DESeq2 workflow tutorial | Differential Gene Expression Analysis | Bioinformatics 101

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Bioinformagician

Bioinformagician

Күн бұрын

Пікірлер: 233
@animatedbiologywitharpan
@animatedbiologywitharpan 2 жыл бұрын
For an Indian PhD student like me (who is not familiar with Bio Info) this channel is a blessing . I will share it with my batchmates. Very nice youTube channel. Keep it up.
@Bioinformagician
@Bioinformagician 2 жыл бұрын
Thank you Arpan, I am glad you find my videos helpful! :)
@AbdullahSharabati
@AbdullahSharabati 2 жыл бұрын
So, even Indians needed tutorials? Sorry it's just a bad joke, please don't mind me, all respect. :)
@animatedbiologywitharpan
@animatedbiologywitharpan 2 жыл бұрын
@@AbdullahSharabati Yes why not we all need help. I actually learn from her channel quite frequently. As Indians, we have developed a culture of Peer Learning.
@AbdullahSharabati
@AbdullahSharabati 2 жыл бұрын
@@animatedbiologywitharpan I totally understand you and know you are meaning, I was just kidding, really, sorry
@AyrodsGamgam
@AyrodsGamgam Жыл бұрын
wow, you made it like a promenade in the park on a nice spring day. Thanks. Plz never stop making these videos, you are a true prophet!
@andreaseriksson4578
@andreaseriksson4578 Жыл бұрын
Thank you for an excellent and pedagogical video on how to operate DESeq2! I had some initial issues, as I had to replace the row numbers with my gene symbols (which where in their own column). But once I had that figured out, for both files (counts and coldata), everything worked smoothly. If someone has the same issue, use this script (for colData; similar process for counts): DF
@江藍-y4c
@江藍-y4c 6 ай бұрын
I very much appreciate that you gave a very clear and concise explanation of the workflow of DESeq2. I've learned a lot from it
@catherinewaaaang
@catherinewaaaang 7 ай бұрын
this is my first time using DESeq2 and your explanations and demonstrations in this video were amazing! tysm
@devinjones7271
@devinjones7271 2 жыл бұрын
SO HELPFUL!!!! I wish I knew about this channel during my phd...
@mayconmarcao4554
@mayconmarcao4554 2 жыл бұрын
Beyond your excellent content, you nailed with your channel name choice (bioinformagician) 🤣😂😂. Thank you!
@luckrols
@luckrols 2 ай бұрын
Thank you and congratulations! Wonderful of you to create and share this fantastic content!
@ChenyiChen-c8o
@ChenyiChen-c8o 8 ай бұрын
Your videos make every step so easy to understand!
@Spirrie2002
@Spirrie2002 5 ай бұрын
Your tutorials are some of the best on you tube for sure! Well done and thank you so much!
@asshimul1168
@asshimul1168 2 жыл бұрын
That's excellent magic indeed. You have done perfectly. Would you please create the next series according to the same data, how to analyze up and down-regulated gene comparison between treated and untreated groups by using box plot or something? It will be helpful as a newbie for me.
@Bioinformagician
@Bioinformagician 2 жыл бұрын
I will surely consider making a video covering downstream processing and visualization of these DEG :)
@Grzegorz-f1b
@Grzegorz-f1b 6 ай бұрын
Now I'm working on projekt and writing application reading disfunction expression From genotypem by cell repair In C++ - This video is very professional and helping Me to understanding data set From IT. Thanks You
@Grzegorz-f1b
@Grzegorz-f1b 6 ай бұрын
DeSeq2 - That I nedeed 💪❤️🙏
@MGRVE
@MGRVE 8 ай бұрын
Great tutorial. One comment: reducing the size of the input is not done primarily because of reducing the computational burden, but to lessen the impact from multiple testing correction.
