How to analyze RNA-Seq data? Find differentially expressed genes in your research.

If you benefit from my tutorial and use the same strategy for data analysis, please CITE my RNA-Seq paper published in "Scientific Reports - Nature": www.nature.com/articles/s4159...
And "PLOS ONE": journals.plos.org/plosone/art...
●Chu, C.P., Hokamp, J.A., Cianciolo, R.E. et al. RNA-seq of serial kidney biopsies obtained during progression of chronic kidney disease from dogs with X-linked hereditary nephropathy. Sci Rep 7, 16776 (2017).
●Brinkmeyer-Langford C, Chu C, Balog-Alvarez C, Yu X, Cai JJ, et al. (2018) Expression profiling of disease progression in canine model of Duchenne muscular dystrophy. PLOS ONE 13(3): e0194485.
------------------------------------
This is a class recording of VTPP 638 "Analysis of Genomic Signals" at Texas A&M University.
No RNA-Seq background is needed, and it comes with a lot of free resources that help you learn how to do RNA-seq analysis. You will learn:
(1) The basic concept of RNA-sequencing
(2) How to design your experiment: library prep, sequencing depth, budgets, statistical power.
(3) The analysis pipeline
(4) Useful resources (all free!!!).
Up-to-date RNA-Seq Analysis Training/Courses/Papers (Updated on Dec 2017)
www.biostars.org/p/174376/
------------------------------------
Other links are listed here:
1. Comparison of different Illumina Truseq RNA Prep kits:
www.illumina.com/documents/pro...
2. Scotty-Power analysis for RNA Seq Experiments:
scotty.genetics.utah.edu/
3. Illumina Sequencing Technology:
• Illumina Sequencing Te...
4. Ensembl FTP download website (include file format description):
useast.ensembl.org/info/data/f...
5. HISAT2: ccb.jhu.edu/software/hisat2/i...
6. HTSeq:
www-huber.embl.de/users/anders...
7. DESeq2 (click on "PDF" for manual):
bioconductor.org/packages/rele...
8. How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?
rnajournal.cshlp.org/content/2...
9. Gene Ontology: pantherdb.org/
10. IPA Webinars:
www.qiagenbioinformatics.com/...
11. Texas A&M Supercomputing Facility-trainings:
sc.tamu.edu/wiki/index.php/HP...
12. Command Line Tools for Genomic Data Science:
www.coursera.org/learn/genomi...
13. Informatics for RNA-Seq Analysis 2016:
• Informatics for RNA-Se...
14. www.rnaseq.wiki/
15. Informatics for RNA Sequencing: A Web Resource for Analysis on the Cloud:
journals.plos.org/ploscompbiol...
16. A survey of best practices for RNA-seq data analysis:
genomebiology.biomedcentral.c...
17. RNA-Seq workflow: gene-level exploratory analysis and differential expression:
www.ncbi.nlm.nih.gov/pmc/arti...
18. RNA-Seq Blog: www.rna-seqblog.com/
19. BioStars: www.biostars.org/
20. RNA-Seq Analysis Tutorial:
github.com/CandiceChuDVM/RNA-...

[@CandiceChuDVM]

If you benefit from my tutorial and use the same strategy for data analysis, please CITE my RNA-Seq paper published in "Scientific Reports - Nature": www.nature.com/articles/s41598-017-16603-y And "PLOS ONE": journals.plos.org/plosone/article?id=10.1371/journal.pone.0194485 ●Chu, C.P., Hokamp, J.A., Cianciolo, R.E. et al. RNA-seq of serial kidney biopsies obtained during progression of chronic kidney disease from dogs with X-linked hereditary nephropathy. Sci Rep 7, 16776 (2017). ●Brinkmeyer-Langford C, Chu C, Balog-Alvarez C, Yu X, Cai JJ, et al. (2018) Expression profiling of disease progression in canine model of Duchenne muscular dystrophy. PLOS ONE 13(3): e0194485.

[@jinli5752]

very useful!Thank you!

[@geraldjoaquin9159]

You prolly dont care but if you are bored like me atm then you can stream pretty much all of the new movies on Instaflixxer. I've been binge watching with my girlfriend recently xD

[@reyanshelian7837]

@Gerald Joaquin definitely, been using InstaFlixxer for since december myself :D

[@AqleemAbbas]

Candice chu Great Lecture

[@janakassem2776]

تاغتس172تدشءى. كشذ1مضشش

[@mariaelenadebellard3715]

This is the BEST video I had seen!!! Finally someone did the step-by-step in an approachable manner. Thanks Candice. Great job!

