For all of those who don't understand the index thing: - you have your genome DNA which you fragment with restriction enzymes (sticky ends) - you create blunt ends and select for a specific size (too long reads are not possible) with purification beads --> what you get is double stranded DNA with specific length - you add a Adenin and a phosphat group to the end of the fragment so that the phosphat and the A are "complementary" to each other at both ends - now you add the predesigned double stranded index molecules consisting of the two flow cell binding sequences + index 1 and 2 + index read primer 1 and 2. To the index read primers a Thymin and a phosphat group are bound - again "complementary" (so only at ONE end) - because of that your DNA fragment and the index molecules can be ligated (A-T binding) - the index is used to say: the fragments with index ATC e.g. is from E. coli whereas AGT e.g. is from B. subtilis Hope this was not too confusing :D
" predesigned double stranded index molecules consisting of the two flow cell binding sequences + index 1 and 2" doesn't the first sentence before the + sign mean index 1 and 2 ? If yes, then why did you mention it again after + .... i'm new to NGS so that is so confusing for me ... if i'm wrong could you amplify that part more ?
I think the cleaving off of the fluorescent signal and removal of terminator during sequencing by synthesis is a key step that is omitted in the video. It becomes very confusing, then, how the previously added nucleotide's flourescent does not interfere with the newly added nucleotide.
Once a nucleotide is added it has a terminator group attached as far as the terminator group is removed it is not allowed to add another base in the strand. Once the fluorescence signals are captured the terminator group is removed and a new base is added with terminator group.
This is so helpful! I was trying to understand what really was going on during the process, which the lecture slides from my lecturer didn't seem to help much. Thanks!
Hello! if I may offer some critique, I actually prefer the original video. The animation might be less fancy, but I found it clearer and more illustrative than this "3D" one.
Actually I agree with greennecko. The reasoning for the index reads (as mentioned by Steven Kim below and Tom Shen above) is explained much better in the original video. Additionally the sample prep portion includes on screen details for the "additional motifs," which are very useful. Lastly, it was not clear to me that the reasoning for removing the reverse strand and then subsequently removing the forward strand was to accomplish the "Paired end reads" until watching the original video. Thanks for the videos they are are helpful!
Could you please tel me more about it ? Also : how do they "add" adaptaters to each end of the fragments (not knowing their sequence) ? Glue 🤔? Thanks !
There are different ways to add adapters. One way is through amplicon PCR, where some of the primer binds to your region of interest, and the rest of the primer adds the indexing binding site. @@martinmazukiewicz
@@martinmazukiewicz The adapters were ligated to the sequence of interest during library preparation step, which prepares DNA or RNA samples to be compatible with a sequencer. Library preparation is before sequencing
Everything is clear except the index 1 read part (3:17), as well as index 2 (3:35). Their purpose was not explained in the video. I read somewhere that unique pairs of index 1&2 allow mixing multiple samples together and sequencing them at the same time. Could you elaborate? I don't understand how that can be accomplished based on this video alone.
from what I understand from the video: 3:17 = deprotect the index 1 side from cleaving and allow it for reading 3:35 = prevent index 2 side from reading. next question is "how these processes allow doing that (protection and blocking reading)?" = I don't know
these sequences are being done with some many at a time. the idices are used as marker for each strand. The computer puts all the strands with the same index together to get a preciser read.
Steven Kim i think index 1 and 2 here are working as indicators for 5' or 3' end of the forward sequence. The index to mix different samples are additional index sequences added to each sample during tagmentation. For example, i add N701 index (a short sequence in Nextera preparation kit )to sample A, N702 index to sample B, and then mix two samples in one lane. During sequence analysis, i could distinguish the two samples for there is a tag added to each sample.
I think they showed it incorrectly. The index read primer is hybridizing to white sequence which is presumably unknown sample sequence. The index and the primer that reads it should be within the blue sequence. Or they could just put the index sequence directly downstream of the initial primer binding site to save the trouble?
I still do not get it why after sequencing the first strand and before proceeding to adding index 2 primer to generate clusters of the other strand index 1 is added again (3:17) to generate a short read. Reading the comments below has not helped. Is anyone able to explain this again? Is this done to signal to the data acquisition computer: "this is the end of sequencing data for the fragments labelled with index 1"?
i can only say, WOW what a discovery!!! sometimes i think how we got here? it is absolutely captivating and i feel euphoric being involved that journey
@@antisocialdistancing3 To everyone still confused watch this th-cam.com/video/oIJaA6h2bFM/w-d-xo.html&ab_channel=Illumina to save yourself some tears. Really helped me out as a beginner. It's illumina's introduction video to sequencing by systhesis.
@@heerababu6 I didn't recieve your reply before but i'm glad to tell you that I passed it thank you 😭💙 I still have 3 left to graduate ! The first one is in 7 hours please wish me luck 🥹💙
Amazing video with clear explanation but i have small doubt, here i found that when the reads are cleaved and washed off their respective indices are not ligated to their respective reads, how will this work in demultiplexing??
