tab or window. I am trying to understand the calculations powered in the "AddModule Score" function of seurat. Seurat function to convert a matrix of normalized nanostring data (and also with a demodata) and I always get this error: as. 3k. In the previous Seurat versions when one used both split by and group by arguments, would get a plot like related issue: #4178 I discovered great difference between log2fc calculated by Seurat FindMarkers function and the script I wrote myself. Usually, the log2fc is In the normalization step, there are two representative tools in seurat (NormalizeData, and SCTransform). I have CSV file which contains around 2000 genes. Hi - I've been struggling all week with trying to install seurat v4. I then want to cluster my data and do a QC Hi, This seems to be a memory-related issue. Although we haven't precisely diagnosed it, we have been able to fix Hi, I'm running into an issue with Seurat::CellCycleScoring() in Seurat V5. Using the CreateSeuratObject, takes an extremely long time, such that I'm not sure if it will ever Hello, Based on previous issues posted here I gather that if I want to recluster a subset of my dataset that has been integrated I should use the "RNA" assay when I subset my data if I wish to rerun the integration. Please let me know if there is a way to achieve this. If you are dealing with multiple samples or experiments, I would definitely expect to have some batch effects due to inter No issues running PCA on the integrated dataset, using PCA for findNeighbors() and runUMAP() I thought it would be insufficient memory but this system has 32gb, running I believe the above will still utilize only the features extracted from SelectIntegrationFeatures rather than using all features that might be noisy (and defeat the purpose of using SCTransform). I have carried out different integrations on my datasets just like in the tutorial e. Code; Issues 378; Pull New issue Have a question about this Just to say I had this exact same issue when I accidentally updated to v5 and tried to revert back to v4. 3, I have two different UMAP visualization results and they are mirrored. Unfortunately, my downstream analysis relies on the count matrix. The CreateSeuratObject command requires either sparse of dense matrix where cells are columns and genes are When I run Seurat::FindClusters using the future package I get the following warning: > breast_cancer_patients_tumor_cells <- FindClusters Sign up for a free GitHub Hi, I am trying to load data that I previously merged from two datasets in python. To remove an Assay from a Seurat object, please set the assay as NULL using the double bracket [[setter (eg. This has created a file sample_DGE. So this is expected performance of the function(s). Recently, our team has encountered significant reproducibility challenges while using Seurat. Are there any plans to integrate I'm trying to follow the steps of the sketch integration vignette with my own data. But when I used the function JoinLayers with "norm_seurat_lis When I run the same R code in my local computer RStudio (R 4. data slot, which is eventually used for dimensionality reduction. I want to create a spatial Seurat object from a published data. When I run the code with my MacBook Pro, it was You signed in with another tab or window. This was a bug, caused by the default behavior of using the counts slot for calculating fold-changes, which doesn't take into account imbalanced sample sizes between the two groups Hi Seurat team, @saketkc #8153 I have two datasets, namely Vasc_1 and Vasc_2. I have a question concerning Seurat 3. singletons parameter which if set to FALSE, will assign all singletons to their own "singleton" cluster. h5") However, my r How can I remover doublet in a subset of Seurat object?. integrated[['integrated']] <- NULL). 4. Sign in Product Sign up for a free I am trying to run the tutorial for 250K Mouse cell atlas example, and run into trouble on the Visualization step. Seurat issue with Hello, Thank you so much for developing Seurat and the constant support! I am looking for some help with the FindConservedMarkers function in Seurat after data integration. , 10 × Visum) or the image-based analysis method (e. However, I'm In seurat V5, trying to subset data, especially data that has already been integrated, straight up does not work. Seurat as. The data is saved as . I was wondering how t Hi Chan, This question has been answered a couple times in github issues (see #1769 and others). Everything runs well until I get to the IntegrateLayers object <- IntegrateLayers(object, method = RPCAIntegration, orig = "pca", You signed in with another tab or window. Code; Issues 377; Pull New issue Have a question about this project? Sign up for a free GitHub account to when running NormalizaData() using the same data, v4 would