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 ?
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!
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.
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 ruclips.net/video/oIJaA6h2bFM/видео.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.
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?
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!
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"?
@@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??
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?
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?
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!
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.
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?
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 ….”
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.
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.
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?
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?
7 лет назад
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?
7 лет назад+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).
7 лет назад
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.
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.
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.
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
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?
Nucleotides introduced during the sequencing step have 3’ blockers to prevent the addition of other nucleotides. This allows the machine to read the light given off and determine which nucleotide was attached. Unused nucleotides are then washed away, followed by the removal of the blocker and the fluorescent dye on the attached nucleotide. The next wave of nucleotides can now be introduced and read.
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.
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)
you have to use long-read sequencing technologies in combination with these. Usually this is done to help align areas with repetitive sequences greater than 150 bps (the typical read length for Illumina).
Hi Shaima, The motifs can be added in many different ways according to what library prep or sequencing method that you use. But the crucial motifs are: Sequencing primer motif - this is used to initiate the sequencing reads Index read motif - this acts as a barcode so you know where the sample came from Regions complementary to the flow cell motif - These motifs act as “hooks” (think Velcro) to the flow-cell in the sequencing machine. These are also often mentioned as P5 and P7 adaptors. I hope that helped, let us know if you have further questions. Thanks!
The length of each read is about 150 bp, since that is the most they can do to maintain accuracy. Not explained in this video, but longer reads start to generate errors due to the error rate of DNA polymerase.
Any video that has been uploaded to RUclips 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 (RUclips, Facebook, Instragram, etc...) permission to share your material.
you can contact your closest hospital. Mostly hospitals bring in funding in a project to buy this machine and they can be contacted by individuals or smaller projects. If you just want to see this machine in action, most hospitals would allow you to follow the procedure (pre-corona)
1:05 "hybridization is enabled by the first of the two types of oligos on the surface..." Does it mean that at that time only one type of oligo is hybridized though there is ssDNA snippets complementary to the other type of oligo too? And if so what's the principle?
Hi @illumina, I believe the question is: 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?
Hi J Liu, Yes, only one type of oligo is hybridized first at that step (purple in this video). This is possible because on the DNA fragment all purple oligos are complementary to the purple on the flow cell, whereas all of the blue oligos on the DNA fragment is the same sequence as the blue on the flow cell (1:08). That is why the blue oligos can only hybridize with the ones at the flow cell after one cycle of polymerization (1:18) Hopefully that answers your question. Thank you.
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 ?
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!
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
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
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:)
woah this is actually so smart
I was thinking exactly the same thing
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
In addition, a description providing the video times of each step would be useful. I.e: Paired end joining @ 2:00
Need some more explanations on index 1 and index 2
who's awake in 2021?
@@antisocialdistancing3 To everyone still confused watch this ruclips.net/video/oIJaA6h2bFM/видео.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.
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.
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
ruclips.net/video/womKfikWlxM/видео.html
He best at speedrun
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"?
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
high quality video, exciting to watch
Did anyone else get the chills at (2:01) or was that just me?
I wonder what is the physical proximity of the oligos to be able to bend.
This explains better than me prof. on class
Can anyone help me
Before the first sequencing, how the reverse strand are cleaved and washed off , leaving only forward strand?
Thank you so much! Visualizing the process while reading about this technique is too difficult, an animated illustration is the perfect solution! ❤️
It's so clear. Thank you for your video.
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??
Can anyone help me to understand what is an NGS assay? Thank you very much in advance.
Thanks to explain NGS😊
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?
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)
This video is very helpful. Thank you.
Thank you so much, it's so great and clear video
My teacher wants me to write about this complex device. But this is difficult. I'm just a high school student. Wish me luck.
How does the actual sequencing work from 2:13? It's not that clear. Does the labelled nucleotide bind and automatically release fluorescence?
yes
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.
Is single cell sequencing the hottest spot in the current sequencing field？
Wow Thanks a lot!!!
How come no polymerase is needed to add the building nucleotides to the cluster?
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!
Thank you for making this simpler!!!
very nice, thank you
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 ❤️
The best lecture vedio
I've go to study this for my genetic engineering exam
It is clear for me, thanks
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?