Dear Illumina group thank you for video, can you please answer my few questions:- 1) at 2:22 and 4:00 reading starts from 5'? 2) is numbers of reads generated per fragment depends on the type of NGS application? 3) If reads are generated from fragments how they are aligned? 4) Do data generated from the read contains sequence of amplicons?
The reference genome is available there to compare these fragments and there are software's like Spades to align these short reads. Since this is not a single read each base is read at least 30 time which allows to chose the best option from the quality score assigned to each read.
Don't know if they meant to show these details, but the letter ordering in the forward and reverse passes is reversed but not complemented- is this an error? in 2:35 you see the ordering (from top to bottom) of AATTCGCATCG and at 3:56 on the reverse read the order is reversed but not complemented i.e. GCTACGCTTAA- shouldn't it have been complemented i.e. (CG,AT)?
So that the fragment becomes attached to the flow cell. In the first step, the fragment of interest hybridizes to the oligo on the flow cell via its complementary adaptor sequence. But it is not attached yet - under denaturing conditions it will come off. The oligo on the flow cell acts as a primer so that polymerase can extend the sequence, using the original fragment as a template. Once that is done, you have the sequence of interest (it would be the compliment of the original fragment) bound to the flow cell. Denature to release the original fragment, and you are ready for bridge amplification.
I have the same question as Stephen Kim, but most of my question is explained by the comments. However, I still wonder why Index 1 is sequenced separately? Why couldn't it be sequenced together with the read; at 3:11 the read was still incomplete but it already got washed away. Thanks!
Hi Thuy, The sequencing read stops being synthesized further due to a blocking mechanism that’s done by the machine. The reason why indices are sequenced separately and not with the sequencing reads is because you would not be able to tell which one is the index and the actual sequencing read if they’re done together. I hope that answers your question, let us know if you need further clarification. Thanks!
I'm wondering whether there is a mistake in the narration. Before read 1 it mentions the reverse strands are cleaved off. Isn't read 1 a copy of the forward/coding strand, thus you'd need the reverse/non-coding strand to remain attached? At least my MiSeq read primers were always designed this way.
Can someone please explain why sequencing the reverse strand is necessary? We just sequenced the given read by synthesizing from the forward oligo. At 3:20, you have the sequence of the entire read and the index. Is there any new information we gain after 3:20? Thanks!
Yeah, so the reason you need to sequence the reverse strand is because as the sequencing progresses, the error rate increases, so you have to go back and sequence in the opposite direction to minimise errors if that makes sense. Otherwise only the start of the sequence would be necessary, but the paired end reads increases the accuracy of the sequencing by allowing you to identify errors.
Morgan Desira not really the answer. Certainly a possibility but I have never worked on any data where people have purposely done that (working in bioinformatics and genomics for 10 years). NB reverse strand isn't necessary, it's an option ('paired-end' vs. 'single-end' sequencing). NB also that in the cartoon the read seems short, but should be ~500bp, and reads are 75-150bp. There is meant to be an 'insert' in the seq library, so you have a 150bp stretch, then unknown insert (usually ~200-250bp) then another 150bp. In highly fragmented DNA e.g. from FFPE material you might get shorter and overlapped reads. In paired-end you would get back two reads, but we know they should be near each other. This makes aligning to the reference genome more accurate. Should mention that if we sequence to a high enough depth, error rate is less of an issue, and depth should be considered in any experimental design.
Hey, i think the reverse strand is importand to match (pair) forward and reverse reads, so to form cotiguous sequences. I mean that forward and reverse reads can have different lengths, a redundancy would assure the ending or begining of a paired read. Later could be of relevance for tracking splicing events. Its a theory. :)
@@brucemoran1063 Thank you Bruce :) I was wondering if the Illumina sequencing they do here was paired-end. Since they never mentioned anything about it in the video I was unsure.. Is that why they used 2 indexes on one strand (one forward and one reverse)?
Hi, I have a few questions that need urgent answers: 1/ How flourescent label in the blocking group are removed from the nucleotide? 2/ How to wash away free nucleotide? 3/ Why do we have to sequece 2 strain of DNA? 4/ What is the function of index 1 and 2 in adapter attached to DNA? Thank you.
You don't have to sequence two strands. This video is showing paired-end sequencing, which sequences both strands. But you can do single-end sequencing, which sequences only one strand. The benefit to paired-end is better coverage of the entire genome.
I have a question. It seems during the first read all the two types of oligos have been occupied through bridging amplification. Why there are free oligos to be used for bridging during the second read?
Thanks! Why don't we cleave and wash off the reverse strand (p7 adaptor) before bridge amplification, won't it be a waste of nucleotides and energy released from hydrolysis?
When sequencing by synthesis (2:35), the animation seems to show that only one of the nucleotide lights up when excited. Why doesn't the fluorophores from all the nucleotides emit light?