@niharikasingh7677
@niharikasingh7677 Жыл бұрын
Your channel is extremely helpful to me and has been a real world saviour for gaining a fundamental understanding of my projects. I am working with gene knockout vs control conditions and will be using your pipeline to do the further analysis. Thanks again and keep up the amazing work!! 💛💛
@niharikasingh7677
@niharikasingh7677 Жыл бұрын
Hi again! I tried to use this method but I'm facing a small error from my end. The Gene IDs are a separate column and hence my no. of rows are not equal to the no. of columns. How did you ensure that the Gene IDs don't get counted as a separate column?
@aymsagagi
@aymsagagi Жыл бұрын
I am having the same problem !
@stretch8390
@stretch8390 2 жыл бұрын
Would you be willing to do a video on more complicated design setups for DESeq?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
Thank you for the suggestion. I will surely consider making a video covering this topic :)
@phakawatchusuth3181
@phakawatchusuth3181 Жыл бұрын
Thank you so much from Thailand!
@mamosangcala6499
@mamosangcala6499 Жыл бұрын
This was super helpful and easy to follow, thank you sooo much 🙌🙏💓. You are a star
@samuelyeo5450
@samuelyeo5450 2 жыл бұрын
Thanks for your tutorial! It was clear, concise and extremely helpful.
@momilan
@momilan 8 ай бұрын
Thank you!!!! -From Las Vegas, Nevada
@johnbaker3296
@johnbaker3296 Жыл бұрын
Hey great stuff! I was wondering, what if you wanted to compare treated vs untreated but per cell line, would you have something in your design when creating your deseq object like (design = cell_line + condition) or is this extracted using contrasts or both?
@audebenigneikuzwe4531
@audebenigneikuzwe4531 2 жыл бұрын
thank youuuuu, you just saved my life literally
@RaquelAjalik
@RaquelAjalik Жыл бұрын
Absolutely amazing! Thank you so much! You are so gifted.
@andrenicolau3824
@andrenicolau3824 2 жыл бұрын
Congratulations for your channel. I'm subscribing because of this video and your clear explanation...
@user-db2os6sr8s
@user-db2os6sr8s 2 жыл бұрын
Medical student who was struggling with this ! You're so kind and helpful, Thx!! And I'm curious about how to export the DESeq2 results into csv file or Excel file to check which gene is on the Upper/Lower right quadrant on the MA plot.
@Bioinformagician
@Bioinformagician 2 жыл бұрын
This is how you can export your results: write.csv(as.data.frame(results), file="results.csv")
@Sadin15
@Sadin15 2 ай бұрын
Question: For the DESeq summary, what constitutes 'low counts," and what is the "mean count < 6" mean?
@Ice84letters
@Ice84letters 5 ай бұрын
Excellent videos, what does it means the following error when doing the DeSeq matrix? "In DESeqDataSet(se, design = design, ignoreRank) : some variables in design formula are characters, converting to factors" thank you very much
@shamimashrafiyan8591
@shamimashrafiyan8591 3 ай бұрын
thanks for the video, If I want to skip "estimate size factor" part, how can I do it? because my data is deseq normalized of expected counts.
@umarsheikh1992
@umarsheikh1992 2 жыл бұрын
Thank you the tutorial, was highly helpful and informative.
@ahmadnajem9762
@ahmadnajem9762 7 ай бұрын
Thank you so much for your videos. I would like to learn the pipeline for RNA seq data analysis and I am wondering if there is any order to follow up on the videos. Thank you so much
@abc_ratio
@abc_ratio Ай бұрын
You don't do shrinkage before getting the MA plot ?
@RicardoRodriguez-yu8ss
@RicardoRodriguez-yu8ss Жыл бұрын
I have learned a lot from your videos! you are the best :)
@freezingtolerance7493
@freezingtolerance7493 Жыл бұрын
Hello. I have an quick question in terms of normalization. Since Deseq2 itself has a normalization algorism, I do not need to do further normalization such as FPKM? Or, before performing Deseq2 run, should I first do normalization my read count data?
@marioperez8302
@marioperez8302 Жыл бұрын
Your videos are wonderful! would you consider expanding on the use of contrast, perhaps a demonstration with a sample with 3 conditions or more exploring the results? Thank you for considering it and keep up the great work you are doing!