[@CandiceChuDVM]

Check out my new video "Grad School 101: Design a great slide presentation": kzbin.info/www/bejne/aX69hJWvZtVlotU

[@jadedavies9898]

This is one of the best tutorials I've seen on any subject, thank you so much for this!

[@sharanyamanoharan3482]

Even after years, i found this video is the best for explaining RNA-Seq analysis from top-bottom. Thanks a lot @Candice Chu.

[@deni9264]

Thank you so much for sharing your expertise on RNA-seq analysis. Clear and practical explanations. Highly appreciated!!

[@fatinnadhirahzulkifle3085]

I am a Bioinformatics degree student from Malaysia and I have no experience on NGS and RNA-seq analysis but watching this video makes me want to learn more. Thank you so much for sharing your knowledge. This video is very helpful :)

[@kaitokaito19]

Thank you so much Candice! This is a very well structured lecture and I absolutely loved it and finally understood the whole analysis pipeline! You are definitely someone I look up to and I hope one day I could present my research as well as you did! Thank you!

[@genemaker4976]

Thank you for a great video Candice, I have just started an Honours Project which includes RNA-seq and I feel like this hour just saved me at least a weeks work!!

[@devanna56]

The best video on RNA seq analysis, also with loads of resources. Thank you.

[@mohamadhasanansarizadeh7420]

Super useful! You know exactly what a beginner needs to know. And that's really interesting you introduce the websites. It keeps us updated. Thanks

[@markmccaulley5210]

What a great video! Aside from the well presented information, your tribute to your cat touches my inner emotions. As a soon to retire internal medicine physician, I am progressing in learning RNA-Seq technology in order to pursue my specific research interests. I appreciate your insights and applaud your kindness to learners and all creatures. Mark McCaulley, .MD, FACP

[@chocol0230]

Thank you for this clearcut guiding in the tremendous world of omics. Though, the last few minutes of your vid were perhaps the most valuable... Big hug, Candice!

[@arresteddoctor3563]

Thank you Candice. Brilliantly done.!

[@macarima]

Thank you for your video. This will be useful for starting RNASeq analysis. And congratulations on your paper!

[@natachamalu]

Thank you so much for this video! It opened my mind to know how to analyze my rna-seq data. It was really perfect! Congrats.

[@BERKKK]

This is very nice and very clear, thank you :)

[@sheliostrow3974]

This is a great video I learned a lot! thanks! and I am so sorry for your cat passing away to early. from one cat lover to another, I know the feeling too well, and send you my deepest condolences.

[@dumindasenevirathna4543]

Thank you Candice for your nice introduction to RNA seq.. i was wondering what and how around a year.. but now I'm in a clear pathway....please continue...

[@cprithiviraj]

This is a great intro seminar! Thank you, Candice.

[@mich5477]

This video gave me a thorough introduction to RNA-Seq. Thanks so much Candice!

[@joshymole]

Thank you Candice! This has been very helpful. I just about to start an RNAseq experiment and its really great to see the workflow from start to finish.

[@CandiceChuDVM]

You are welcome. You can now read the full research article at www.nature.com/articles/s41598-017-16603-y

[@paulxu2205]

Hi, Candice, thanks a lot for your pretty useful and funny video. If I had a teacher like you, I would conquer RNA-Seq data analysis much earlier.

[@jamesh508]

This was really helpful for me. Thank you.

[@DrKaranSingh-gc7yk]

Terrific information for RNA-Seq data analysis.

[@aisyahaziz238]

Very nice presentation. Thanks for sharing your experience and guidance.

[@hungnguyensg]

Many thanks Candice Chu for your super clear explanation and your sweet voice :)

[@kiplimosimon1429]

Excellent talk. I highly appreciate the good work. Currently working on rnaseq work.

[@azuandaud8208]

Thanks a lot, Candice Chu...Really appreciate it!

[@ModupehB]

Great recommendations and resources presented. Thank you.

[@Family_FSRA]

Thanks a lot for your detailed explanation. I got the idea how to proceed on this now.

[@olimikaboyo1648]

Thank you for the ellaborate explanation and resources. Most interesting talk I have heard about this topic (RNA-seq)

[@sunnyyoda5511]

Such a great video, i luv this. Thank you Candice

[@xunjialiu6828]

Very good video. Thanks Candice Chu.

