Finally somebody explaining how the technique actually works. Over videos are so vague and assume you already understand the function of all the parts. Thanks so much
@@diego80mi as long as the DNA oligo lacks a 3' cap the DNA polymerase should always replicate 5' to 3', orientation in space does not matter, as the DNA polymerase will replicate 5' to 3'
This is the best explanation on NGS. I had no clue, done so many researches but cant understand clearly. But this explanation is just awesome. Thank you so much.
From now on you can become a member of this channel! You will have access to some cool emojis like or . This is of course voluntary, but the financial support helps me a lot.
I have a question, so at the end of the vid is DNA read of only 1 small cut fragment, how do we combine all fragments that have been cut at 0:31 1 little fragment is analyzed by overlaid fragments result from PCR, then what about the whole thing, I mean restriction enzyme will cut it nicely and no overlaid could happen
I have heard some conflicting ideas on what Hybridisation is. One thing I read seemed to imply it was the process of binding the oligonucleotide adaptors to the cut up DNA, however is that not just library formation. Then other places and your video have implied it is when the oligonucleotide adaptors bind to the complementary oligonucleotides on the flow cell.
What is NGS sequencing? For that what we do first is fragment our DNA. Each fragment will then be attached to two adapters, a side chain nucleotide binding sequence and an oligonucleotide that will bind to the flow cell. The DNA is denatured and the fragments then attach to the flow cell. Here DNA amplification by PCR results. The unattached atrand is washed away. Hence, bridge amplification is carried out where the strand attached via both it's ends to the flow cell and the PCR is carried out. In this way, both the strands are attached to the flow cell. After several rounds we have the amplified DNA. Having both forward and reverse strands. The reverse strands are washed away and the forward strands undergo sequencing by synthesis Here each strand has primer attached and one by one a fluorescently labeled nucleotide attaches to the DNA, which is detected each time a nucleotide is incorporated, once the DNA fragments are replicated and nucleotide for one particular strand is detected along with for other strands, bioinformatics tools are used to determine the sequence of entire fragments by placing the sequences side by side and determining the sequence overlaps. The sequence are compared to reference genome
Hi! This video is great but I still struggle to understand the process. At 01:29, we have a single-stranded forward fragment which is hybridized to the blue oligos and then PCR occurs and the fragment is amplified. In this step, is not happening the same with the reverse fragments, which are hybridized to the red oligos and amplified? What changes in the second step with the bridge building, why is it necessary? I'd really appreciate if someone can help me understand! 🙂
There are multiple ways to do so. But in many cases the "tweezers" scientists use are enzymes, that bind to specific or in other cases more unspecific DNA and can delete (cut out) base pairs or even add (insert) new ones. A cool example of how to manipulate DNA is the CRISPR Cas9 system... See here: th-cam.com/video/4knIPgD6h1U/w-d-xo.html
Don't know the exact case scenario... but in case you sequence cells of an animal such as "Fish X", you may use the reference genome from the most closely related animal of which the sequence is known/published!
Thanks a lot for this incredible explanation. Anw, I have some questions : 1. What is the purpose of DNA fragmentation? Is there any specific site for this cutting? 2. Regarding the adaptor, would you mind to explain more about the constituent of this? Is it like primer where we should design it first? How the adaptor can recognize the end of the fragmented DNA?
The DNA is fragmented to enhance the speed of the sequencing (it's faster to sequence a lot of tiny bits, than a really long chain), and to reduce the "mistake" rate of the polymerase. plus, the polymerase can fall off if the sequence is extremely long
Remarkably explained! Tho I have a question. If we want to seq the foreign DNA in one go...why do we discard the Reverse based strands, cos if we would keep them, the sequencing will only mark out the initial forward strand and that's what we are aiming for! Still confused.
So in order to 'see' the DNA, you need to see the fluorescence, so you need the leading strand to be fluorescent, therefore needing copies of the template strand.
Just a request there Henrik. It'd be lovely if there'd be fewer rare, field specific, Latin/Greek/French words in these kinds of videos. Makes it really hard to understand for a layman. So I would really love if you could try and translate these into common Germanic words that everyone knows. Examples: Ligation, DNA Adaptors, sequence, complimentary, hybridize, denaturation, polymirazed chain reaction, synthesized, olegol? nucleotides (genuinely don't know what you said there). I could go on and on. I hope you get the gist of what I'm saying. Would really help a lot of people understand what's going on much more easily.