8 ปีที่แล้ว
Only complementary nucleotides emit the light, e.i. only those that are actually added to the strain
I understand what you're saying, but why only complementary nucleotides? Is there something that disables the fluorophores?
8 ปีที่แล้ว +6
But the animation is quite unaccurate there. They are just added within one cycle and the light is emmited upon illumination of external source of light and not immediatelly. Each nucleotide serves also as a terminator, so it stops the reaction, which can continue only after the external illumination (which affects the structure of just added nucleotides in order to allow further synthesis).
8 ปีที่แล้ว
Yeah, got it. Well, I don't know. They are either transformed during the addition (which enables their fluorescence) or before illumination the rest is washed away I guess.
From what I understand, the incorporation of the nucleotide releases the fluorophore, which is the process that excites fluorescence. The fluorophores bound to the unincorporated nucleotides are completely inert, so all bases can be present in the solution without confusion of the fluorophores. Hence why the animation shows the fluorophores flashing upon incorporation.
If the oligos on the flow cell are only complementary to the adaptors on the forward strand fragments, how do they determine which is the forward strand when the adaptors are ligated? Do they just sequence both strands at the same time and then use known sequences to determine which is the forward strand?
On 0:43 what are the remaining colors: yellow and blue? Red are incides, green correspond to sequence binding site, and purple ones are complimentary to oligos on a flow cell. But what about yellow and blue?
One primer mix only contains either P5 or P7 oligo sequence. Bridge amplification works because there are two differing oligo sequences that are attached to the flow cell. This requirement makes two primer sequences necessary.
very informative video, thank you :) How would you use NGS to identify Amyotrophic lateral sclerosis (ALS) with focus on RNA binding proteins TDP43, FUS, TAF15? What would be the approach?
So question about experience, in terms of jobs asking to do things such as experience in PCR, NGS library prep, etc., is experience actually necessary? from being a bit exposed to it and reading it, it just seems as if you just need to follow protocols and instructions in order to do the procedures? Or can anyone elaborate on this?
Reading recipes doesn't make you a chef. You have to understand all the ins and outs, be able to find and fix errors and adapt protocols for specific experiments.
I wish there was no music. It's too loud and the pitch is so high because of the harmonics that you can't omit it. At least if you have anything at all to do with music
Since each random fragment has a different sequence, how do the adapters bind to the fragments? Especially if the fragmentation is done using mechanical shearing? I have other such basic questions. Can anyone point me to a source that will introduce me to NGS, please?
Hi Joe, Adaptor ligation/attachment is very highly dependent on the library preparation kit that you use. We actually compiled a list of different methods to prepare your sample for sequencing in our Sequencing Methods Explorer www.illumina.com/science/sequencing-method-explorer.html. For more basic resources, these are ones that we find to be very helpful: www.illumina.com/content/dam/illumina-marketing/documents/products/illumina_sequencing_introduction.pdf rnaseq.uoregon.edu/ Thanks! And do let us know if you find good ones out there in your search.
Thankyou this video was very helpful. Just 2 questions for clarification: If the reverse strand is washed away, then when the fluorescence labeled nts bind (to template strand) and emit their light signal, what the computer is reading is actually the reverse or complement of the template sequence? And also how is only the reverse strand washed away, how is it specifically targeted? 2) Given a cluster, since one flow cell lawn is amplified to hold one gene fragment when the computer reads the signal from synthesis, its all of that one gene fragment...In other words the coverage of the fragment is dependent on the number of adapters tethered to the lawn of that flow cell ?
You might have a 'pooled sample library' i.e. a library made up of multiple genomic samples. The index reads are unique to each sample and therefore allow all the sequencing reads for a particular sample to be collated during the data analysis.
Bridge amplification happens through multiple steps of thermal and chemical processes. The exact details are too complex for a comment section. I recommend contacting Illumina tech support for these detailed questions. It is however slightly explained in this video th-cam.com/video/J487Pqyswpo/w-d-xo.html
I had the exact same doubt for a long while. It's quite simple really. Ligases and enough adapter sequences are added to the pool of fragments and the enzyme takes care of the rest.
At 1:52 , since the original starnd is washed away, how is it possible for its complementary strand is folding over to hybridize to the second type of oligo? that strand wont be complementary to the second type of oligo but will have exactly the same sequence with that oligo! I am confused. In some other sources, they mention that once you add the fragment both ends hybridize with thte two oligos and then we have the PCR extension which makes more sense. Anyone can help?
Hello, thank you for the nice video. It is indeed very helpful. One question that has been addressed before by another person (@quickfastgoninja) in the comments: Considering that both oligo complements ('purple' and 'blue') are present in the flow cell: what is preventing the "blue" sequences from hybridizing when the DNA is flowed across the cell? From the animation, it seems that purple:purple hybridization would be equally as likely as simultaneous blue:blue hybridization. Are the "blue" adapters somehow blocked from hybridization when the samples are first flowed across the cell?