finish it soon but v5 will keep running and never stop(at least 10 hours). packages('Seurat', 'SeuratObject') then HI @Aaron-sqw, When we find variable features with an object with 2 layers for example, we identify each layer's variable features, find the common variable features, and then supplement until n variable features are There are batch effects between my samples. I have installed FIt-SNE and FFTW. I have the exactly same problem with the vignette code (so not giving an example of my own here). g. Approach 1: Just re-run PCA, We previously have ran code using Seurat pipeline before successfully. I have problems with the subsetting of seurat objects after I create them from matrix, feature You signed in with another tab or window. 'Seurat' aims to enable users to identify and interpret sources of heterogeneity from single cell Also, if the scran normalized data is log transformed, make sure that the values are in natural log, and not log2. Skip to content. Search code, repositories, users, issues, pull requests Search Clear. I am currently working with single cell (scRNAseq) and spatial transcriptomics Hi, I have been still working on the scRNA seq of the data published in GSE120575. I've also added I really like this package, and this is my first issue with it. The authors have submitted the matrix count, the low res image, tissue position list. I think that the following is the correct interpretation of the margin setting, but I am not sure. You can then create a vector of cells including the sampled cells and the remaining cells, then subset your Seurat Hello, I also wanted to reduce a Seurat object to only the counts layer and a single dimension from the many it was composed of (CCA and RPCA integrations) for export, and encountered the same problem as everyone with Hello Seurat Team, In the violin plots draw using VlnPlot, I need to rotate the x-axis labels, as the names overlap rendering them unreadable. Sign up for a free GitHub account to open an issue and contact its maintainers Hi Seurat team, I noticed a difference between when I run FeaturePlot() and VlnPlot() without specifying that the slot/layer to use should be 'scale. You signed out in another tab or window. I am able to load the image using the Read10X_Image() function. Sign up for a free GitHub account to open an issue and contact its maintainers This is the code of merging both SCT and Harmony. gz and GSE139555_all_metadata. gz from here through R and I would like to convert them to a ready-to-use Seurat object. Write better code with AI Sign up for a free GitHub account to open an issue and contact Hi, I'm using the Seurat v5 vignette for integration. This comes in handy if you have a very large number of I am using Seurat to cluster data that previously has been filtered, aligned and turned into DGE by the Drop-Seq alignment pipline from Drop-seq tools. You switched accounts on another tab Hi Seurat team, Using Seurat v5. packages('BiocManager' I don't have any issues with other Hi, You can do use the FindMarkers command, specifying the names of the subclusters you would like to compare and the name of the variable containing the subclustering information. Please read #2724 for more details. Is Hi Howard, if that object was created in Seurat v4, some of the dimension reduction names might be invalid when now loaded into Seurat v5. After I run HTODemux, almost 90% of my cells are classified as doublets. I use subset function to generate a smaller seurat object from SCTransform integrated big seurat object. While the cluster identity and markers identified using FindConservedMarkers are consistent across Contribute to satijalab/seurat-wrappers development by creating an account on GitHub. However, if I have multiple slide and not all clusters are You signed in with another tab or window. by() Sign up Hi, I'm reanalyzing samples using seurat v4. 6. However, because there are 20 samples in the reference object, there are 20 SCT models and this caused a failure in running Hi there, I'm sorry if I am asking the obvious, but I am still confused about the margin argument for CLR. Reload to refresh your session. I used the exact same script shown in vignette for preprocessing, Hi, I'm trying to use the new integration function in Seurat v5, specifically the FastMNNIntegration method. You switched accounts on another tab I am trying to use the SketchData function with the Leverage Score method, and the computation will not go through. We recommend using BPCells to convert the matrices in your Seurat object to on-disk matrices to store your data on disk instead (tutorial here. 0, and following the guided clustering tutorial step-by-step, when I get to the step 'pbmc <- FindNeighbors(pbmc, dims = I have been subsetting a cluster from a Seurat object to find subclusters. I also tried rebuilding the reference object using v5. However when I use the as. Contribute to satijalab/seurat development by creating an account on GitHub. I have used Seurat and Harmony both to correct the batch effects. You switched accounts After some deeper reading on Closed Issues, I think that #1421 articulated my questions the best. Sign in Product GitHub Copilot. The old Hi, Not member of dev team but hopefully can be helpful. by' parameter set to show 4 different samples types in one violin plot. ch. However, I receive this error: mic. cell_data_set() function I get I am currently facing an issue while using Seurat v5. Automate any workflow Codespaces. 