Thank you for that kind of nice feedback! Yes, I definitely agree with you - I guess the videos are not really designed for a pure layman... most people watching the videos are biology students themselves (so I honestly kind of expect them to know) Nevertheless, I take this point and might think twice before using a specific term. Thanks!
I have a doubt. Bridge building occurs with both forward (5' to 3') and reverse sequences (3' to 5') right? So when, a bridge is formed by reverse sequence, the amplification of forward sequence is continuous and when a bridge is formed by forward sequence (5' to 3'), the amplification of the reverse sequence is discontinuous (3' to 5') as we see in traditional DNA replication by the formation of Okazaki fragments?
Very good explanation, but I have one little question: How can it be that the two different Adaptors bind to the two different ends, and not one adaptor to both ends? Is the cutting done by a restriction Enzyme, and therefor it always has the same Nucleotides at the end?
1. It's cos one is Forward primer and the other is reverse primer that's why they join different ends. 2. Yes the restriction is done by a single restriction enzyme, so that the sticky ends are poduced uniformly, and yes the overlapping sequences indicate that they are cut by Restriction endonuclease that's why they overlapp and give us full strand of interest. Remember! We didn't know what the DNA seq was to begin with, it can be some sort of Virus or other microbe but we don't know which!
I feel stupid cuz everyone's like.. this is the best explanation I've ever found! And I *still* don't know what the hell he's talking about im gonna fail rofl
Who else came here cause the illumina video was confusing
😂👋
Me😂😂😂
Meeee
yess 😂
Me😂😂
Finally somebody explaining how the technique actually works. Over videos are so vague and assume you already understand the function of all the parts. Thanks so much
all the other vids on youtube just kinda goes on a rampage abt how innovative and amazing it is like a promotion or smth. So thank u so much my guy
Incredible well explained. Non of my uni professors have been able to explained that well
That’s what I want to say
Can u pls tell me wt he explained in last I didn't understand that word
same haha... finally got it
💗💗💗💗💐💐🥺🥺
One thing to keep in mind is that adaptors are stuck with the 5’ end to the flow cell so the polymerase can synthesize DNA in 5’ to 3’ direction
Ooooh right thank u
shouldn't it be the other way around? DNA polymerase synthesize from 5', so the adaptor stuck onto the plate should be at the 3' end, shouldn't it?
@@diego80mi as long as the DNA oligo lacks a 3' cap the DNA polymerase should always replicate 5' to 3', orientation in space does not matter, as the DNA polymerase will replicate 5' to 3'
This is the best explanation on NGS. I had no clue, done so many researches but cant understand clearly. But this explanation is just awesome. Thank you so much.
I wish I found this video earlier lol, spent 2 semesters on this and none of my professors explained it well enough
Now everything make sense to me
yeah, unbelievable how much that matters and how few people are actually competent to teach..
Just want to give you a shout- out and thank you for such an easy-to-understand overview of this process. It is definitely helpful.
Thank you!
thank you very much sir. This is more well explained than the official Illumina videos.
simple, clear, and comprehensive! Great job!
Excellent, very well explained. Clearing all the concepts one by one
A perfect and clear explanation for the NGS!
Incredible video 🥳🥳
I watched 3 4 lengthy videos of ngs but no one explained with such ease
Which topics should I cover in my next videos? Make suggestions below this comment and like the ones you are most interest in
Gene cloning, micro array, protein assay
@@SK-pi6hr Do you mean Western Blot with Protein Assay? Or do you mean some concentration determination of protein (e.g. Bradford Assay)
@@henrikslab concentration determination of protein
@@SK-pi6hr Your wish is granted! Upload: Today :)
Make sure to subscribe to the channel here
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Thank you, your animation and explanation are very clear. I finally understand what happened.
Great video. The only one that allowed me to understand this difficult topic
This is the best and simple explanation ever in this topic. Great job.
From now on you can become a member of this channel! You will have access to some cool emojis like or .
This is of course voluntary, but the financial support helps me a lot.
This is truly incredible. Good work.
this is incredibly clear and easy, thank you very much
Mein Professor hat dein Video für die Erklärung genutzt. Sehr stark, weiter so!