I think the machine goes through different cycles of temperature. rising the temperature "denatures" the DNA, breaking any double-strand bonds. then, the unwanted DNA molecules are washed away, while the DNA strands attached to the flow cell are retained by mechanical force (ie the fact that they are bound to the actual cell)
Any video that has been uploaded to TH-cam can be shared and used as long as you don't claim they are yours. As you ArcGen upload your video's or pics on imgur, instagram etc... you give them (TH-cam, Facebook, Instragram, etc...) permission to share your material.
In 2:27 we can see, that synthesis goes from purple adapter to blue one, which means, purple must be 5' end. Also in 3:16 the same direction of synthesis occurs. After read product washed off in 3:26, template_X folds over and binds second oligo on the flow cell - that means that if purple adapter on template_X was 5' end, then purple adapter on flow cell in 3:33 must be 3' end. Then how is that possible, that in 3:39 the synthesis goes from 3' end to 5' end? If i missed something, just remind me please... :D but i've watched it few times and can't find it correct. BTW nice video. Actually, in 1:36 synthesis goes from blue oligo on flow cell, so blue oligoes on flow cell must be 5' ends. That means, that in 2:27 synthesis goes from 3' end to 5' end, which shouldn't be possible.
The adapters are not specific to a 5' or 3' end. If this was the case, the ends of the dsDNA (for example, at 1:24) would not have the same colors as its complementary strand.
Imagine being the person that put all this together and someone else was like… “it’s missing an audio track of seven homeless guys beating aluminum pipes and playing the one last working key on a piano at the same volume as the explanation ….”
does anybody know if the fluorescent molecule is attached to the nucleotide and before the next nucleotide is added does it has to be removed in order to get a monochromatic signal?
For all of those who don't understand the index thing:
- you have your genome DNA which you fragment with restriction enzymes (sticky ends)
- you create blunt ends and select for a specific size (too long reads are not possible) with purification beads --> what you get is double stranded DNA with specific length
- you add a Adenin and a phosphat group to the end of the fragment so that the phosphat and the A are "complementary" to each other at both ends
- now you add the predesigned double stranded index molecules consisting of the two flow cell binding sequences + index 1 and 2 + index read primer 1 and 2. To the index read primers a Thymin and a phosphat group are bound - again "complementary" (so only at ONE end)
- because of that your DNA fragment and the index molecules can be ligated (A-T binding)
- the index is used to say: the fragments with index ATC e.g. is from E. coli whereas AGT e.g. is from B. subtilis
Hope this was not too confusing :D
" predesigned double stranded index molecules consisting of the two flow cell binding sequences + index 1 and 2" doesn't the first sentence before the + sign mean index 1 and 2 ? If yes, then why did you mention it again after + .... i'm new to NGS so that is so confusing for me ... if i'm wrong could you amplify that part more ?
I think the cleaving off of the fluorescent signal and removal of terminator during sequencing by synthesis is a key step that is omitted in the video. It becomes very confusing, then, how the previously added nucleotide's flourescent does not interfere with the newly added nucleotide.
100% what i thought
Once a nucleotide is added it has a terminator group attached as far as the terminator group is removed it is not allowed to add another base in the strand. Once the fluorescence signals are captured the terminator group is removed and a new base is added with terminator group.
exactly
Thank you!! That was the part I didn't understand:)
This is so helpful! I was trying to understand what really was going on during the process, which the lecture slides from my lecturer didn't seem to help much. Thanks!
Thanks, I always wondered how plumbuses are made.
She forgot to rub the fleeb
lol, I was thinking the exact same thing. The whole thing seemed highly improvised.
OMG THANK YOU SO MUCH THIS MADE MY NIGHT
Henry Farmery =c
this is one of the best videos for sequencing by synthesis. thanks a lot
The loud, repetitive dinging in the background is unfortunately quite distracting.
Amen. High-pitch harmonics and a loud track. I muted it and switched the subtitles on.
Flames
@Felix Ronan you’re right we don’t care
is it jus me or is this repetition so heckin confusing to follow .... one thing repeated like 10 times god i hate my degree
Can MRNA PASS THE BLOOD BRAIN BARRIER it's blood plus new message makers I THINK SHEDDING IS POSSIBLE TOO
In addition, a description providing the video times of each step would be useful. I.e: Paired end joining @ 2:00
Hello! if I may offer some critique, I actually prefer the original video. The animation might be less fancy, but I found it clearer and more illustrative than this "3D" one.
Actually I agree with greennecko. The reasoning for the index reads (as mentioned by Steven Kim below and Tom Shen above) is explained much better in the original video. Additionally the sample prep portion includes on screen details for the "additional motifs," which are very useful. Lastly, it was not clear to me that the reasoning for removing the reverse strand and then subsequently removing the forward strand was to accomplish the "Paired end reads" until watching the original video. Thanks for the videos they are are helpful!
Hi @greenneko , could you post the original video link please? Thanks
th-cam.com/video/womKfikWlxM/w-d-xo.html
The old video is a lot better! It actually explains more, like how the adapters, primers are generated, the Index explanation is a lot better!
