I can't believe it. I was actually crying and panicking when I clicked on the video because of all that stress caused by "I don't know, I don't understand" thoughts. And your intro just killed me XD
I've learned more from this 18-min long video, than my whole course in transcriptomics. You are so good at explaining these complex processes. Thank you!
I know the feeling. Transcriptomics is so complex that most people, including those working in the field, seem to get "Betriebsblind" and unable to explain what they are actually doing. Josh Starmer is awesome, I just whish we had instructors like that in every research facility.
@@statquest same for me, I've understood this concept really easily thanks to you ( better than what I've understood in a 2h genetic engineering class )
Hey Josh, I just started doing my RNA sequencing and your video was very useful for me to get the basic idea behind the RNA sequencing. I spent hours and hours reading papers and didn't understand a thing about it. I really appreciate what you have done. I think your channel will play a big role in my research. Hats off your efforts in coming up with useful informative videos like this.
Awesome! That's great news. Since I made that video, I updated my PCA video to be easier to understand, so if you're interested in that, check out: ua-cam.com/video/FgakZw6K1QQ/v-deo.html
@Highlander Yeti same for me! Just found out we're going to do RNAseq, immediately went on UA-cam search instead of the classical science databases, came out here! I was already looking into (often expensive) courses, but it is all online! This is the future of learning. I should have had stuff like this 12 years ago to study Biomedical Sciences. That's why I'm on a mission helping future/current Biomeds and life scientists discover channels like this. Already BIG thanks from my part, StatQuest! I've yet to check out the rest of your stuff, but I WILL!
First you got me through my statistics course. Then you pulled me through my machine learning courses. Now, you will save me in my graduate genetics lab. Thank you so much Josh.
8:48 Two semesters of Bioinformatics and all I wanted was the sequence "GATTACA" to appear on any one slide during a lecture. Thanks, StatQuest for making my wish finally come true!
Call this a 'gentle introduction' if you want. It is really just a high level overview of the process. Such presentations are important to building students mental models of any subject, and honestly are frequently overlooked in many subjects in my experience. Great stuff.
Hey! I'm Brazilian, and I've been learning English and molecular bioinformatics. I'm grateful for this video. Thanks a lot, your pronunciation is easy to understand and you have good didactics too.
So the "+" symbol used to be a section where individuals could ad more info about their sequence. This is back when we had far smaller output. Since then, data has grown so much that we have developed tools to go through the data more efficiently. In the end, the "+" is simply an artifact from the early days of these files.
I'm not so sure about that, I think it was like that at the origin of this method, but in the new type of document layout, the + symbol could stand for "+ strand", which is often more investigated than the "- strand" of the double helix. I could be wrong, for sure, but I remeber like that
I am seeing this video in December 2021 and I can't thank you enough for this incredible concept clearing video of RNA-seq. Very Concise and subtle explanation, Besides your ENGLISH is really understandable for other language speakers. I am gonna check another playlist too.
This is actually one of the easiest-to-understand videos I have ever watched about this subject. Thank you for making it so that I can actually understand the information being taught!
@@statquest same comment! patiently explained without assuming we're all experts, written text of what you are saying verbally, simple, pedagogical, and systematic. It's great teaching, and so happy it's free!
Hey JOSH…First thanks for making such great videos over the years. I see that you even left your job as a professor and have devoted full time to make this stuff since 2017. Appreciate it as it is difficult to break your comfort zone. One thing I would say after reading books on mastery is that all elite masters have this skill to explain a tough concept in 2 minutes and they can drag the same concept for hours and hours. From thousands of comments on your videos, majority says you make things extremely simple which is a validation of mastery. The second aspect of elite masters are after working for about a decade and mastering a field then they bring innovation or a new thing (a new concept, theory, a formula, anything that was never explained). I’m sure this would be that phase and stat quest is part of that, and I hope many things would come along with your creativity which you have exploited to make this elite content. There are masters but very few have this skill to make things simpler. Many people might comment that you have mastery or talent in statistics or in music but I can say without any doubt that your talent lies is making complex things easier (the medium could either be through a video or through a book don’t matter), but that’s your core talent which you have used. So I wish you keep using this rare talent and bring more contribution to all the humans. I will certainly support statquest by purchasing the guides and encourage all who have benefited from this content. Thanks again and lastly is there any email if I could send you a message or share something of value. Will appreciate it.
