Reading single-cell data in R: H5AD, loom, MEX, AnnData formats | Bioinformatics for beginners

[@tahadinc1302]

i love your videos, the way you explain them is also very straight forward and easy to understand. Please keep making videos and thank you

[@Ojaswini-Pathak]

Brilliantly done video. Simply too good.

[@ghaishoutak4087]

im getting the following error > Convert( "MCA2.0_Fig1_adata.h5ad", dest = "h5seurat", overwrite = TRUE) Warning: Unknown file type: h5ad Warning: 'assay' not set, setting to 'RNA' Creating h5Seurat file for version 3.1.5.9900 Adding X as data Adding raw/X as counts Error in `[[.H5File`(source, features.source) : An object with name var does not exist in this group

[@IlariaMulas-p1i]

amazingggg !! thank you so much for the precise tutorial

[@ChoonghoLee]

very nice!!

[@zlj8435]

Love your classes! And for .loom format, the link says: This XML file does not appear to have any style information associated with it. The document tree is shown below. Wondering how to solve this, thanks again!

[@Bioinformagician]

Can you paste the exact error?

[@zlj8435]

@@Bioinformagician This XML file does not appear to have any style information associated with it. The document tree is shown below. ExpiredToken The provided token has expired. Request signature expired at: 2021-12-28T23:03:06+00:00 Like this. Thank you for your help!

[@fardaddanaeefard8247]

@@zlj8435 I'm facing same error. Any help on solving this issue will be a big help

[@nathansuek2571]

@@Bioinformagician does this xml file need to be reuploaded? this is such a great tutorial and would help to be able to run the data alongside

[@mrinalsubash8358]

@@nathansuek2571 I am getting the same error

[@mx8128]

Hi, thanks for the tutorial. I have problem reading loom. it says " could not find function "Connect"". Can you help me with this, thanks a lot!

[@Bioinformagician]

Did you load 'SeuratDisk' package before running the command? Also, what system are you using? What are your R and Rstudio versions?

[@mx8128]

@@Bioinformagician thank you for replying!! yea i didn't load 'SeuratDisk' package, now it's working!

[@Collection_of_online_tutorials]

Can you make one video about how to create a seurat obj from txt data

[@chrisdoan3210]

Thank you for a helpful video! I load 'SeuratDisk' package and I got this error: Error in library(SeuratDisk) : there is no package called ‘SeuratDisk’. Would you please tell me why?

[@Bioinformagician]

That is because SeuratDisk is not installed. Install SeuratDisk package first, load the library before running the code. You should not be getting this error then.

[@cr7wakk8]

many many thanks to your video and it helps me a looooottt!! however there was an error when i was reading h5ad data like this : Convert("GSE246613_combined_RTPDv4_scvi_celltypist.h5ad", dest = "h5seurat", overwrite = TRUE) seurat_anndata

[@MasiaMaksymowicz]

I also got this error..

[@mostafaismail4253]

Can you do it in scanpy ? 1-how import single cell data from different types of files like .mtx,h5 ....? 2- how to run the pipeline in scanpy ?

[@Bioinformagician]

My comfort language to code in currently is R and hence I pick packages and tools that could be run using R. Nevertheless, I will surely cover these when I try my hand at python packages :)

[@petatownsent]

Hi Ms. Patel, Great work, you are doing here! Much appreciation. I want to ask a couple of questions about the following : 1. what is the description of the file " adata_SS2_for_download.h5ad "? 2. what is the origin and reference website for the data file " adata_SS2_for_download.h5ad" ? I have conducted an analysis using the data file and from my limited understanding, it is about CVID and Control treatment groups. If you could educate me about the matter or if there are no restrictions about access to the data, please paste a link with information about the data file. Thanks so much.

[@carloseduardobuss6772]

Hi. Could you upload the Loom file again? Please. It's not available to download. Thank you. Error message: This XML file does not appear to have any style information associated with it. The document tree is shown below. ExpiredToken The provided token has expired. Request signature expired at: 2021-12-28T23:03:06+00:00

[@IreneZaalberg]

Thank you so much for your videos, they've been a huge help. In this case, I run into a problem that I haven't found a solution to through googling. I have now tried for two unrelated .h5ad files for different papers, to load them as seurat objects in R. The Convert() step works fine, but in the LoadH5Seurat() step in both cases, I get the error: "Too many values for levels provided". Do you have any advice how I can solve this?

[@garggopal2001]

link of .loom file in invalid

[@paulacohen7970]

Howdy! Clinician living in a PhD world and very lost - I have some pilot scRNAseq data that was processed in to a cloupe format for 10x Loupe Browser. And now I'm just lost on how to identify the clusters and do any meaningful comparisons. Suggestions?