The animation could have mentioned the terminator caps, which are crucial for this technology. Besides that, great explanation.
Could you please tel me more about it ? Also : how do they "add" adaptaters to each end of the fragments (not knowing their sequence) ? Glue 🤔? Thanks !
There are different ways to add adapters. One way is through amplicon PCR, where some of the primer binds to your region of interest, and the rest of the primer adds the indexing binding site. @@martinmazukiewicz
@@martinmazukiewicz The adapters were ligated to the sequence of interest during library preparation step, which prepares DNA or RNA samples to be compatible with a sequencer. Library preparation is before sequencing
Everything is clear except the index 1 read part (3:17), as well as index 2 (3:35).
Their purpose was not explained in the video.
I read somewhere that unique pairs of index 1&2 allow mixing multiple samples together and sequencing them at the same time.
Could you elaborate? I don't understand how that can be accomplished based on this video alone.
from what I understand from the video: 3:17 = deprotect the index 1 side from cleaving and allow it for reading
3:35 = prevent index 2 side from reading.
next question is "how these processes allow doing that (protection and blocking reading)?" = I don't know
these sequences are being done with some many at a time. the idices are used as marker for each strand. The computer puts all the strands with the same index together to get a preciser read.
Steven Kim i think index 1 and 2 here are working as indicators for 5' or 3' end of the forward sequence. The index to mix different samples are additional index sequences added to each sample during tagmentation. For example, i add N701 index (a short sequence in Nextera preparation kit )to sample A, N702 index to sample B, and then mix two samples in one lane. During sequence analysis, i could distinguish the two samples for there is a tag added to each sample.
I think they showed it incorrectly. The index read primer is hybridizing to white sequence which is presumably unknown sample sequence. The index and the primer that reads it should be within the blue sequence. Or they could just put the index sequence directly downstream of the initial primer binding site to save the trouble?
@@Valariel I agree with you , illumina accidently or delibretely make the issue ambiguous.
Only one word: AMAZING
I still do not get it why after sequencing the first strand and before proceeding to adding index 2 primer to generate clusters of the other strand index 1 is added again (3:17) to generate a short read. Reading the comments below has not helped. Is anyone able to explain this again? Is this done to signal to the data acquisition computer: "this is the end of sequencing data for the fragments labelled with index 1"?
i can only say, WOW what a discovery!!! sometimes i think how we got here? it is absolutely captivating and i feel euphoric being involved that journey
I am now understanding how DNA sequencing works clearly!
explain to me
woah this is actually so smart
I was thinking exactly the same thing
Next Generation Sequencing is really amazing!
영어잘하시나봐요 부럽
학교에서 배운 내용이라서...한국어로 이해하고 들어서 덜 어려웠어요..ㅎㅎ
@@dewsdays8908 죄송한데 같은내용 한글로 설명된곳 아는곳 있으긴가요?? ㅠ
Need some more explanations on index 1 and index 2
who's awake in 2021?
@@antisocialdistancing3 To everyone still confused watch this th-cam.com/video/oIJaA6h2bFM/w-d-xo.html&ab_channel=Illumina to save yourself some tears. Really helped me out as a beginner. It's illumina's introduction video to sequencing by systhesis.
Thank you so much! Visualizing the process while reading about this technique is too difficult, an animated illustration is the perfect solution! ❤️
Wait a minute…this isn’t a Minecraft speed run
Wish I came here from minecraft 🥲
I have my final exams in an hour and i'm cryinggg 😭
@@crystalrose8658 how was your exam.
Lmao that's why the word Illumina seemed familiar 😂
@@heerababu6 I didn't recieve your reply before but i'm glad to tell you that I passed it thank you 😭💙
I still have 3 left to graduate ! The first one is in 7 hours please wish me luck 🥹💙
Data analysis is the real D and A
underrated comment
Amazing video with clear explanation but i have small doubt, here i found that when the reads are cleaved and washed off their respective indices are not ligated to their respective reads, how will this work in demultiplexing??
Did anyone else get the chills at (2:01) or was that just me?
high quality video, exciting to watch
This explains better than me prof. on class
when could I exert my effort to a company like this?🤯 I'll do my best in college first.✊
Dear Illumina group thank you for video, can you please answer my few questions:-
1) at 2:22 and 4:00 reading starts from 5'?
2) is numbers of reads generated per fragment depends on the type of NGS application?
3) If reads are generated from fragments how they are aligned?
4) Do data generated from the read contains sequence of amplicons?
The reference genome is available there to compare these fragments and there are software's like Spades to align these short reads. Since this is not a single read each base is read at least 30 time which allows to chose the best option from the quality score assigned to each read.
For Denovo sequencing the overlap sequencing is performed and base call is 60x to get more accurate data
He best at speedrun
It's so clear. Thank you for your video.
I wonder what is the physical proximity of the oligos to be able to bend.