Oh wow. Thanks for the explanation! I didn't really understand this concept until now. I've watched this video 3 times now because it is so comprehensive. Thanks a lot.
Hey. I'm a clinician trying to do a basic sciences post doc. I haven't touched my biology notebooks in 10 years... and your Video makes it look... like I could maybe, one day, perhaps... UNDERSTAND transcriptomics. (Also makes me feel like an idiot, but in a gentle and kind manner). Thanks
My God Lord this was heaven sent. Any you guys are hilarious! I don't think it's "excessive self promotion" at all, I'm going to watch your PCA one right now!
Thank you for the clear explanations. This was the first time I learned how the machine reads the bases by taking pictures from the top with single base annealing intervals. It was very informative. I am heading to watch your other videos. Many thanks!
Normally I get bored very quickly from explanation but hey this didn't feel like 18 mins at all :D Very useful content and you explain the ideas in pretty well organized way. Thank you that was really helpful ✔
These are great, I’ve been inundating myself with media about genomics and bioinformatics, but a lot of the specific techniques and terminology were still going over my head. Thanks for the thorough, but no-nonsense explanations!
"+" sign in raw data indicates the forward strand. If paired-end sequencing was performed there will be files with"+" or "-" signs that indicate forward or reverse strands, respectively.
this makes sense… i think its easy for the machine to figure out in the beginning since probably the oligo adaptors, which bind to the flow cell floor, are probably colour-coded for forward and reverse adaptors, and the adaptors themselves, are also probably colour coded, and the camera can pick up on this at the start, which means the algorithm can interpret forward and reverse strand locations and write it to the file.
@@statquest I want to say a hesitant yes :D I remember reading about orientation representation in NGS somewhere: It is almost universally + because only the forward/reverse strand is kept as one of the final steps of sequencing.
According to the "+" in fastQ, I have heard once, that in this line it used to be a repeat of the sequence name without "@" at the begining, to ensure that quality regards to the sequence above, but to lower file weight it has been switched to the "+". That's what I have heard, but it would be worth to approve somehow this information, I have no sources to do that.
I was taught in my course in metagenome and microbiome analysis, that the + line exists so you can put comments there! Never seen one with comments though.
MrErPeeeg is correct; but also it signals the end of the sequence lines. FastQ spec: @
+[]
A requirement is that the following '+' is optional, but if it appears right after '+', it should be identical to the following '@'. maq.sourceforge.net/fastq.shtml
The reason is that FASTQ is a combination of FASTA and QUAL files. Each of those has identifier followed by data. FASTA has sequence, QUAL has quality scores. interleaving these two pieces of information (2 lines from each file "zippered in") gives us 4 lines where 2 of the lines are identical. Later on, when machines got smarter, the third line became obsolete (and expensive space-wise) but is retained in a stunted form for backward compatibility.
I loved this explanation!!!. It is the most didactic I have seen of all. Congratulations and many thanks. It was very useful. I really appreciate what you have done.
I'm just starting my internship for my bioinformatics studies. Just watched the intro and feel so blessed to have a Video like this. So... thanks a lot ^^
Hi！This might be too late to ask. Firsst of all, thanks for your video, I have no experience with RNA sequencing and struggled to follow through during my bioinformatics class in Graduate school, this video is helping me understand!!! I was wondering what does a sample actually refer to. I though about them as a group of amplified target DNA being sequenced and read. But at 10:55 there is an explanation about bulk RNA sequencing sample that made me confused. Would you mind clarifying this? Thank you very much!
It really depends. A sample could be RNA from an individual tissue from an individual person, or it could be pooled from that person, or it could be a pool of people. So, "sample" refers to whatever unit the experiment is based on.
Thank you so much for this video. It is a great starting point for me, and I have a feeling that I'll be coming back to your channel. Keep up the great work! :)
Dear Josh, can you please make a video on small RNA-seq, I think you are the best teacher to explain small RNA-seq in detail. Hope you consider. Thank You.
at 13:35 when you state that 'we counted the reads per gene' are you referring to read depth - meaning how much (quantity) of the mrna i.e. gene is being expressed?