@Bioinformagician
@Bioinformagician Жыл бұрын
I will surely consider making a video on contrasts. Thanks for the suggestion.
@kjeyaprakash2638
@kjeyaprakash2638 5 ай бұрын
Sorry if my question is wrong. You have done DEseq with raw gene counts. is not required to convert these id to gene name and normalize to FPKM or TPM for further analysis?
@naveedkhan-fi6ux
@naveedkhan-fi6ux Жыл бұрын
it was very easy and informative..... but I really wish you could also work on rice genome
@jgitau001
@jgitau001 2 жыл бұрын
Very well explained, thank you preparing this video. . .
@stanyang4321
@stanyang4321 Жыл бұрын
very well explained. Can you tell how to plot heatmap for the data you analyzed in this video ?
@danielaalejandragonzalez72
@danielaalejandragonzalez72 27 күн бұрын
Thaanks for your amazing tutorial. I am in the NCBI >Geo dataset part to retrieve the information related to the dataset, but I can't know which file I should download to have it as counts_data.csv. Any leads will be deeply appreciated
@Sadin15
@Sadin15 Жыл бұрын
Thank you so much! This was incredibly helpful.
@manavgandhi2503
@manavgandhi2503 2 жыл бұрын
Hello, I really loved the walkthrough of DESeq2. Definitely going to follow your channel. I have a question. I have a counts matrix with following groups: control, treatment 1, treatment 2, treatment 3. First, I need to draw comparison between each treatment and control as reference and then compare between treatments. While setting the factor level, if I use control as the reference, then will it draw comparisons as follows: control vs treatment 1, control vs treatment 2 and control vs treatment 3? I believe I can use the contrast function for getting the comparison between treatments? Thank you!
@Bioinformagician
@Bioinformagician 2 жыл бұрын
I am glad my video has been helpful! Regarding your question, yes you can use contrast to make a comparison between treatments.
@manavgandhi2503
@manavgandhi2503 2 жыл бұрын
@@Bioinformagician Thank you. Could you also confirm if setting the reference as control for factors would draw the same comparisons that I wrote?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
@@manavgandhi2503 Yes, setting control as the reference will allow you to make comparisons between control and treatment levels. Only difference being, you will be able to see the order reversed i.e. "condition_treatment1_vs_control", which essentially gives you genes up/down regulated in treatment 1 compared to control.
@manavgandhi2503
@manavgandhi2503 2 жыл бұрын
@@Bioinformagician Got it. Thank you!
@abhisheksawalkar1018
@abhisheksawalkar1018 Жыл бұрын
Greatly explained. Thanks
@Paachi8651
@Paachi8651 21 күн бұрын
I have one doubt Deseq is the only tool to identify deg in NGS RNA-seq data(illumina).? Can we use R studio limma to identify DEG for same dataset NGS RNA seq data?(illumina )?
@sanjaisrao484
@sanjaisrao484 Жыл бұрын
Mam help. Which is better mam, ballgown or DEseq2 ?
@sachithrak.yaddehige6251
@sachithrak.yaddehige6251 Жыл бұрын
It was very helpful and clear. Thank You
@bobyang8491
@bobyang8491 2 жыл бұрын
Thanks a lot for making this video! This is very very help ful!!!
@adeyemioluwaseun334
@adeyemioluwaseun334 Жыл бұрын
Easy to understand video. thank you
@smritimohanty3483
@smritimohanty3483 Жыл бұрын
Hey. Thanks for the video. Can you just lemme know that whether you took one data or two different types of data?
@poulamigoswami8008
@poulamigoswami8008 2 жыл бұрын
When we are performing de novo, how to make counts and sample info files? Can you suggest me any tool for making those files
@deepshikharathore4182
@deepshikharathore4182 7 ай бұрын
kindly share a video on how to perform differential expression analysis of transcriptome data from TCGA database
@joseantonioduarteconde8743
@joseantonioduarteconde8743 2 жыл бұрын
Thanks for your useful video! I am a beginner and I have some problems. When I upload my airway package (It is done well) I do not get to obtain the files.csv in my file folder. It looks like nothing happens. Is there another way to get them?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
Can you give me exact commands you ran to get the data?