0. data It seems that this issue is a result of incompatible updates of CRAN and Bioconductor packages. You switched accounts Hi, I am pretty new to Seurat and cell hashing. Seurat assumes that the normalized data is log transformed using natural log (some functions in Seurat So I am analyzing a scRNA seq from homogenized tissue from different samples, I've two questions here, 1 - Do I integrate by sample or by sequencing run? I have no information whether the samples were processed hi @ziyuan-ma, A new layer has been added to Seurat V5, and the slot slot used to extract gene expression data in previous versions seems to no longer work in V5. parquet to csv format earlier. Sign up for a free GitHub account to I have done this in the past with older versions of Seurat where I had applied the integration workflow that creates an assay slot called "integrated". combined, method = Hello, I am trying to load visium HD data using the following command: Load10X_Spatial(data. 0 version in both The documentation is fine, accessing any of the data layers is straight forward. I would like to use the SCTransform workflow Thanks for using Seurat! It appears that this issue has gone stale. I have merged all of them into one big Seurat object You signed in with another tab or window. 7K" and "TCS. Luckily, I got a post that explains the fundamentals very well. Provide feedback We read every piece of feedback, Seurat provides a great workflow for data integration. Hello, I want to delete one named as 'integrated' assay in my big Seurat object. dir - what does your file structure look like? Yes, it is. What that means is that prior to I am interested in trying out Scanorama to see how it fares with our data, but I have the issue that we have been working with our data and doing our processing and analysis in Seurat in R. First, GetAssayData has been superseded by LayerData so suggest moving to that when using V5 structure Hopefully you are doing well. If the counts have already been Here is my workaround code, not very clean but it works for me! Step1: identify your uploaded image dimension used for spaceranger (not the ones in the spaceranger output image folder, the resolution is different): satijalab / seurat Public. I want to use Monocle3 to perform single-cell trajectory analysis. You switched accounts I am using as. For example, the FindMarkers() command has a features argument that you can use to perform DE only on I have waited for more than 30 mins. I have used the following code to make Seurat object, and since the data Hi, Option 1 has recently been added to the develop branch as a group. I saw a lot of issues & your paper about SCTransform (Hafemeister and Satija, 2019). Recently I am stuck with SCTransform steps. Hi, I guess you can randomly sample your cells from that cluster using sample() (from the base in R). Can you please Interfaces for HDF5-based Single Cell File Formats - Issues · mojaveazure/seurat-disk These are from replicate biological samples but cells are not exactly the same ones. csv files are missing the headers provided by other formats - if that's not the case, that would explain the problem you're encountering. dir = localdir, filename="filtered_feature_bc_matrix. I've tried googling numerous solutions, but none of them Hi, these days I run the integration for 24 scRNA-seq datasets, I filter features and cells for each dataset and then merged them. 2) and on Code Ocean R 4. 1: The sub-samples was SCTransformed before merging, after It seems like Read10X_Image is finding more than one tissue_positions file in the specified image. Other's have commented on the object size, but that's not really a Seurat issue, that's just single-cell in a Hi, UpdateSeuratObject() should work with the current versions of Seurat, SeuratObject, and Matrix. That is not the conundrum I've run into. , coming from different published studies), each with multiple samples. But I have several Hello Seurat Team, I would like to slim down my multi-modal object to keep only the RNA-seq based elements. Seurat uses pearson residuals for all downstream tasks and not the corrected counts.
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