Love the song! Also, thank you for explaining the fragments part. I didn't know that I don't fully understand the rationale behind fragmentation until you explained it. It's an important side note to understand RNA-seq.
4:15 I have a question: if this is a DNA fragment, shouldn't it be double-stranded? And after, in consequence, when the probes attach themselves to the nucleotides, won't the former attach to nucleotides in both strands? Is this something that is known/on purpose, for example, for increasing the confidence of a read? Thank you in advance!
As mentioned around the 3-minute mark of the video, it was discussed that when creating cDNA, it is possible to observe fragmentation of RNA. You mentioned that this is a technical limitation. In that case, for example, if there is one mRNA and it is fragmented, the number of mRNAs would increase depending on the number of fragments. I'm curious to know how this issue is being addressed. I'm asking this question now as I've started studying. Thank you.
There are lots of ways to normalize the number of fragments sequenced per gene. For example: ua-cam.com/video/TTUrtCY2k-w/v-deo.html and ua-cam.com/video/UFB993xufUU/v-deo.html
If the concentration is too high or too low, the machine will not work properly. If the fragments are too long or too short, the machine will not work properly.
@@statquest Thanks for the reply! Would you mind elaborating on a molecular scale what's going on? I'm a 4th year biology student doing a presentation on RNA-seq this week
@@danielgladish2502 Unfortunately I don't really how the details (I'm a computational person). However, this might help: blogs.iu.edu/ncgas/2021/09/17/introduction-to-illumina-sequencing/
You're the first person to ask about that. I wish I could tell you I would cover it soon. However, I have a long over due promise to make a series on neural networks and another series on time series and mixed models... So it might be a while to get to this. :(
Are isoforms of mRNA distinguishable and identified /quantified by this method of sequencing. I read that something like 95% of our mRNA can be isoforms.
Hello congratulations for the video. Very clear and well explained. Thank you! I'd like to have some clarifications: 1) minute 9:14 when you say "the genome fragments that matched the reads fragments will determine a location (chromosome and position) in the genome" basically means that for reads aligned/mapped we know their position with respect to the reference genome, or are you referring to something else? 2) minute 9:29 - I don't understand why breaking read sequences into small fragments. In the example (9:45) you say that the first fragment will not match beacuse of the A mismatch, but also with some mismatches reads can map on the reference genome. So why make fragments is a better choice. Moreover fragments too small improve multimapping, not good for gene expression profiling. Could you clarify my misunderstanding? 3) Finally i ask you with what program have you created these images. Thanks!
Support StatQuest by buying my book The StatQuest Illustrated Guide to Machine Learning or a Study Guide or Merch!!! statquest.org/statquest-store/
I can't believe it. I was actually crying and panicking when I clicked on the video because of all that stress caused by "I don't know, I don't understand" thoughts. And your intro just killed me XD
Bam! :)
Me too!! I was breaking down internally from exam stress, then i watched the intro and it was like a breath of fresh air :,,,,)
I've learned more from this 18-min long video, than my whole course in transcriptomics. You are so good at explaining these complex processes. Thank you!
Hooray! :)
I know the feeling. Transcriptomics is so complex that most people, including those working in the field, seem to get "Betriebsblind" and unable to explain what they are actually doing. Josh Starmer is awesome, I just whish we had instructors like that in every research facility.
@@statquest same for me, I've understood this concept really easily thanks to you ( better than what I've understood in a 2h genetic engineering class )
Hey Josh, I just started doing my RNA sequencing and your video was very useful for me to get the basic idea behind the RNA sequencing. I spent hours and hours reading papers and didn't understand a thing about it. I really appreciate what you have done. I think your channel will play a big role in my research. Hats off your efforts in coming up with useful informative videos like this.
Awesome! That's great news. Since I made that video, I updated my PCA video to be easier to understand, so if you're interested in that, check out: ua-cam.com/video/FgakZw6K1QQ/v-deo.html
@Highlander Yeti same for me! Just found out we're going to do RNAseq, immediately went on UA-cam search instead of the classical science databases, came out here! I was already looking into (often expensive) courses, but it is all online! This is the future of learning. I should have had stuff like this 12 years ago to study Biomedical Sciences. That's why I'm on a mission helping future/current Biomeds and life scientists discover channels like this. Already BIG thanks from my part, StatQuest! I've yet to check out the rest of your stuff, but I WILL!
