Hey, I just want to say a big thank you for all the effort you guys are putting in doing free content in yt, and also in all the amazing bioinformatic/genomic videos in your channel.
Hi, it is really nice. It would be great if you could demonstrate how to create barcode, matrix and genes gz data files from the raw data obtained from 10x or alumina machine output.
Really good tutorial! thank you! how about comparing scRNA-seqs from patients with a certain disease Vs controls? will you consider making a tutorial about that?
This is a wonderful tutorial! It would be great if you could show how to use the subset function (i.e. if we only wanted to look at CD45- cells). Thanks for the strong work.
Hi Liquid Brain! Thank you so much for the great tutorial. I have a question: at7:55, you executed a few lines together and then the progress bar showed up on the left side of the codes and the plots showed up in your script window. How did you make Rstudio show plots on your script window? This looks much more efficient than going through each plots on the Plots window on the right bottom window.
Oh, do you mean executing the code in a r markdown format? Just create a r markdown file and place the code in the code think and execute them. You can also just download the file directly from my github page ~
Thanks for the wonderful tutorial. I could understand why the plot is so. I wonder if you could do one tutorial for Visium spatial analysis with Seurat too? The vignette provided by Satija's lab is kinda confusing for me.
Hi.. I would like to thank you for sharing this wonderful workflow.. I would also like to know how do you subset the cells as epithelial, fibroblasts, myeloid lineages? Could you please explain that
Hi Mangala, it relies on the biological knowledge to match the cluster markers (obtained from the FindMarkers function) with the known cell type markers, for example, CD14 amd CD8A are known as immune cell markers…another popular way is to run gene set enrichment analysis for the cluster markers against Molecular Signatures Database (MSigDB), hope it helps. -Lind
Hi liquid brain, can you please help me in knowing this tutorial from the very basic, I don't know anything like UMAP, PCA or other. May be some resource that is useful??
Great run of the Seurat Vignette. It is easy for a single sample/folder. Can you summarize for to analyze 3 groups with 2 samples each? [Total = 6 files] :-) Help me in integration/ creating matrix. Thanks!
Thank you very much for the informative tutorial! Is it possible to manually filter two cell subsets based on the expression of a specific gene, then do differential gene expression analysis? For example, Can we filter cells with high gene A expression vs cells with low gene A expression, then analyze differential gene expression between these two cell subsets? Thank you!
Hi, I have actually just made a new video on the subsetting of clusters based about names. You will need to build new meta data for the HIGH and LOW in your merged Seurat object. Once you are done you can use the FindMarkers() to get the DEGs between the two. Perhaps have a look at the video and see if that helps, if not please feel free to sent me a script you are working on and I can try to see if I can help from that
Hi, thanks for the video. Nice and clear. I'm wondering sometimes GEO datasets don't always give genes barcodes and matrix files, they give some raw files orsome other files. Could you tell me or advise me a tutorial or tool that I should look into? thanks
@@LiquidBrain sorry but I can't see my previous answer, it disappeared. basically I had put a link to a dataset where there was a huge RAW file or some sort of final analysis. I said that in that case I believe I have to go through cellranger to process the data in that case. Not sure why my post disappeared, I'll put the link in another answer just in case.
In general: features = name of genes matrix = expression number of those genes barcode = sample (of each cells) Basically, you can think of them coming together to form a matrix with the expression data inside the table; with rows as the genes name and; with columns as the name of each cell. They are all in a tab delimited format as generated another software (such as cellranger).
Can you do it ( the full pipeline analysis in scanpy ) ? 1- how to read the different file format of single cell .mtx .h5 .... Etc ... In scanpy 2- down stream analysis pipeline in scanpy
Scanpy has always been in the pipeline, making it run as a tools is actually much easier than turning it into a video. so I think it might take a while to publish that video
Sometimes the data is not available for download from the Seurat website (I would assume.is the traffic). For that, I have another copy of the data in my github repo (where the script is located)
Hi, I didn't understand, or it was not clear to me, how did you name cells at the end: "Naive CD4 T", "Memory CD4 T", "B" and so on, and why did you name them so, why didn't you leave them as GZMK, FCGR3A, or they just have such names?