[@sunnyyoda5511]

Excellent job, this was very clear!

[@cokuilda8461]

Very easy to understand and very helpful! thank you so muchhhh!!

[@fangweihan3586]

Thank you! Very helpful! Appreciate your work!

[@sameermudbhari69]

Thank you for the wonderful presentation

[@ayo4757]

Awesome work!! Thanks for all the info

[@ellangoramasamy1989]

Good tutorial for DEG finders. Thanks Candice

[@prakashd4]

Thank you for slow and nice presentation

[@juliaescandonbrehm5187]

Good job, thanks for the talk, enjoyed it and got a lot of tips from you!

[@amandap8215]

Thank you so much for this talk. It really helped me figure out what tools to explore using for my reserach. And I’m so sorry that your cat passed away.

[@rodongo5221]

Very informative. Kudos!

[@TheRednaxela100]

Thank you so much, easy to understand with great resources :)

[@amonoracheal9118]

Thank you so much for a clear and great presentation

[@sajilck8246]

Hi, Thank you so much for the informative presentation. I have few questions. Is it possible to calculate t & p values without replicates? Why some FastQc report gives boxplots without boxes and only lines?

[@zhengchensu5330]

Very nice tutorial, thank you so much!

[@guilingma9613]

Great presentation for starters. Thanks a lot.

[@himalayanplanespotter2021]

Thank you for this very informative video. It helped me a lot in my project.

[@johnalexander9861]

Very useful Candice, thank you for the video and useful resources -definitely helpful - ooo my pug died a while back, I know how you feel...stay blessed!

[@niceday2015]

Thank you very much for all the details. And I like your lecture very much.

[@helent.862]

Thank you, very helpful video!

[@threadtag]

Thanks, very useful

[@minasattari805]

Thank you so much for this useful video.

[@baidyanathdemandal6930]

Thanks a lot for this nice tutorial. Do you have any suggestions for the fungal RNA Seq analysis or I can still follow the protocol mentioned in the tutorial.

[@ming-jieluo8157]

You are great helpful. It's really interesting that I've already read your post on Biostar 2 weeks before. I thought this guy is really help. Wooh, see you again here in youtube.

[@koterm]

Thank you so much for your work!

[@yzp8193]

Thanks for the upload, very useful for me.

[@YTChannel_fun]

Thanks a lot for this video. Very useful for beginners like me.

[@anilkhar]

Liked your way of disseminating information. Keep it up.

[@RyonBang]

Great presentation.!!

[@bionic-world6754]

Thank you for this informative session. It was very helpful for me. I wanted to ask if you have resources available, can I get all.deseq2.R script. I am doing some work with plants around 30 samples.

[@lydouo]

看到一半突然看到繁體中文的google介面才驚覺你是台灣人! 謝謝您的講解 祝您在美國一切順利! 希望有天也能到達您的高度~

[@mariamkhaled6873]

Thank you very much for this video. I benefited greatly from this and will definitely try to use the same approach for my project. I'm so sorry you lost your cat; I lost mine 2 weeks ago and I know how it fells

[@tinyikonkuna1479]

Definitely using this my literature review thanks

[@utopianreality]

This is very useful, thanks!

[@dpkmcbl]

Thanks for the video. I need to use a published E. coli RNA seq data to see the particular region of interest. This is to find out the RNA reads in a region of interest. Do you have any video tutorial to do this alignment with the genome and visualize this data?

[@elliottkillian]

Great job teaching

[@StoicLoic7]

Thanks for the video! It was helpful to me. I also shared it on the r/bioinformatics subreddit as I think others might also benefit from it. The ressources you provide are great, both in quantity and in quality.

[@CandiceChuDVM]

Thanks for sharing :)

[@dongshengzhang3193]

It is very useful. Thank you a lot.

[@gangliu4658]

Awesome, thank you very much

[@hyrunnisa997]

This was really great to help me understand the entire process. I really enjoyed it. Is there a way for me to do this without having access to these packages like hisat2 and HTseq? I don't think I can download them on my computer and even if I did the flies that will be analyzed by this are too big to download. what are my option if I want to do an independent project?

[@w-berheazeba9464]

Thank you，very much helpful ！

[@aramiskessleragostini]

Thank you, your video help me alot

[@timothyhsu9418]

Hi, thank you for your video, a cat person study dogs, interesting.

[@doaarefat3317]

Hi,Thank you so much Candice! This is a very well structured lecture and I absolutely loved it, I am beginner and I need more help to understand RN sequence analysis.