@vetlove4056
@vetlove4056 4 ай бұрын
How did you take that geneids to the serial number ?? Please guide mee
@Juliossca
@Juliossca 2 жыл бұрын
This is so nicely explained, thanks for your videos :3
@grsbiosciences
@grsbiosciences 2 жыл бұрын
What are technical replicates and biological replicates madam
@Bioinformagician
@Bioinformagician 2 жыл бұрын
I have explained this in one of my previous video - kzbin.info/www/bejne/iWKzlIdrp9VrmZY
@alvaroruiztabas5627
@alvaroruiztabas5627 2 жыл бұрын
Congratulations for the video. Very helpful. I am having problems with the DESeq2 installation, R tells me that the path is not writeable. Any help? Thanks
@Bioinformagician
@Bioinformagician 2 жыл бұрын
Can you paste the exact error?
@alvaroruiztabas5627
@alvaroruiztabas5627 2 жыл бұрын
@@Bioinformagician Of course, when I run "BiocManager::install("DESeq2")", in the end of the run the console show "There were 16 warnings (use warnings() to see them)" and hen I run "warnings()", R shows 1: In install.packages(...) : installation of package ‘png’ had non-zero exit status 2: In install.packages(...) : installation of package ‘curl’ had non-zero exit status 3: In install.packages(...) : installation of package ‘openssl’ had non-zero exit status 4: In install.packages(...) : installation of package ‘RCurl’ had non-zero exit status 5: In install.packages(...) : installation of package ‘RcppArmadillo’ had non-zero exit status 6: In install.packages(...) : installation of package ‘httr’ had non-zero exit status 7: In install.packages(...) : installation of package ‘GenomeInfoDb’ had non-zero exit status 8: In install.packages(...) : installation of package ‘Biostrings’ had non-zero exit status 9: In install.packages(...) : installation of package ‘GenomicRanges’ had non-zero exit status 10: In install.packages(...) : installation of package ‘KEGGREST’ had non-zero exit status 11: In install.packages(...) : installation of package ‘SummarizedExperiment’ had non-zero exit status 12: In install.packages(...) : installation of package ‘AnnotationDbi’ had non-zero exit status 13: In install.packages(...) : installation of package ‘annotate’ had non-zero exit status 14: In install.packages(...) : installation of package ‘genefilter’ had non-zero exit status 15: In install.packages(...) : installation of package ‘geneplotter’ had non-zero exit status 16: In install.packages(...) : installation of package ‘DESeq2’ had non-zero exit status I don't really know how to fix it. Thanks!!
@anamikapandey4769
@anamikapandey4769 2 жыл бұрын
@@alvaroruiztabas5627 install all the dependencies one by one, your problem will be resolved
@CynthiaFrancis-sv4rc
@CynthiaFrancis-sv4rc 4 ай бұрын
This was great! Thank you.
@soniabachamp347
@soniabachamp347 2 жыл бұрын
Thank you so much for preparing this video for us. It was extremely useful! I will definitely subscribe to your channel!
@AdekunleAjiboye-x1x
@AdekunleAjiboye-x1x Жыл бұрын
Hi. Thanks for the video. Please how do I view all the genes (expressed and unexpressed)?
@acramulhaquekabir5852
@acramulhaquekabir5852 2 жыл бұрын
Are you planning to do a video on Gene enrichment analysis?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
Yes, I surely have plans to make a video on it.
@awesomemiso
@awesomemiso 2 жыл бұрын
Thank you, awesome explanation, I am now a subscriber :)
@OuanhPhomvisith
@OuanhPhomvisith 11 ай бұрын
Hello, thank you so much for sharing the very helpful vdo. I want to know that when you load read counts data, the head of your read counts table shows only 8 columns of sample data excluding gene_id column, but when I do with my data, it still shows 9 columns (9 variables) including gene_id column. So, how can I do as you did? @Bioinformagician
@ahmedal-mammari9639
@ahmedal-mammari9639 2 жыл бұрын
thank you so much for this very help videos, can you plz explain why you didn't do cpm tpm rpk rpkm before DESQ?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
DESeq2 requires raw un-normalized read counts as it performs its own set of normalization steps. CPM, TPM, RPKM are all normalization methods that DESeq2 does not use.