Darf ich fragen an welcher Uni, welcher Professor? :D
Such a amazing esiyer undesirable way of teaching ❤️ we need teacher like u . Great 👍🙏
this should be illuminas explanation video. very well done 👍🏽
Great video! Crisp explanation! Much appreciated!
God bless you, Henrik!
Incredibly clear ! Thank you !
Besser als mein Prof. es je erklärt hat, Danke für das Video :)
Your lectures are wonderful!!!
Thank you. Simple and clear. Now I know how it works!
I have a question, so at the end of the vid is DNA read of only 1 small cut fragment, how do we combine all fragments that have been cut at 0:31 1 little fragment is analyzed by overlaid fragments result from PCR, then what about the whole thing, I mean restriction enzyme will cut it nicely and no overlaid could happen
Great explanation - simple and effective!
You save my exam thank you ❤️❤️
thank you so much Henrik. This was perfect!
This is indeed good and helpful, thank you so much
clear and easy to understand. Thank you !
Very cool and informative Henrik 👏👍🙏
I have heard some conflicting ideas on what Hybridisation is. One thing I read seemed to imply it was the process of binding the oligonucleotide adaptors to the cut up DNA, however is that not just library formation. Then other places and your video have implied it is when the oligonucleotide adaptors bind to the complementary oligonucleotides on the flow cell.
Thank you so much, you explained it super clearly!
perfect explanation, thank you
This was an incredible explanation! Thank you so much
thank you for your clear explanation
sehr gut gemacht hinnerk
Thank you! This was very helpful :)
Thank you for the video. This made my concept much clear now!
This is the best explanation✨
Amazing explanation and video! Thank you.
Super clear. Thank you.
very well explained, thank you
Great video, fast and detailed!
Very short and very understandable! Thank you for good work!
your explanation was perfect but i'm definitely failing my exam tomorrow
If you still read it: All the best for the exam!
Very well defined, To the point. Thank you.
Thumbs up man, it's incredible. Help more.
Very well explained. Thanks!
really great helpful explanation thank you much
What is NGS sequencing?
For that what we do first is fragment our DNA.
Each fragment will then be attached to two adapters, a side chain nucleotide binding sequence and an oligonucleotide that will bind to the flow cell.
The DNA is denatured and the fragments then attach to the flow cell. Here DNA amplification by PCR results. The unattached atrand is washed away.
Hence, bridge amplification is carried out where the strand attached via both it's ends to the flow cell and the PCR is carried out. In this way, both the strands are attached to the flow cell. After several rounds we have the amplified DNA. Having both forward and reverse strands.
The reverse strands are washed away and the forward strands undergo sequencing by synthesis
Here each strand has primer attached and one by one a fluorescently labeled nucleotide attaches to the DNA, which is detected each time a nucleotide is incorporated, once the DNA fragments are replicated and nucleotide for one particular strand is detected along with for other strands, bioinformatics tools are used to determine the sequence of entire fragments by placing the sequences side by side and determining the sequence overlaps. The sequence are compared to reference genome
straight to the point
Thank you!! This was so helpful, my uni lecture was quite confusing and the illumina video just made it so much worse lol
You're a saviour! Thanks
Amazing explanation 🤩🤩🤩 thank you so much!
THANK. YOU. SO. MUCH. Really clear
Very well explained. Please also explain depth of read and coverage
Superb explanation
Hi! This video is great but I still struggle to understand the process. At 01:29, we have a single-stranded forward fragment which is hybridized to the blue oligos and then PCR occurs and the fragment is amplified. In this step, is not happening the same with the reverse fragments, which are hybridized to the red oligos and amplified? What changes in the second step with the bridge building, why is it necessary? I'd really appreciate if someone can help me understand! 🙂
Yes I was wondering the same thing
Thank you so much for sharing. It really helped.
Thanks so much, really well done!
Very good explanation. Thank you
I'm liking the Next Generation Sequencing (illumina)
very clear explanation thank you very much
I love the explanation
Great video! very clear thank you
How do you manipulate DNA? I suppose you cannot grab them using tweezers, right?
There are multiple ways to do so. But in many cases the "tweezers" scientists use are enzymes, that bind to specific or in other cases more unspecific DNA and can delete (cut out) base pairs or even add (insert) new ones. A cool example of how to manipulate DNA is the CRISPR Cas9 system...