Don't know if they meant to show these details, but the letter ordering in the forward and reverse passes is reversed but not complemented- is this an error? in 2:35 you see the ordering (from top to bottom) of AATTCGCATCG and at 3:56 on the reverse read the order is reversed but not complemented i.e. GCTACGCTTAA- shouldn't it have been complemented i.e. (CG,AT)?
no these are meant to be parallel strands
helloo, why do we do the step before bridging ( the replication and detachment of one strand..) thanks !
So that the fragment becomes attached to the flow cell.
In the first step, the fragment of interest hybridizes to the oligo on the flow cell via its complementary adaptor sequence. But it is not attached yet - under denaturing conditions it will come off. The oligo on the flow cell acts as a primer so that polymerase can extend the sequence, using the original fragment as a template. Once that is done, you have the sequence of interest (it would be the compliment of the original fragment) bound to the flow cell. Denature to release the original fragment, and you are ready for bridge amplification.
I have the same question as Stephen Kim, but most of my question is explained by the comments. However, I still wonder why Index 1 is sequenced separately? Why couldn't it be sequenced together with the read; at 3:11 the read was still incomplete but it already got washed away. Thanks!
Hi Thuy,
The sequencing read stops being synthesized further due to a blocking mechanism that’s done by the machine. The reason why indices are sequenced separately and not with the sequencing reads is because you would not be able to tell which one is the index and the actual sequencing read if they’re done together.
I hope that answers your question, let us know if you need further clarification.
Thanks!
I'm wondering whether there is a mistake in the narration. Before read 1 it mentions the reverse strands are cleaved off. Isn't read 1 a copy of the forward/coding strand, thus you'd need the reverse/non-coding strand to remain attached? At least my MiSeq read primers were always designed this way.
Can anyone help me
Before the first sequencing, how the reverse strand are cleaved and washed off , leaving only forward strand?
Can someone please explain why sequencing the reverse strand is necessary? We just sequenced the given read by synthesizing from the forward oligo. At 3:20, you have the sequence of the entire read and the index. Is there any new information we gain after 3:20?
Thanks!
Yeah, so the reason you need to sequence the reverse strand is because as the sequencing progresses, the error rate increases, so you have to go back and sequence in the opposite direction to minimise errors if that makes sense. Otherwise only the start of the sequence would be necessary, but the paired end reads increases the accuracy of the sequencing by allowing you to identify errors.
Morgan Desira not really the answer. Certainly a possibility but I have never worked on any data where people have purposely done that (working in bioinformatics and genomics for 10 years).
NB reverse strand isn't necessary, it's an option ('paired-end' vs. 'single-end' sequencing). NB also that in the cartoon the read seems short, but should be ~500bp, and reads are 75-150bp. There is meant to be an 'insert' in the seq library, so you have a 150bp stretch, then unknown insert (usually ~200-250bp) then another 150bp. In highly fragmented DNA e.g. from FFPE material you might get shorter and overlapped reads.
In paired-end you would get back two reads, but we know they should be near each other. This makes aligning to the reference genome more accurate.
Should mention that if we sequence to a high enough depth, error rate is less of an issue, and depth should be considered in any experimental design.
Hey, i think the reverse strand is importand to match (pair) forward and reverse reads, so to form cotiguous sequences. I mean that forward and reverse reads can have different lengths, a redundancy would assure the ending or begining of a paired read. Later could be of relevance for tracking splicing events. Its a theory. :)
@@brucemoran1063 Thank you Bruce :)
I was wondering if the Illumina sequencing they do here was paired-end. Since they never mentioned anything about it in the video I was unsure..
Is that why they used 2 indexes on one strand (one forward and one reverse)?
4:39 - to resolve ambiguous alignment
Wow ❤ nicely explained 👏
The second and third phase is that a primer?
I mean, oligos = adaptor = primer right?
@JS L the adaptor contains a binding site for the primer, an index and the complement to the flow cell (which has a oligonucleotide)
Hi, I have a few questions that need urgent answers:
1/ How flourescent label in the blocking group are removed from the nucleotide?
2/ How to wash away free nucleotide?
3/ Why do we have to sequece 2 strain of DNA?
4/ What is the function of index 1 and 2 in adapter attached to DNA?
Thank you.
You don't have to sequence two strands. This video is showing paired-end sequencing, which sequences both strands. But you can do single-end sequencing, which sequences only one strand. The benefit to paired-end is better coverage of the entire genome.
I think the terminator is only fluoroscent when bond, also after excitation it is "removed" or deactivated.
Thanks to explain NGS😊
How does the actual sequencing work from 2:13? It's not that clear. Does the labelled nucleotide bind and automatically release fluorescence?
yes
Aveces no me gusta mi carrera y otras veo este tipo de cosas y me enamoró más de mi carrera y de la ciencia jajaja ❤️
Can anyone help me to understand what is an NGS assay? Thank you very much in advance.
I have a question. It seems during the first read all the two types of oligos have been occupied through bridging amplification. Why there are free oligos to be used for bridging during the second read?