Why do the nucleotides have to bind sequentially?
Hi. What would happen if instead of put 5% of PhiX, I put 60% and the library is the correct amount? Thanks.
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?
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?
I am wondering what the background music in this video is?
Ive pressed the replay button 6 times
How do you control the reed's length?
well explained..thanks
thank you
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 ….”
What is the purpose of the indices in the adaptors mentioned at 0:40?
who else is here because they have an assessment
very helpful..
at 1:26 why the original dna strand removed?
is it not same as newly formed?
plz reply asap
Thanks 🤗
I've heard for some applications generation of the reverse read is not necessary, if this is true what would those applications be?
How the Oligo fold over ?
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.
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.
Wait the name is IlluminaHD‘s!
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.
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.
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.
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 !
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
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
I understood nothing!
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
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
what about ion torrent which is kinda better?
During sequencing step, how only one nucleotide could be incorporated ? Why not several nucleotides ?
Thanks 😊
Nucleotides introduced during the sequencing step have 3’ blockers to prevent the addition of other nucleotides. This allows the machine to read the light given off and determine which nucleotide was attached. Unused nucleotides are then washed away, followed by the removal of the blocker and the fluorescent dye on the attached nucleotide. The next wave of nucleotides can now be introduced and read.
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.
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 happen if we dont have the reference gene to align back the contig seqs?
you have to use long-read sequencing technologies in combination with these. Usually this is done to help align areas with repetitive sequences greater than 150 bps (the typical read length for Illumina).
0:43 what's indices?
anyone know what end is the 3', purple or blue in the video?
Pretty sure it's arbitrary - purple or blue could be either 5' or 3'. Typically a forward strand is 5' to 3' though.
What kind of motifs are added? (0.42). I am not able to understand that. Can someone please write that for me.
Hi Shaima,
The motifs can be added in many different ways according to what library prep or sequencing method that you use. But the crucial motifs are:
Sequencing primer motif - this is used to initiate the sequencing reads
Index read motif - this acts as a barcode so you know where the sample came from
Regions complementary to the flow cell motif - These motifs act as “hooks” (think Velcro) to the flow-cell in the sequencing machine. These are also often mentioned as P5 and P7 adaptors.
I hope that helped, let us know if you have further questions.
Thanks!
How is the read length determined? At 4:10
The length of each read is about 150 bp, since that is the most they can do to maintain accuracy. Not explained in this video, but longer reads start to generate errors due to the error rate of DNA polymerase.
does the extension of the primer with a nucleotide also require a polymerase? (2:15)
Hi Waldo,
Yes, the extension of the sequencing primer requires a polymerase
What is index?
Hi @illumina do you allow the showing of your videos in lectures on NGS for educational purposes?
Any video that has been uploaded to RUclips 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 (RUclips, Facebook, Instragram, etc...) permission to share your material.
You go straight into it with the unexplained technical jargon don't you
This isn't for us students
@@nero91 this is like third year stuff m8
study a degree in genetics and youll understand
Please drop the music
I passed my college watching RUclips videos
can someone link me the music?
This should be a meme
No entiendo nada ni tiene subtitulos
Rip Roche
Where can I hire use of this machine in the UK?
you can contact your closest hospital. Mostly hospitals bring in funding in a project to buy this machine and they can be contacted by individuals or smaller projects. If you just want to see this machine in action, most hospitals would allow you to follow the procedure (pre-corona)
1:05 "hybridization is enabled by the first of the two types of oligos on the surface..."
Does it mean that at that time only one type of oligo is hybridized though there is ssDNA snippets complementary to the other type of oligo too?
And if so what's the principle?
Hi @illumina,
I believe the question is: 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?
Hi J Liu,
Yes, only one type of oligo is hybridized first at that step (purple in this video). This is possible because on the DNA fragment all purple oligos are complementary to the purple on the flow cell, whereas all of the blue oligos on the DNA fragment is the same sequence as the blue on the flow cell (1:08). That is why the blue oligos can only hybridize with the ones at the flow cell after one cycle of polymerization (1:18)
Hopefully that answers your question. Thank you.
Deckow Shoal
I do that at work everyday
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Ameisin
Yeah,, I dont understand the concept enough to invest in it. Theranos feel!!