See here: th-cam.com/video/4knIPgD6h1U/w-d-xo.html
I have a question: Why is a copy done before the Bridge Amplification? Isn't the Bridge Amplification enough for amplifying my DNA?
You saved me! Thankyou so muchh😭😭❤️
Thank you so much sir.... You are my savior!
This is the greatest explanation I have seen about the procedure. Kudos!
If we sequence it for the first time,and we will have no reference genome,then what will do? Plzz must reply me
Don't know the exact case scenario... but in case you sequence cells of an animal such as "Fish X", you may use the reference genome from the most closely related animal of which the sequence is known/published!
Thank you! Clear explanation.
Thanks a lot for this incredible explanation. Anw, I have some questions :
1. What is the purpose of DNA fragmentation? Is there any specific site for this cutting?
2. Regarding the adaptor, would you mind to explain more about the constituent of this? Is it like primer where we should design it first? How the adaptor can recognize the end of the fragmented DNA?
The DNA is fragmented to enhance the speed of the sequencing (it's faster to sequence a lot of tiny bits, than a really long chain), and to reduce the "mistake" rate of the polymerase. plus, the polymerase can fall off if the sequence is extremely long
doesnt shorter reads increase the error rate?@@keb_in
Remarkably explained! Tho I have a question. If we want to seq the foreign DNA in one go...why do we discard the Reverse based strands, cos if we would keep them, the sequencing will only mark out the initial forward strand and that's what we are aiming for! Still confused.
Thank you so much, this was super well explained!
very good. now you can go to my exam and maybe pass
Well explained 👏
Straight to the point
Use good English with good explanation .
Thanx✌👍👏
So in order to 'see' the DNA, you need to see the fluorescence, so you need the leading strand to be fluorescent, therefore needing copies of the template strand.
Just a request there Henrik. It'd be lovely if there'd be fewer rare, field specific, Latin/Greek/French words in these kinds of videos. Makes it really hard to understand for a layman. So I would really love if you could try and translate these into common Germanic words that everyone knows.
Examples: Ligation, DNA Adaptors, sequence, complimentary, hybridize, denaturation, polymirazed chain reaction, synthesized, olegol? nucleotides (genuinely don't know what you said there). I could go on and on. I hope you get the gist of what I'm saying. Would really help a lot of people understand what's going on much more easily.
Thank you for that kind of nice feedback! Yes, I definitely agree with you - I guess the videos are not really designed for a pure layman... most people watching the videos are biology students themselves (so I honestly kind of expect them to know)
Nevertheless, I take this point and might think twice before using a specific term. Thanks!
@@henrikslab Thanks for your response and for considering my advice!
no one teaches better than a guy with German accent
Does the Flow cell already have the oligo adapters attached, or is that something we do prior to adding are strand for replication?
I finally understand, thank you so much!
Thank you Thank yo sososososososso much!!! you explained it super clearly!!!!!!
I’m high school student..
How is DNA synthesized in the 3 to 5 direction in the reverse strand?
Please let me know about reverse strand!!
Best Video.. ThankYou so much sir.
amazing tutorial, so well explained, made a very complex subject very simple :)
Amazing, thank you!
Is there any difference purpose between DNA amplification and Bridge Amplification?
I have a doubt. Bridge building occurs with both forward (5' to 3') and reverse sequences (3' to 5') right? So when, a bridge is formed by reverse sequence, the amplification of forward sequence is continuous and when a bridge is formed by forward sequence (5' to 3'), the amplification of the reverse sequence is discontinuous (3' to 5') as we see in traditional DNA replication by the formation of Okazaki fragments?
Very good explanation, but I have one little question: How can it be that the two different Adaptors bind to the two different ends, and not one adaptor to both ends?
Is the cutting done by a restriction Enzyme, and therefor it always has the same Nucleotides at the end?
1. It's cos one is Forward primer and the other is reverse primer that's why they join different ends.
2. Yes the restriction is done by a single restriction enzyme, so that the sticky ends are poduced uniformly, and yes the overlapping sequences indicate that they are cut by Restriction endonuclease that's why they overlapp and give us full strand of interest. Remember! We didn't know what the DNA seq was to begin with, it can be some sort of Virus or other microbe but we don't know which!
Very informative.
I feel stupid cuz everyone's like.. this is the best explanation I've ever found! And I *still* don't know what the hell he's talking about im gonna fail rofl