Amazing video! Better than any textbooks or even my professor😂
First you got me through my statistics course. Then you pulled me through my machine learning courses. Now, you will save me in my graduate genetics lab. Thank you so much Josh.
No way! That’s totally crazy. Good luck with your lab! :)
8:48 Two semesters of Bioinformatics and all I wanted was the sequence "GATTACA" to appear on any one slide during a lecture. Thanks, StatQuest for making my wish finally come true!
Yes!!!! :)
This is one of the best videos for explaining RNA-seq. You are fantastic. Thank you very much.
Thank you! :)
Call this a 'gentle introduction' if you want. It is really just a high level overview of the process. Such presentations are important to building students mental models of any subject, and honestly are frequently overlooked in many subjects in my experience.
Great stuff.
Thanks!
Hey! I'm Brazilian, and I've been learning English and molecular bioinformatics. I'm grateful for this video. Thanks a lot, your pronunciation is easy to understand and you have good didactics too.
Muito obrigado! :)
This is the clearest explanation in this sequencing videos. Usually explanation of such technology is hard, but they did a great job.
Wow, thanks!
I think it's great how there's an effort to speak and pronounce clearly; It really helps when you're not a native English speaker!
Thanks!
Great explanation! I'am bachelor's degree from biology of Universitas Indonesia. It's very useful for me to start learn RNA-seq scope. Big thankyou!!
Glad it was helpful!
Fantastic video. This is the kind of gentle explanation students (including myself) need. Many thanks from Sweden!
Glad you enjoyed it!
The intro song really fits me lol. I'm literally crying and watching StatQuest right now.
Bam!
I'm writing my bachelor's thesis about RNA-seq and usage of this method in the viruses life cycle, u helped me a lot in understanding this. Thank you!
So the "+" symbol used to be a section where individuals could ad more info about their sequence. This is back when we had far smaller output. Since then, data has grown so much that we have developed tools to go through the data more efficiently. In the end, the "+" is simply an artifact from the early days of these files.
I'm not so sure about that, I think it was like that at the origin of this method, but in the new type of document layout, the + symbol could stand for "+ strand", which is often more investigated than the "- strand" of the double helix.
I could be wrong, for sure, but I remeber like that
thank you for making this video so accessible by having captions for everything
Josh Starmer I love you, man. I have been spending hours reading and have only added stress and headaches after
Thank you so much
Happy to help!
I am seeing this video in December 2021 and I can't thank you enough for this incredible concept clearing video of RNA-seq. Very Concise and subtle explanation, Besides your ENGLISH is really understandable for other language speakers. I am gonna check another playlist too.
Thank you!
You stop my worring , stress and anxiety! thank you God and bless Josh Starmer!
Thank you very much! :)
This is actually one of the easiest-to-understand videos I have ever watched about this subject. Thank you for making it so that I can actually understand the information being taught!
Glad it was helpful!
@@statquest same comment! patiently explained without assuming we're all experts, written text of what you are saying verbally, simple, pedagogical, and systematic. It's great teaching, and so happy it's free!
@@daedrmr2dae Thank you!
I LOVE gentle intros
double bam! :)
Simple, concise, and straight forward. Well worth the 18 minutes.
Thank you! :)
As a scientist with no previous experience in big data (who will need to be tuned in for my new research position), all I can say is THANK YOU.
Thanks!
Hey JOSH…First thanks for making such great videos over the years. I see that you even left your job as a professor and have devoted full time to make this stuff since 2017. Appreciate it as it is difficult to break your comfort zone.
One thing I would say after reading books on mastery is that all elite masters have this skill to explain a tough concept in 2 minutes and they can drag the same concept for hours and hours. From thousands of comments on your videos, majority says you make things extremely simple which is a validation of mastery. The second aspect of elite masters are after working for about a decade and mastering a field then they bring innovation or a new thing (a new concept, theory, a formula, anything that was never explained). I’m sure this would be that phase and stat quest is part of that, and I hope many things would come along with your creativity which you have exploited to make this elite content.
There are masters but very few have this skill to make things simpler. Many people might comment that you have mastery or talent in statistics or in music but I can say without any doubt that your talent lies is making complex things easier (the medium could either be through a video or through a book don’t matter), but that’s your core talent which you have used. So I wish you keep using this rare talent and bring more contribution to all the humans.