@amus21455
@amus21455 2 жыл бұрын
really helpful! Thank you so much! But would love to know the explaination of each command. Like what do "~", "," do in the command. Thankssssssss
@liviagozzellino7266
@liviagozzellino7266 2 жыл бұрын
Hi, thank you for your explanation!!! Very useful video :) I only have one question: once I got the results, how do I select the most differentially expressed genes? Let's say I wanna view only the top 40 genes.
@Bioinformagician
@Bioinformagician 2 жыл бұрын
You can sort your results by largest log fold changes and lowest adjusted p-values. The first 40 genes on your list are the ones which are most differentially expressed. NOTE: log fold changes can be positive or negative, if you sort log fold changes descending, you will only get top genes with largest positive fold changes. If you wish to get top differentially expressed genes irrespective of the direction, you can sort by taking absolute log fold change values.
@CooktoCope
@CooktoCope 2 жыл бұрын
Can you please let me know from where did you get the metadata file?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
You could fetch metadata associated with your samples using GEOquery's phenoData() function.
@CooktoCope
@CooktoCope 2 жыл бұрын
@@Bioinformagician Thank you so much. I prepared the metadata file accordingly. I am doing Bioinfo for the first time. Your channel came in handy. Thanks👍
@munibabashir6951
@munibabashir6951 2 жыл бұрын
Informative video. Thanks I have a query regarding data analysis if you could please help me in that. I have a data set for tumors that I downloaded from cancer data portal so now I have gene expression data and clinical data for both tumors. I want to compare the gene expression of both tumors but I am no getting from where I should start, how can I compare these tumors by using DESeq2. Please guide me. Thank you
@Bioinformagician
@Bioinformagician 2 жыл бұрын
A couple of questions - 1. What data have you downloaded - RNA-Seq reads or quantified expression values? 2. What is the format of the data - are these raw counts or normalized expression values?
@ayeshatariq8774
@ayeshatariq8774 Жыл бұрын
Hi, thank you for this amazing video. I am currently doing a gene expression analysis. Even though I have the same row and col names in my counts and coldata I am still getting the FALSE arguments for all(colnames(Counts) %in% rownames(Coldata)) can you please help with that?
@juliangrandvallet5359
@juliangrandvallet5359 2 жыл бұрын
Thank you! How can I plot now the heatmap? something like > heatmap(as.matrix(res)) ??
@furkankurtoglu_sys_bio
@furkankurtoglu_sys_bio 2 жыл бұрын
Thank you very much! Such a great video!
@julioavazquezm6294
@julioavazquezm6294 2 жыл бұрын
Hello there, thank you so much for all amazing tutorials. Quick question: If I trying to analyze a counts normalized matrix (median of ratio DESeq2) Do I need to run DEGA? or log2 to that counts matrix?. Thank you so much for your help, I naive bioinformatician
@toanphanvan9739
@toanphanvan9739 2 жыл бұрын
Fot the "set factor level" step, What should I do if I have 3 levels and I want to compare gene expression between three level?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
You could do pairwise comparisons first and then take an intersection between them.
@kobrarahimi9164
@kobrarahimi9164 2 жыл бұрын
well done! wait for more videos.
@tankkar9995
@tankkar9995 2 жыл бұрын
May I ask how to download the CountData file in this tutorial please.... I tried looking at the study page but i just couldnt find a way to download the csv file.
@Bioinformagician
@Bioinformagician Жыл бұрын
Here is the code to get the data: github.com/kpatel427/KZbinTutorials/blob/main/getData.R
@ethanvouzas6255
@ethanvouzas6255 8 ай бұрын
Supremely useful!!!