In the end of the read 1, the forward strand is washed for now you can amplify and read the reverse strand.
This is also used for whole genome sequencing correct?
I've go to study this for my genetic engineering exam
Thanks! Why don't we cleave and wash off the reverse strand (p7 adaptor) before bridge amplification, won't it be a waste of nucleotides and energy released from hydrolysis?
When sequencing by synthesis (2:35), the animation seems to show that only one of the nucleotide lights up when excited. Why doesn't the fluorophores from all the nucleotides emit light?
Only complementary nucleotides emit the light, e.i. only those that are actually added to the strain
I understand what you're saying, but why only complementary nucleotides? Is there something that disables the fluorophores?
But the animation is quite unaccurate there. They are just added within one cycle and the light is emmited upon illumination of external source of light and not immediatelly. Each nucleotide serves also as a terminator, so it stops the reaction, which can continue only after the external illumination (which affects the structure of just added nucleotides in order to allow further synthesis).
Yeah, got it. Well, I don't know. They are either transformed during the addition (which enables their fluorescence) or before illumination the rest is washed away I guess.
From what I understand, the incorporation of the nucleotide releases the fluorophore, which is the process that excites fluorescence. The fluorophores bound to the unincorporated nucleotides are completely inert, so all bases can be present in the solution without confusion of the fluorophores. Hence why the animation shows the fluorophores flashing upon incorporation.
If the oligos on the flow cell are only complementary to the adaptors on the forward strand fragments, how do they determine which is the forward strand when the adaptors are ligated? Do they just sequence both strands at the same time and then use known sequences to determine which is the forward strand?
Ive pressed the replay button 6 times
The best lecture vedio
This video is very helpful. Thank you.
Wow Thanks a lot!!!
Thank you so much, it's so great and clear video
On 0:43 what are the remaining colors: yellow and blue?
Red are incides, green correspond to sequence binding site, and purple ones are complimentary to oligos on a flow cell. But what about yellow and blue?
It is clear for me, thanks
very nice, thank you
What does Reduced Cycle Amplification means?
Is single cell sequencing the hottest spot in the current sequencing field？
I've heard for some applications generation of the reverse read is not necessary, if this is true what would those applications be?
Question: Do you do a second read when analyzing "16s rRNA Gene" DNA fragments? Or is this only for full genome analysis?
Yes , you do. You use the second read because you need to sequence the full amplicon, around 300 bps.
Thank you for making this simpler!!!
Thanks for the upload!
Great video
Why are two primer mixes necessary? Why is not one primer mix sufficient?
One primer mix only contains either P5 or P7 oligo sequence. Bridge amplification works because there are two differing oligo sequences that are attached to the flow cell. This requirement makes two primer sequences necessary.
I have one question... your DNA is sequenced by ilumina miseq is of 5MB size. U have to get 10x coverage . How much data do u need?
What is the purpose of the indices in the adaptors mentioned at 0:40?
How come no polymerase is needed to add the building nucleotides to the cluster?
My teacher wants me to write about this complex device. But this is difficult. I'm just a high school student. Wish me luck.
This could be applied to cosmetic without surgery if applied concurrent with Active MRI and Lighting treatment and coating optic gel
very informative video, thank you :) How would you use NGS to identify Amyotrophic lateral sclerosis (ALS) with focus on RNA binding proteins TDP43, FUS, TAF15? What would be the approach?
maybe with CHip-seq?
it's just the sequencing technique, what you want to do with it specifically is up to you
you have to do immunoprecitation of proteins bound to RNA, and then sequence the pull-down
So question about experience, in terms of jobs asking to do things such as experience in PCR, NGS library prep, etc., is experience actually necessary? from being a bit exposed to it and reading it, it just seems as if you just need to follow protocols and instructions in order to do the procedures? Or can anyone elaborate on this?
Reading recipes doesn't make you a chef. You have to understand all the ins and outs, be able to find and fix errors and adapt protocols for specific experiments.
@@alexandercook6459 does it make you a cook, though? is that how you did it?
I wish there was no music. It's too loud and the pitch is so high because of the harmonics that you can't omit it. At least if you have anything at all to do with music
Since each random fragment has a different sequence, how do the adapters bind to the fragments? Especially if the fragmentation is done using mechanical shearing? I have other such basic questions. Can anyone point me to a source that will introduce me to NGS, please?
Hi Joe,
Adaptor ligation/attachment is very highly dependent on the library preparation kit that you use. We actually compiled a list of different methods to prepare your sample for sequencing in our Sequencing Methods Explorer www.illumina.com/science/sequencing-method-explorer.html.
For more basic resources, these are ones that we find to be very helpful:
www.illumina.com/content/dam/illumina-marketing/documents/products/illumina_sequencing_introduction.pdf
rnaseq.uoregon.edu/
Thanks! And do let us know if you find good ones out there in your search.