I will certainly support statquest by purchasing the guides and encourage all who have benefited from this content. Thanks again and lastly is there any email if I could send you a message or share something of value. Will appreciate it.
Wow! Thank you very much!!! BAM! :)
Oh wow. Thanks for the explanation! I didn't really understand this concept until now. I've watched this video 3 times now because it is so comprehensive. Thanks a lot.
Glad it was helpful!
Less than half way through, already learned more than an entire online course!
bam!
Thank you for this. My current molecular bio professor recommedned we see it before our first class, and it was well worth the 18 minutes!
That's awesome! I'm glad you liked the video. :)
Thank you so much for the video, I have been having headache to grasp the RNA seq concept, now I am set for transcriptomics module.
bam! :)
This is really well-explained! You got a suscriber! Hope you continue making videos
Thank you very much! :)
Very impressed how you explain pseudo-alignment! So clear and so concise! Amazing!!
I'm working on a new video that will do a whole analysis pipeline from start to finish and will include pseudo-alignment.
Thank you for this video! It help me a lot, I didn't find anywhere a clear explanation of this content.
Glad it was helpful!
Hey. I'm a clinician trying to do a basic sciences post doc. I haven't touched my biology notebooks in 10 years... and your Video makes it look... like I could maybe, one day, perhaps... UNDERSTAND transcriptomics. (Also makes me feel like an idiot, but in a gentle and kind manner). Thanks
Good luck! :)
this is the clearest explanation on the internet about rna seq, everywhere else makes it sounds like gobbledygook
Hooray! I'm glad you like my video. :)
This is honestly amazing... the best explanation on UA-cam for RNA-seq!!
how grateful i am to the universe to find this 20hours before my interview
Good luck! :)
I am also revising before my interview :D
@@aleksandrat.5058 good luck!
Thanks for this tutorial. It's much better than others I've viewed.
Thanks! :)
Bro, super love the various title songs you used for each videos, that attract me to the statistic world! Thank you!
Hooray!!! :)
this 18-min video is way more concise than 2-years study at Uni.
Thanks!
My God Lord this was heaven sent. Any you guys are hilarious! I don't think it's "excessive self promotion" at all, I'm going to watch your PCA one right now!
Thank you for the clear explanations. This was the first time I learned how the machine reads the bases by taking pictures from the top with single base annealing intervals. It was very informative. I am heading to watch your other videos. Many thanks!
Glad it was helpful!
This is an amazing video! I have learnt a lot. We would like to see more videos on bioinformatics.
The intro song gets me every time ha. But honestly thanks for breaking down the concepts for a newbie like me, keep up the good work
This technique finally makes sense! Thank you so much, this really helped a lot. Very clearly explained and easy to follow.
1:27 I love that you did this
you are literally saving my entire life rn
Happy to help! :)
Normally I get bored very quickly from explanation but hey this didn't feel like 18 mins at all :D Very useful content and you explain the ideas in pretty well organized way. Thank you that was really helpful ✔
Thank you very much! :)
Really helpful video, and extremely easy to understand for beginners, many thanks!
Glad it was helpful!
My report on RNA-Seq is due in 74 hours and I’m just starting this. Fingers crossed.
Good luck!
These are great, I’ve been inundating myself with media about genomics and bioinformatics, but a lot of the specific techniques and terminology were still going over my head.
Thanks for the thorough, but no-nonsense explanations!
This is such a good video. I felt lost until now. Thank you
bam! :)
"+" sign in raw data indicates the forward strand. If paired-end sequencing was performed there will be files with"+" or "-" signs that indicate forward or reverse strands, respectively.
Thanks!
this makes sense… i think its easy for the machine to figure out in the beginning since probably the oligo adaptors, which bind to the flow cell floor, are probably colour-coded for forward and reverse adaptors, and the adaptors themselves, are also probably colour coded, and the camera can pick up on this at the start, which means the algorithm can interpret forward and reverse strand locations and write it to the file.
Wow this is such a god send for my presentation tomorrow
Good luck!
For 7:30, the + symbol is the orientation I believe. That is, the direction from 3' to 5' or vice versa in which the sequence is read. Great video.
So is it sometimes a '-'?