[@realdealsa]

Great content, please enlighten me. I'm working on Nanostring Geomx DSP .dcc files, I'm struggling to load them into r using the recommended code , please help

[@ShahanaDilruba-y9h]

very helpful and well explained video. Thanks a lot!! Is it possible to use the seurat object for DESeq2 analysis?

[@kokosali5423]

Thank you very much! I had one problem, my data are pooled under one CSV file, how can I import it in this cas. Thank you again!

[@lanternofthegreen]

Is there a database to download scRNAseq files (like SRA for bulk RNAseq) or do we have to look for different company websites, etc.?

[@Bioinformagician]

You can ample of single-cell data on 10X genomics platform. Apart from that, NCBI GEO also stores a lot of single-cell datasets as well.

[@lanternofthegreen]

@@Bioinformagician Thank you. I was trying to ask about raw data, instead of count matrices. Do scRNAseq studies produce fastq files as well or do they use different formats? If they are in fastq format, can we download them from SRA?

[@tranphilong2229]

Hello, thank you for your instruction, however, this link seem to be die " adult-hem-organs-10X-bone-marrow.loom:" Could you kindly fix this?

[@cnbmonster1042]

I like your video. But .loom format data is unable to download because the download key is expired.

[@tushardhyani3931]

Thank you for this video !!

[@subatsamat4899]

Would you please helpe me how to install seuratdisk packages. I couldn't download it 😢

[@Nipsoj]

hello, thank you for the very informative video. I was wondering if we could read single cell count data into a seurat object from a .tsv file?

[@Bioinformagician]

Yes, read the tsv file. Ones you have the counts into a data.frame in R, you can provide that data.frame to counts parameter in CreateSeuratObject function. Something like this: counts.df

[@alpr1864]

Hey Although I have downloaded the right file and even unzipped the file, R gives me this. Do you know what the solution is? Error: Cannot find expression matrix at C:\Users\Mahdi\Downloads aw_feature_bc_matrix aw_feature_bc_matrix\matrix.mtx.gz >

[@Bioinformagician]

The error means it cannot find the "matrix.mtx.gz" file in this path - "C:\Users\Mahdi\Downloads aw_feature_bc_matrix aw_feature_bc_matrix". Can you double-check your file path?

[@alpr1864]

@@Bioinformagician You were right! Can you please help me with this problem, as well? After running the codes of MTX, I face this error! Error in url(description = uri) : URL scheme unsupported by this method

[@alpr1864]

Solved! The problem was from the new version of R. For windows users, you can install R version 4.0.5 (2021-03-31). For more info: github.com/satijalab/seurat/issues/5687

[@Bioinformagician]

Awesome! This will help others who are also facing the same issue. Thanks so much!

[@alpr1864]

@@Bioinformagician My pleasure!

[@Bananalane]

Hi, I got the Library (seuratdisk), However I can't run any scripts after that. all errors since readRDS.... What others proceudures or data downloads we have to do before following your tutorial? Thanks again!

[@Bananalane]

How do I install the "seuratdisk"?

[@AyrodsGamgam]

hi, what do you do when memory is exhausted? Thanks, see error mssg below > seurat_loom

[@Ojaswini-Pathak]

HI, Where do I download the SeuratDisk package from. I was able to download Seurat as it was straight forward. Thanks.

[@danieladediran3228]

Am not sure, is the install.package("SeuratDisk") not working or check Satija lab documentation on how to do that

[@anamikapandey4769]

please suggest, while i am loading SeuratDisk library ,i am getting the following errors error: package or namespace load failed for ‘SeuratDisk’ in get(method, envir = home): lazy-load database '/home/gkp/R/x86_64-pc-linux-gnu-library/4.1/Seurat/R/Seurat.rdb' is corrupt In addition: Warning messages: 1: In .registerS3method(fin[i, 1], fin[i, 2], fin[i, 3], fin[i, 4], : restarting interrupted promise evaluation 2: In get(method, envir = home) : restarting interrupted promise evaluation 3: In get(method, envir = home) : internal error -3 in R_decompress1 please tell what should i do now?

[@Md.NaiemHossain-t7x]

Thanks a lot for making such an informative video.

[@mansisingh190]

I am having troubles downloading the Seurat package on Mac and have tried everything, would you know how to get around this?

[@Bioinformagician]

What errors do you see?