[@mabillycoxhbdias9329]

Thank you a lot!

[@TommyCarstensen]

Thanks for the upload. Shame about the sound at times. Can you elaborate on why at 20:09 you chose HISAT2 (instead of STAR) for alignment and HTseq (instead of StringTie) for quantifying expression levels and DESeq2 (instead of Ballgown) for differential expression? Thanks!

[@CandiceChuDVM]

Hey Tommy, sorry for the late reply. I have been working on finishing my PhD and getting a new job lately. I wasn't very into STAR at the time I was doing the analysis, but now I am aware that more and more people are using STAR for alignment. As for the differential analysis, I briefly mentioned the reason in my paper (see the first comment). Michael Love, the creator of DESeq2, is on Bioconductor ALL THE TIME. He almost immediately answered all my questions. Alternatively, you can use EdgeR, but DESeq2 is more conservative so you get less false postive genes.

[@nicoleseng2877]

Thank you, is very useful.

[@williammo4450]

Good explanation.

[@leixiao169]

Candice, thanks for the video, do you mind sharing the "Presentation.R" script in your video (around 38:35 min)?

[@booJay]

Hi Candice, firstly, thank you for your video. I'm watching in 2021, but maybe like you I'm stuck in 2016 as my dog and best friend passed away about a month and a half before your cat (1996.01.09-2016.08.14), so I sympathize deeply. Life will never be the same, but I'm trying to throw myself into research to see what I can learn about what it all means...

[@Itshamsul]

Very interesting lecture:)

[@LiliMontreal]

nice tutorial!

[@yixu7765]

Thanks, helps a lot!

[@TheCookieSweety]

Could you provide the R-code for your analysis? I am quite new to R, but I would love to try and follow.

[@hateprotoss]

Hi Candice, thanks for upload the video. I have a question, do you know the source where I can upload example of RNA-seq data to practice?

[@CandiceChuDVM]

Do you mean download RNA-seq for practice? See this: www.biostars.org/p/111040/

[@oneminuteasmr6486]

Thank you for this video!! Very helpful and informative. Could you please make one for differential expression analysis using ballgown? I'm following the pipeline- HISAT STRINGTIE BALLGOWN. Thank you very much 💕

[@ashokmandal2064]

Hello, thank you so much for this video i am also planning to do my thesis on RNA Seq can i stay in touch with you

[@kojomawuli8663]

Hey Candice I’m wondering if your answer to the question about why RNA-seq is used instead of Protein profiling, you mentioned the current technology isn’t sensitive enough to profile all the proteins. But I think RNA- seq has the same problem. Are you able to sequence all the RNA molecules in present in that condition?

[@raphaeltavares6278]

Thanks a lot Chu! =)

[@fabbiotec]

YOU ARE SO SWEETTTTT

[@YusufKhanzhc]

hi, nice video , can you share code by link or mail. Have you used mixmodel and poisson for DE genes. any video related to that.

[@feitu6403]

Awesome talk for the new player in RNA seq! I am just wondering why KZbin recommends your channel to me later.

[@wenxiahe8251]

Dear Candice! Thank you so much and may I ask if I can have the complete R script on your vedio. Some of them has been out of the screen.

[@ardykharabian3453]

Hi Candice Thanks for nice tutorial. Is there any tool able to integrate results of proteomics with RNA-Seq?

[@stillsummer]

I am not doing proteomics analysis but you might be able to benefit from this article: www.ncbi.nlm.nih.gov/pmc/articles/PMC3637682/

[@ardykharabian3453]

Thanks for the article. So helpful! Another question; Can a missing biological replicate affect the transcripts abundance and differential expression pattern of genes? How a missing replicate can be dealt during the differential expression analysis?

[@emantoraih7700]

Thank you for this informative session. can I execute all the steps in R instead of python?

[@CandiceChuDVM]

I did not use python. I use Linux system in the supercomputer and then use R for differential analysis.

[@jonbas6]

Thanks a lot! :D

[@yzfmonster]

Bravo!

[@yuanhou616]

yzfmonster I

[@zesttenorista5235]

Im bioinformatic noob i wanna know @30:12. Is where index located? Do i need to create these files before runnign hisat2 right? And right now in manual it is . Is it the same? Thank you in advance

[@CandiceChuDVM]

Yes. is the index file. You need to create the index by yourself or download it from HISAT2's website: ccb.jhu.edu/software/hisat2/manual.shtml