@bzaruk
@bzaruk 2 жыл бұрын
how would you do a differential expression between multiple cell lines? do them in pairs and then find the shared highly differentially expressed genes? or is there a way of doing it in one analysis?
@aliciagarciaalonso6930
@aliciagarciaalonso6930 Жыл бұрын
Same issue!! I guess one may be able to do this comparisons in one go using the 'contrast' parameter of the result function? But I haven't checked it out...
@manuelsokolov
@manuelsokolov Жыл бұрын
Hi! I have one question, if data is given in the TPM format can you still apply the DESeq2? Does it only work with raw data? Thank you!
@jkim9931
@jkim9931 2 жыл бұрын
I enjoy your videos! I have one question about your design. It seems like there are two categorical variables - cellLine and dexamethasone in the colData table. Is there some reason you didn't include the cellLine variable in the design matrix?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
You are right, I could have used both. However, I wanted to keep it simple for this tutorial and explain how the DEseq2 works for one design factor (i.e. dexamethasone) and so my goal for this analysis was to study the effect of treatment on gene expression. I could have used complex designs like ~ cellLine + dexamethasone, if I wanted to test for the effect of dexamethasone while controling for the effect of cellLine. But in this case, to keep it intuitive I chose to demonstrate with one factor. Hope that answers your question. Thank you! :)
@jkim9931
@jkim9931 2 жыл бұрын
@@Bioinformagician Thanks for the explanation. I was thinking about that. This is a tutorial video so it doesn't have to be more difficult. I think forming a design matrix is involved in linear models which is another topic to explain. Thanks!
@Bioinformagician
@Bioinformagician 2 жыл бұрын
@@jkim9931 That is true, I have tried to explain linear models in my previous video (kzbin.info/www/bejne/ZpOVZaCmr7KSa68). But yes, it can be a whole separate video in itself!
@justsoil15
@justsoil15 Жыл бұрын
when i use my data, i have error "more columns than column names". I check your file and see 2 files are same format. Why can you read file without error?
@jahanshanzida7697
@jahanshanzida7697 2 жыл бұрын
You did a great job
@mithunrock5427
@mithunrock5427 2 жыл бұрын
Great video can i know what is your qualification and what do you do?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
I have talked about my qualifications and what I do in one of my videos - kzbin.info/www/bejne/r5WbfWqZhc98Z7s
@mithunrock5427
@mithunrock5427 2 жыл бұрын
@@Bioinformagician Hy myself Mithun I have done my undergraduate in bioinformatics and currently pursuing my postgraduate in bioinformatics in Reva University and I am interested to do my PhD in US so I think u can guide me can I have u r mail id or insta Id so I can contact you about this.
@Bioinformagician
@Bioinformagician 2 жыл бұрын
@@mithunrock5427 Hi Mithun, you can reach out to me with your questions on LinkedIn/email, you can find the details to contact me in the description of every video.
@PharmaAI-LearningCenter
@PharmaAI-LearningCenter 2 жыл бұрын
Hi, I am new in this domain so please tell about how you got expression data for GSE52778 and how to club all 8 sample data in one csv file.
@Bioinformagician
@Bioinformagician 2 жыл бұрын
The data was already merged and provided by the authors. However, if you are interested to learn how to merge datasets, I have previously covered it. Here's where you can find it - kzbin.info/www/bejne/fqPFlpR7f9Z-mbs
@fizzahzulfiqar2884
@fizzahzulfiqar2884 Жыл бұрын
@Bioinformagician very helpful. can you please make vedios on functional annotation of RNA seq data. It would be very helpful
@humerainayat2858
@humerainayat2858 2 жыл бұрын
I am getting this error when I am trying to create dds Error: unexpected '=' in: "dds
@Bioinformagician
@Bioinformagician 2 жыл бұрын
"dds
@ytz-i3h
@ytz-i3h Жыл бұрын
so wonderful!!!! thanks a lot!
@shilpisehgal5613
@shilpisehgal5613 2 жыл бұрын
Keep up the good work.