I created a volcano plot by enhance volcano.But in the plot instead of showing the gene symbol some kind of number are shown.So how can i solve this problem. EnhancedVolcano(exp1, x = "logFC", y = "adj.P.Val", lab = rownames(exp1), FCcutoff = 0.6, pCutoff=0.01, cutoffLineType = 'twodash', cutoffLineWidth = 0.4, pointSize = 1, legendPosition = 'none', title = "", subtitle = "", caption = "", labSize = 3, ylim = c(0, 6)) + theme_pubr(legend = "none"). exp1 is my dataset and the first column of that dataset is gene symbol.
CAnt download raw data, it gives following error: AccessDenied Access Denied 548G3NM2GXQM8VFW D2RiKgdI96sfQm0QmNeVWqsEwQvGcDbGHYpOZq0WxcYcjBEhl6nJkQ1MxPE+dkkf6lkhsNYPCSc= Can you help?
Hey, I just want to say a big thank you for all the effort you guys are putting in doing free content in yt, and also in all the amazing bioinformatic/genomic videos in your channel.
Big thanks to Liquid Brain! This tutorial really saved my time. Looking forward more wonderful videos like this!
This has clearly explained the workflow!
Amazing tutorial! Finally a reliable channel.
Thanks for this great intro to Seurat!
This is outstanding. Nice presentation!
wow. that's what I needed. thanks guys.
Well done, very clear presentation, help me to understand more about all these bioinformatic data. Thanks.
Hi, it is really nice. It would be great if you could demonstrate how to create barcode, matrix and genes gz data files from the raw data obtained from 10x or alumina machine output.
Really good tutorial! thank you! how about comparing scRNA-seqs from patients with a certain disease Vs controls? will you consider making a tutorial about that?
Thanks, Let me look into it. Seems like an interesting idea
This is a wonderful tutorial! It would be great if you could show how to use the subset function (i.e. if we only wanted to look at CD45- cells). Thanks for the strong work.
I have finally made a video about it :) (th-cam.com/video/zZvt0elwvTQ/w-d-xo.html)
Thank you!
Thank you so much!
Hi Liquid Brain! Thank you so much for the great tutorial. I have a question: at7:55, you executed a few lines together and then the progress bar showed up on the left side of the codes and the plots showed up in your script window.
How did you make Rstudio show plots on your script window? This looks much more efficient than going through each plots on the Plots window on the right bottom window.
Oh, do you mean executing the code in a r markdown format? Just create a r markdown file and place the code in the code think and execute them. You can also just download the file directly from my github page ~
Thanks for the wonderful tutorial. I could understand why the plot is so. I wonder if you could do one tutorial for Visium spatial analysis with Seurat too? The vignette provided by Satija's lab is kinda confusing for me.
Thanks, I will look into them
Hi.. I would like to thank you for sharing this wonderful workflow.. I would also like to know how do you subset the cells as epithelial, fibroblasts, myeloid lineages? Could you please explain that
Hi Mangala, it relies on the biological knowledge to match the cluster markers (obtained from the FindMarkers function) with the known cell type markers, for example, CD14 amd CD8A are known as immune cell markers…another popular way is to run gene set enrichment analysis for the cluster markers against Molecular Signatures Database (MSigDB), hope it helps. -Lind
@@LiquidBrain Thank you very much
Thanks for the useful video. I was wondering if you can do an InferCNV video.
Thanks, not in the pipeline yet, but let ma have a look at that 😃
Hi liquid brain, can you please help me in knowing this tutorial from the very basic, I don't know anything like UMAP, PCA or other. May be some resource that is useful??
Great run of the Seurat Vignette. It is easy for a single sample/folder. Can you summarize for to analyze 3 groups with 2 samples each? [Total = 6 files] :-) Help me in integration/ creating matrix. Thanks!
Hi, you can have a look at the new video uploaded.on this topic (integration in Seurat)
Thank you very much for the informative tutorial!
Is it possible to manually filter two cell subsets based on the expression of a specific gene, then do differential gene expression analysis?
For example, Can we filter cells with high gene A expression vs cells with low gene A expression, then analyze differential gene expression between these two cell subsets? Thank you!