@@statquest I want to say a hesitant yes :D I remember reading about orientation representation in NGS somewhere: It is almost universally + because only the forward/reverse strand is kept as one of the final steps of sequencing.
im so glad I found ur channel to potentially save my course
Hooray and good luck! :)
i love you. cozy stat vids. this is good for the soul
Thanks! :)
According to the "+" in fastQ, I have heard once, that in this line it used to be a repeat of the sequence name without "@" at the begining, to ensure that quality regards to the sequence above, but to lower file weight it has been switched to the "+". That's what I have heard, but it would be worth to approve somehow this information, I have no sources to do that.
I was taught in my course in metagenome and microbiome analysis, that the + line exists so you can put comments there! Never seen one with comments though.
MrErPeeeg is correct; but also it signals the end of the sequence lines. FastQ spec: @
+[]
A requirement is that the following '+' is optional, but if it appears right after '+', it should be identical to the following '@'. maq.sourceforge.net/fastq.shtml
The reason is that FASTQ is a combination of FASTA and QUAL files. Each of those has identifier followed by data. FASTA has sequence, QUAL has quality scores. interleaving these two pieces of information (2 lines from each file "zippered in") gives us 4 lines where 2 of the lines are identical. Later on, when machines got smarter, the third line became obsolete (and expensive space-wise) but is retained in a stunted form for backward compatibility.
Too many ads
This is an incredibly brilliant playlist. Thank you sp much for your videos!
Glad you like them!
wonderful i always wondered how the dot like thing tell you about sequence ,lovely you cleared my basic doubt
bam!
I loved this explanation!!!. It is the most didactic I have seen of all. Congratulations and many thanks. It was very useful. I really appreciate what you have done.
Thank you so much!!! :)
Thank you for sharing, it is very useful to increase my knowledge of RNAseq
Glad it was helpful!
I'm just starting my internship for my bioinformatics studies. Just watched the intro and feel so blessed to have a Video like this. So... thanks a lot ^^
Hooray!!! Good luck with your internship. :)
someone give this man a medal please
Thanks! :)
Hi！This might be too late to ask. Firsst of all, thanks for your video, I have no experience with RNA sequencing and struggled to follow through during my bioinformatics class in Graduate school, this video is helping me understand!!! I was wondering what does a sample actually refer to. I though about them as a group of amplified target DNA being sequenced and read. But at 10:55 there is an explanation about bulk RNA sequencing sample that made me confused. Would you mind clarifying this? Thank you very much!
It really depends. A sample could be RNA from an individual tissue from an individual person, or it could be pooled from that person, or it could be a pool of people. So, "sample" refers to whatever unit the experiment is based on.
Your channel is a blessing i might clear an interview after watching all this xD
Good luck! Let me know how it goes. :)
You are totally amaizing! Thank you so so much for so complete explanations . Some details are diff to find or figure out.
Thank you! :)
Thank you so much for this video. It is a great starting point for me, and I have a feeling that I'll be coming back to your channel. Keep up the great work! :)
Your videos help me immensely! Thank you!
Thanks!
Thank you so much for this very gentle intro to RNA-seq. It helped me with my defense!
Hooray!!! Congratulations on your defense! I hope it went well. :)
Excellent course! Thank you for making these stuff so clear! It's much easier to understand!
Hooray! I'm glad the video was helpful. :)
Dear Josh, can you please make a video on small RNA-seq, I think you are the best teacher to explain small RNA-seq in detail.
Hope you consider.
Thank You.
I'll keep that in mind.
Hey Josh, amazing content, incredible way of teaching.
P.S. 'excessive self promotion' killed me! Thanks for being ausum.
Thank you! :)
Great description. Thank you.
Additional information/description about the sequence may be put in the third line that starts with the +
Thank you very much! :)
Perfect for the lay-person!! Thanks much
You're welcome!
Amazing content ! Easy to understand and complete as possible for YT! Keep it up! Cheers !
Hey, really good video!
This was very educational, straight to the point and very much coherent.
Thanks
Thank you!
best explanation for this on youtube.... Thanks man. You're the best.
Clear, concise, well done! Loved it!
Thank you very much! :)
I just loved it! You made it so simple! Thank u so much!
Thank you! :)
Thank you! This is the best video I have ever seen! ❤️
Awesome!