@yipan3694
@yipan3694 2 жыл бұрын
Hi, do you mind sharing your .csv files?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
Unfortunately, I don't have these files saved, I got these files for the tutorial using this script - github.com/kpatel427/KZbinTutorials/blob/main/getData.R
@prabirbarman877
@prabirbarman877 2 жыл бұрын
Error in `[.data.frame`(countData, , rownames(colData)) : undefined columns selected
@adekunleajiboye1244
@adekunleajiboye1244 Жыл бұрын
Hi I’ve been trying to reach you. Please when will you be available for a meeting?
@ashasanu1988
@ashasanu1988 2 жыл бұрын
Hello Madam Thanks for you video, I am having 12 samples, in that 3 controls, 3 one trtmt, 3 another trtmt, 3 another trtmt,. So like this when there are 4 conditions, how to perform DESeq of those?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
Did you try following my video or the DESeq2 vignette? Could you tell me what you tried and what didn't work?
@saswatsatapathy658
@saswatsatapathy658 Жыл бұрын
I get stuck at 7:44 when we put "design = ~ dexamethasone" - Its shows an error of "some values in assay are negative"!! Can someone help here
@paradoxoftrips
@paradoxoftrips Жыл бұрын
Hi, I am PhD student struggling to use the R studio. would you please help me to perform differential abundance analysis for my data, please let me know, I will mail you the data. I would be very grateful to you.
@alexyang274
@alexyang274 2 жыл бұрын
absolutely great videos
@excelobiageli9446
@excelobiageli9446 2 жыл бұрын
Nice video! Really helped, but I have 3 sample groups or levels. I have done pairwise comparison between the levels, but I don't know how to get final results
@Bioinformagician
@Bioinformagician 2 жыл бұрын
You can just intersect DE genes between both those comparisons.
@excelobiageli9446
@excelobiageli9446 2 жыл бұрын
Okay, but how will my final result be like? Like the table containing the degs, will it still contain the original number of samples? And what values would it contain? That is where I am confused
@Bioinformagician
@Bioinformagician 2 жыл бұрын
@@excelobiageli9446 So for each pair-wise comparison, you will have a data.frame with differentially expressed genes, log fold change values, p-values, min.pct and q-values. You will have 2 such data frames from each comparison. You can filter differentially expressed genes based on p-values and/or q-values and log fold changes, and intersect genes column from both data.frames. So what you end up with a vector of genes differentially expressed from both comparisons.
@excelobiageli9446
@excelobiageli9446 2 жыл бұрын
@@Bioinformagician okay!! Thank you very much
@shilpisehgal5613
@shilpisehgal5613 2 жыл бұрын
Could you please make a video on how to collapse technical replicates? Thanks in advance.
@Bioinformagician
@Bioinformagician 2 жыл бұрын
Collapsing technical replicates could be done by collapseReplicates() function in DESeq2. I will surely plan on making a short video explaining it. Thanks :)
@shilpisehgal5613
@shilpisehgal5613 2 жыл бұрын
@@Bioinformagician Thank you so much. I am looking forward to it.
@devinjones7271
@devinjones7271 2 жыл бұрын
In my metabarcoding dataset, I have multiple factors that I am interested in testing for differences. Does the factor level matter? Would I need to re-run DESeq with different factors for each of my different questions?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
If I am understanding your question correctly, factor level does not matter. All levels are compared with a reference. Even then you could create contrasts between different levels. contrast in results() will allow you can compare any two groups you want.
@devinjones7271
@devinjones7271 2 жыл бұрын
@@Bioinformagician Great! So if I am interested in looking at time, sex, age, season, and a few other factors downstream analysis, I can just use something like sequencing run for my design to normalize across different sequencing runs and then pull that normalized data for other analyses?
@tushardhyani3931
@tushardhyani3931 2 жыл бұрын
Thank you for this !!
@prachimishra5517
@prachimishra5517 2 жыл бұрын
While creating dds, I am getting a error that "count matrix should be numeric, currently it had mode: Character. Can you please tell me how to resolve.