Hi, I have actually just made a new video on the subsetting of clusters based about names.
You will need to build new meta data for the HIGH and LOW in your merged Seurat object. Once you are done you can use the FindMarkers() to get the DEGs between the two. Perhaps have a look at the video and see if that helps, if not please feel free to sent me a script you are working on and I can try to see if I can help from that
@@LiquidBrain Thank you so much! I applied your integration code and considered the two subsets as 2 samples for integration.
Can we make a sub cluster from seurat clusters and plot them independently ? for example can we just plot and B cells and its marker genes as heatmap?
That's sounds like a good video to make, let me try to make one on this topic
@@LiquidBrain plese also see if its possible to make a sub cluster of 2 or more clusters from a umap
Hi, thanks for the video. Nice and clear. I'm wondering sometimes GEO datasets don't always give genes barcodes and matrix files, they give some raw files orsome other files. Could you tell me or advise me a tutorial or tool that I should look into? thanks
Hi would you mind to show me a GEO example (theGSE ID) so I’ll try work on it?
@@LiquidBrain sorry but I can't see my previous answer, it disappeared. basically I had put a link to a dataset where there was a huge RAW file or some sort of final analysis. I said that in that case I believe I have to go through cellranger to process the data in that case. Not sure why my post disappeared, I'll put the link in another answer just in case.
@@LiquidBrain GSE155960 (my link was again discarded
Could you give us if possible a little insight on the data used? What each file represents?
In general:
features = name of genes
matrix = expression number of those genes
barcode = sample (of each cells)
Basically, you can think of them coming together to form a matrix with the expression data inside the table; with rows as the genes name and; with columns as the name of each cell. They are all in a tab delimited format as generated another software (such as cellranger).
@@LiquidBrain thank you!
Can you do it ( the full pipeline analysis in scanpy ) ?
1- how to read the different file format of single cell .mtx .h5 .... Etc ... In scanpy
2- down stream analysis pipeline in scanpy
Scanpy has always been in the pipeline, making it run as a tools is actually much easier than turning it into a video. so I think it might take a while to publish that video
@@LiquidBrain anther thing, ATAC-seq and chip-seq , what about these topics , can u make a video tutorial about them ?
Is the raw data still available for training? The raw data page shows ERROR. I am not sure if it expires or it is just my problem.
Sometimes the data is not available for download from the Seurat website (I would assume.is the traffic). For that, I have another copy of the data in my github repo (where the script is located)
@@LiquidBrain Many thanks!
Hi, I didn't understand, or it was not clear to me, how did you name cells at the end: "Naive CD4 T", "Memory CD4 T", "B" and so on, and why did you name them so, why didn't you leave them as GZMK, FCGR3A, or they just have such names?
hi! it was not possible to download the raw data. does the link not work anymore? Satija lab's link does not work neither...
Perhaps they are down at that moment, I just tested and it was working for me at this moment
I created a volcano plot by enhance volcano.But in the plot instead of showing the gene symbol some kind of number are shown.So how can i solve this problem.
EnhancedVolcano(exp1, x = "logFC", y = "adj.P.Val",
lab = rownames(exp1),
FCcutoff = 0.6, pCutoff=0.01, cutoffLineType = 'twodash',
cutoffLineWidth = 0.4, pointSize = 1, legendPosition = 'none',
title = "", subtitle = "", caption = "", labSize = 3, ylim = c(0, 6)) +
theme_pubr(legend = "none").
exp1 is my dataset and the first column of that dataset is gene symbol.
Would you mind sending the script to.me in email?
@@LiquidBrain plz give your mail id
@@johirislam8174 will you please help me for one thing..
I always came over here with some hope to learn but that initial point about dataset 😣....
The data should be back now :)
i can not access to raw data. why?
Perhaps they are down at that moment, I just tested and it was working for me at this moment
CAnt download raw data, it gives following error:
AccessDenied
Access Denied
548G3NM2GXQM8VFW
D2RiKgdI96sfQm0QmNeVWqsEwQvGcDbGHYpOZq0WxcYcjBEhl6nJkQ1MxPE+dkkf6lkhsNYPCSc=
Can you help?
last steps was not clear to me ..