Thankyou this video was very helpful. Just 2 questions for clarification:
If the reverse strand is washed away, then when the fluorescence labeled nts bind (to template strand) and emit their light signal, what the computer is reading is actually the reverse or complement of the template sequence? And also how is only the reverse strand washed away, how is it specifically targeted?
2) Given a cluster, since one flow cell lawn is amplified to hold one gene fragment when the computer reads the signal from synthesis, its all of that one gene fragment...In other words the coverage of the fragment is dependent on the number of adapters tethered to the lawn of that flow cell ?
thank you for your reply. Looking forward to it.
Why we have a Index Read?
You might have a 'pooled sample library' i.e. a library made up of multiple genomic samples. The index reads are unique to each sample and therefore allow all the sequencing reads for a particular sample to be collated during the data analysis.
AN index read
who else is here because they have an assessment
Why do the nucleotides have to bind sequentially?
What causes the strand to bend to form the bridge?
Bridge amplification happens through multiple steps of thermal and chemical processes. The exact details are too complex for a comment section. I recommend contacting Illumina tech support for these detailed questions. It is however slightly explained in this video th-cam.com/video/J487Pqyswpo/w-d-xo.html
well explained..thanks
Hi. What would happen if instead of put 5% of PhiX, I put 60% and the library is the correct amount? Thanks.
Yeap, but the question is : how do they "add" adaptaters to each end of the fragments (not knowing their sequence) ? Glue 🤔?
I had the exact same doubt for a long while. It's quite simple really. Ligases and enough adapter sequences are added to the pool of fragments and the enzyme takes care of the rest.
@@siddharthwastaken Ligases ! That was the missing point... Thanks !
I have a question for this video.
can you explain to me how to the ends of template are deprotected and protected?
Thank you!
The specific chemicals used in this reaction is Illumina proprietary information and cannot be disclosed to the public
At 1:52 , since the original starnd is washed away, how is it possible for its complementary strand is folding over to hybridize to the second type of oligo? that strand wont be complementary to the second type of oligo but will have exactly the same sequence with that oligo! I am confused. In some other sources, they mention that once you add the fragment both ends hybridize with thte two oligos and then we have the PCR extension which makes more sense. Anyone can help?
Hello, thank you for the nice video. It is indeed very helpful.
One question that has been addressed before by another person (@quickfastgoninja) in the comments:
Considering that both oligo complements ('purple' and 'blue') are present in the flow cell:
what is preventing the "blue" sequences from hybridizing when the DNA is flowed across the cell? From the animation, it seems that purple:purple hybridization would be equally as likely as simultaneous blue:blue hybridization. Are the "blue" adapters somehow blocked from hybridization when the samples are first flowed across the cell?
I think the machine goes through different cycles of temperature. rising the temperature "denatures" the DNA, breaking any double-strand bonds. then, the unwanted DNA molecules are washed away, while the DNA strands attached to the flow cell are retained by mechanical force (ie the fact that they are bound to the actual cell)
What is the purpose of the index reads?
at 1:26 why the original dna strand removed?
is it not same as newly formed?
plz reply asap
Wait the name is IlluminaHD‘s!
How do you control the reed's length?
I am wondering what the background music in this video is?
Hi @illumina do you allow the showing of your videos in lectures on NGS for educational purposes?
Any video that has been uploaded to TH-cam can be shared and used as long as you don't claim they are yours. As you ArcGen upload your video's or pics on imgur, instagram etc... you give them (TH-cam, Facebook, Instragram, etc...) permission to share your material.
Why a read 2 (4:00) is nessesary? How come just read 1 (2:23) is not sufficient?
Thank you for this :D
In 2:27 we can see, that synthesis goes from purple adapter to blue one, which means, purple must be 5' end. Also in 3:16 the same direction of synthesis occurs. After read product washed off in 3:26, template_X folds over and binds second oligo on the flow cell - that means that if purple adapter on template_X was 5' end, then purple adapter on flow cell in 3:33 must be 3' end. Then how is that possible, that in 3:39 the synthesis goes from 3' end to 5' end? If i missed something, just remind me please... :D but i've watched it few times and can't find it correct. BTW nice video.
Actually, in 1:36 synthesis goes from blue oligo on flow cell, so blue oligoes on flow cell must be 5' ends. That means, that in 2:27 synthesis goes from 3' end to 5' end, which shouldn't be possible.
The adapters are not specific to a 5' or 3' end. If this was the case, the ends of the dsDNA (for example, at 1:24) would not have the same colors as its complementary strand.
very helpful..
Does anyone know how only the reverse strands are removed from flow cell?
Imagine being the person that put all this together and someone else was like… “it’s missing an audio track of seven homeless guys beating aluminum pipes and playing the one last working key on a piano at the same volume as the explanation ….”
I understood nothing!
I don't doubt it is an exact exposition, but it is also obscure.
does anybody know if the fluorescent molecule is attached to the nucleotide and before the next nucleotide is added does it has to be removed in order to get a monochromatic signal?
Yes, thats right. They might do this by bleaching, for example, between the addition of subsequent nucleotides.