@Bioinformagician
@Bioinformagician 2 жыл бұрын
Your counts data matrix might have some values as character, you need to convert all values to numeric. You can do that by running: apply(counts_data, 2, as.numeric)
@prachimishra5517
@prachimishra5517 2 жыл бұрын
Now it is showing, NAs introduced by coercion
@Bioinformagician
@Bioinformagician 2 жыл бұрын
@@prachimishra5517 Is it possible for you to email me a screenshot of your counts matrix and the commands you are running on my email? You will find my email in the description of the video. Thanks!
@kobrarahimi9164
@kobrarahimi9164 2 жыл бұрын
I have a question, you chose 10 for filtering genes. Is there any method to find this number? I used 10 for my data analysis, I got a lot of reads that was differentially expressed. and I think it is wrong. please help me, if there is method for finding filtering threshold.
@Bioinformagician
@Bioinformagician 2 жыл бұрын
I followed the threshold provided in the vignette. There is no method to find a threshold, it depends on how relaxed or stringent you want to be while filtering for genes. However, you need to be careful as having a higher threshold might filter out genes that may be differentially expressed but are not highly expressed. You said you found a lot of reads differentially expressed, can you explain why you think it is wrong?
@kobrarahimi9164
@kobrarahimi9164 2 жыл бұрын
@@Bioinformagician I got the data from ncbi and working on it. In the summary that was provided with the data, it is written that they are comparing knockdown strain with wild strain in both irradiation and unirradiation condition. They found 31 genes that was differentially expressed. Unfortunately, they did not submit the protocol in supplementary data. When I run this command with threshold 10, I get more than 3000 genes. I dont know where is my problem.
@kobrarahimi9164
@kobrarahimi9164 2 жыл бұрын
And when I set the pvalue
@Bioinformagician
@Bioinformagician 2 жыл бұрын
If you have retrieved the data from NCBI GEO, you will find associated publication. Read through the paper to find out what thresholds they used and why. Without those, it is really difficult to re-create what they found. You should also be interested in "why" those thresholds were used, it is important to be able to justify your choices.
@rajatranjan974
@rajatranjan974 2 жыл бұрын
How can I start learning Bioinformatics from scratch ? what are the major skills required to be expert in Bioinformatics?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
I think the best way to introduce yourself to bioinformatics is to take an online course. There are a lot of starter courses offered on platforms like Udemy or Coursera. There are various online bioinformatics workshops available as well - www.ecseq.com/workshops/workshop_2022-03-A-Practical-Introduction-to-NGS-Data-Analysis-Online-Course.html Skills required to be an expert? I don't know either, I am figuring it out too. lol
@maniskarki
@maniskarki 2 жыл бұрын
Hi, can I get the dataset used for this video?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
This is the code to get data - github.com/kpatel427/KZbinTutorials/blob/main/getData.R Did you try that?
@fabioseiva-uenp9155
@fabioseiva-uenp9155 2 жыл бұрын
Congrats for your videos! They are really, really very useful and well explained. Just one question, maybe you can help me; Do you know how can I find, using R, the gene names or symbols from the ENSG numbers?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
I am in the process of making a video on it. Please stay tuned! :)
@fabioseiva-uenp9155
@fabioseiva-uenp9155 2 жыл бұрын
​@@Bioinformagician Thank you for your prompt reply. You can bet I'll be tuned. Also, could you tell me if it is possible to get the gene names from the ASHG numbers?
@Bioinformagician
@Bioinformagician 2 жыл бұрын
@@fabioseiva-uenp9155 I haven't dealt with ASHG numbers, can you tell me what database are they associated with?
@fabioseiva-uenp9155
@fabioseiva-uenp9155 2 жыл бұрын
@@Bioinformagician Ok, I am learning about using datasets, extracted from GEO, so maybe I am asking the wrong question. The dataset I am referring to is GSE55191. After extracting the data, the ID I have is based on ASHG. Sorry for not being more specific, but if you could take a look and answer me, I would be extremely grateful.
@Bioinformagician
@Bioinformagician 2 жыл бұрын
@@fabioseiva-uenp9155 Found the mappings - www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL24530
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