Your introduction song made me smile 😄
Hooray!!! Thank you! :)
Hi qt
at 13:35 when you state that 'we counted the reads per gene' are you referring to read depth - meaning how much (quantity) of the mrna i.e. gene is being expressed?
It's a relative quantity.
This is so clear! I'm so happy I checked this. Thank you!
You’re welcome! :)
Super helpful and informative, thank you!! You are amazing at explaining things!
:)
Love the song! Also, thank you for explaining the fragments part. I didn't know that I don't fully understand the rationale behind fragmentation until you explained it. It's an important side note to understand RNA-seq.
Glad you liked it!
4:15 I have a question: if this is a DNA fragment, shouldn't it be double-stranded? And after, in consequence, when the probes attach themselves to the nucleotides, won't the former attach to nucleotides in both strands? Is this something that is known/on purpose, for example, for increasing the confidence of a read?
Thank you in advance!
In these experiments we denature the DNA to make it single stranded.
@@statquest thank you! Great video btw, very explicative and understandable :)
@@josueibarra4718 Thanks! :)
Ya I even can watch Statquest when I am bored ... Great video
Nice!!! :)
As mentioned around the 3-minute mark of the video, it was discussed that when creating cDNA, it is possible to observe fragmentation of RNA. You mentioned that this is a technical limitation. In that case, for example, if there is one mRNA and it is fragmented, the number of mRNAs would increase depending on the number of fragments. I'm curious to know how this issue is being addressed. I'm asking this question now as I've started studying. Thank you.
There are lots of ways to normalize the number of fragments sequenced per gene. For example: ua-cam.com/video/TTUrtCY2k-w/v-deo.html and ua-cam.com/video/UFB993xufUU/v-deo.html
thank you so much for the awesome simple explaining
Thanks!
Hi there! I was wondering why you go about verifying fragment length and library concentration as stated at 4:06 ?
If the concentration is too high or too low, the machine will not work properly. If the fragments are too long or too short, the machine will not work properly.
@@statquest Thanks for the reply! Would you mind elaborating on a molecular scale what's going on? I'm a 4th year biology student doing a presentation on RNA-seq this week
@@danielgladish2502 Unfortunately I don't really how the details (I'm a computational person). However, this might help: blogs.iu.edu/ncgas/2021/09/17/introduction-to-illumina-sequencing/
@@statquest Cheers mate!
good explanation and graphics keep up the good work
Thank you! :)
Fantastic explanation! Thank you!
Thanks!
Thank you very much for your videos! They are indeed clearly explained.
You’re welcome!
Thank you for explaining all the concept in a simple way for easy understanding. Could you please also post a video on pseudo-alignment in RNA-Seq
You're the first person to ask about that. I wish I could tell you I would cover it soon. However, I have a long over due promise to make a series on neural networks and another series on time series and mixed models... So it might be a while to get to this. :(
@@statquest Thank you so much for the reply. It is a well worth the wait.
Are isoforms of mRNA distinguishable and identified /quantified by this method of sequencing. I read that something like 95% of our mRNA can be isoforms.
Definitely.
@@statquest Thank you!
Hello congratulations for the video. Very clear and well explained. Thank you!
I'd like to have some clarifications:
1) minute 9:14 when you say "the genome fragments that matched the reads fragments will determine a location (chromosome and position) in the genome" basically means that for reads aligned/mapped we know their position with respect to the reference genome, or are you referring to something else?
2) minute 9:29 - I don't understand why breaking read sequences into small fragments. In the example (9:45) you say that the first fragment will not match beacuse of the A mismatch, but also with some mismatches reads can map on the reference genome. So why make fragments is a better choice. Moreover fragments too small improve multimapping, not good for gene expression profiling. Could you clarify my misunderstanding?
3) Finally i ask you with what program have you created these images.
Thanks!
Super helpful and clear! Thanks!
Glad it was helpful!
Thanks! Revising for my viva and this helps streamline my thoughts!! Off to watch PCA analysis-your shameless self promotion worked keep it up!!
Awesome! Make sure you check out the new and revised PCA video as well: ua-cam.com/video/FgakZw6K1QQ/v-deo.html
Same!!!
This is utterly fantastic, thanks a lot for making these!
Thank you Sir for this information. you made my day
Thanks!