[@priyaamadhukaran4745]

Love your videos-needs more background info as beginners like me are struggling. You did show and provide link to sample data in different formats. How did you download them and where did you save them will be helpful. The black window titled inputformat _single cell 80x x 24x how do we get it? is it necessary to have the source file name coded on it for R -studio to recognize the files? I downloaded the rds file and save in my desktop and then followed all the steps in R studio from 5. but I get error msg > source("~/.active-rstudio-document") Error in eval(ei, envir) : object 'ependymal_cells.rds' not found. If you could provide details that would be helpful. Thanks

[@Bioinformagician]

The window titled inputformat _singleCell is the console in RStudio where you see the output of the code. The title "~/Desktop/demo/Inputformat _singleCell" is the project working directory. To get an idea of what a project working directory is, check this link out - intro2r.com/work-d.html You are getting an error because your R is unable to find where the file "ependymal_cells.rds" is located on your system. Since you mentioned your file is saved on Desktop, your command should look something like this - readRDS("~/Desktop/ependymal_cells.rds").

[@healthnut4936]

Hello, I love your videos. I am new to sc analysis. I have >10 files and two condition types and they are all in .h5 format. What do I do? Do I Read10X_h5() them individually and analyze them individually? Thanks! any help would be appreciated.

[@Bioinformagician]

What is the goal of your analysis? Do you want to compare across conditions? If yes, then filter and remove doublets for each dataset individually and then integrate datasets by condition.

[@hossainadamzad5907]

Thanks a lot for this great content, i have question:what is the difference between normal matrix and "dgCMatrix" actually???

[@Bioinformagician]

dgCMatrix is basically a class of sparse numeric matrices

[@taysaraujocamilo2313]

You are excelent! Thanks

[@nourlarifi1689]

thank you very much

[@HermesMarcialRomeroLuque]

nice video

[@saheljahangiri3453]

like your work buddy!

[@mahamoussa5712]

You are amazing!!!!!!

[@vishnushukla7502]

Can we make umap from processed CSV file? Please help

[@Bioinformagician]

What data does your processed CSV have?

[@vishnushukla7502]

@@Bioinformagician first column is the geneID and other 20 columns are their logFC values...1st row is the name of each cell type

[@Bioinformagician]

​@@vishnushukla7502 You will need to wrangle your data and run some commands. Linking some articles that might help: 1. cran.r-project.org/web/packages/umap/vignettes/umap.html 2. jtr13.github.io/cc21fall1/efficient-dimension-reduction-with-umap.html 3. datavizpyr.com/how-to-make-umap-plot-in-r/

[@vishnushukla7502]

@@Bioinformagician thanks...now I have my processed .mtx file (barcode, feature and matrix) with me...can you tell me how make umap plot directly from .mtx file in R

[@vishnushukla7502]

@@Bioinformagician I already made the seurat object now..thanks a lot

[@namartakalia8563]

hello I am biologist and trying to learn single cell analysis from scratch. could you please help let me know the necessary software to download and share their links

[@グライ-q6g]

Simply too good

[@laiumiunix]

QUestion: I convert a h5ad into h5seurat, now Im trying to load it into rstudio, I got this error: Validating h5Seurat file Initializing RNA with data Adding counts for RNA Adding feature-level metadata for RNA Error in match.arg(arg = layer, choices = Layers(object = object, search = FALSE)) : 'arg' should be one of “counts”, “data”, “scale.data

[@laiumiunix]

the h5ad I got it from the gutatlas website, I know the log counts are not available in the h5ad file

[@anamikapandey4769]

inspite of running the given commands for uploading hdf5 file .., i am still getting the error 'file not found', even if i have checked my directory and corrected it. please suggest😊

[@Bioinformagician]

The error suggest it cannot find the file in the path you have provided. Please ensure you have spelled the file name correctly.

[@anamikapandey4769]

@@Bioinformagician thank you again😀

[@nayeemanushrat3174]

Hi, very useful tutorial, thanks a lot! I have a problem loading "Seuratdisk" library. >>SeuratDisk is not currently available on CRAN>> so I tried to install it from GitHub [if (!requireNamespace("remotes", quietly = TRUE)) { install.packages("remotes") } remotes::install_github("mojaveazure/seurat-disk")], but it says "Permission denied" Can you help me with this, thanks a lot!

[@Bioinformagician]

What system are you using? Can you paste in the entire console output that throws 'permission denied' error? Also, can you try devtools::install_github("mojaveazure/seurat-disk") instead? Thanks!

[@nayeemanushrat3174]

@@Bioinformagician Thanks for your reply, I will try and let you know soon! 😄

[@robertlogan9733]

Come study bioinformatics with me! enc.edu/program/bioinformatics/

[@9447452569]

for the data adult-hem-organs-10X-bone-marrow.loom it is showing like this "ExpiredTokenInvalid argument.The provided token has expired. Request signature expired at: 2021-12-28T23:03:06+00:00" Can you help me with this.