+Illumina Inc Have to agree with Tiago's comment. I have had multiple professors attempt explanations of this with little success comparatively. Thank you!!
This is just mindblowing. Truly amazing how far we've come in the last 25 years in the field of genomics. To think that incremental improvements in research over the years were capable of combining to make such a clever method of sequencing. Im still left speechless.
+Gustavo Loureiro It allows for a copy of your DNA profile to be made, and from this medicines unique to you are possible, rather than as at present, a generalised one size fits all.
thank you so much for this explanation of the illumina sequencing ,but i didn't understand something, flowcell oligo are complementary only to the region mentioned on the video " 0:42 min" or to all the sequence composed of "region complementary to flowcell oligo+index+ sequence primer binding site " i was confused because in 1:08 it is shown that all sequence is complementary to the oligo on the flowcell . I'm waiting for your response thank you .
I'm not sure about where the fluorophore is binded on the nucleotides. It can't be binded on the 3'-OH or it would stop the polymerase reaction. Anybody knows?
I don't get something. How to you make sure that only one fragment gets caught in a single cluster? If 2 fragments get caught in the cluster, wouldn't that PCR into 2 different sequences, messing up the reading later?
If you mean " If 2 fragments get caught in the same spot, so as to generate a mixed cluster instead of a uniform one", the answer is Yes, it can happen. They arrange the concentration of DNA fragments in such way that generally only one molecule will strand to every small spot at the flow cell, and every cluster created from it occupies only a tiny spot. Occasionally a couple of fragments will fall together mixing the signal but that's small exception. Each part of the original DNA molecules is represented many times. A computer deduces the overall sequence from the majority of signals, so a small number of mixed signals doesn't really change anything.
Why do you need to sequence indices? I thought its known already, since you add it in the beginning. Also, why even care to continue sequencing, if the template portion has been sequenced by synthesis?
Pavel Ryzhov you can pool many samples e.g. different microbiomes and reduce costs due to massive parallelization. second sequencing is for verification of first sequ.
Pls, if some1 could explain, i know im Missing something. How does the polymerase stop between each fluorescent nucleotide? Can a new fluorescent nucleotide hybridise next to another fluorescent nucleotide? Do the nuclotides still have the OH group that lets the polymerase extend the strand? How does emission of previous fluorescent nuclotides not overlap with emission of the 'last added', essentially how do you know the color of the last added nucleotide? Do they measure intensity of the light and can tell an increase in one of the 4 fluorescent nucleotides when already having say 30 of the same nucleotide hybridised prior to the last one added'?
Hello, nice video, I've always wondered how can the image software to know the identity of each new base added to the growing strand, given that this chain is composed by a mixture of many fluorescent nucleotids?
Very good explanation! The only thing I didn’t understand was the part with the index part. What happens exactly when the second primer binds at the first read and why? 😅
0:36 the upper original strand should be 5 prime to 3 prime, so the primer should bind on the right bottom side , instead of on the top left side (as shown in the video) thus the amplification can go from 5 prime of the primer to 3 prime of the primer, right? In this video it seems the amplification is going from 3 prime of the primer to 5 prime.
Maybe stupid question. But for what can I use this technique, except for sequencing a complete genome. Can I sequence shorter areas and compare different individuals or something like this, to identify the mutated gene f.e.? I am practising for an upcoming exam and I really wanna understand what these techniques can be used for, because my prof asked very specific and practical questions.
How is the order of the bases specified , does every cycle assume that the next base shows up is up next in the sequence? And the order in which the bases light up from the flow cell, is the same order for the alignment? What’s the point of getting loads of the same read? Does the genome have different sequences for the same gene? What
You should label 3' and 5' and make the colors of the DNA strands different colors and not just blue. Not labeling and using the same colors makes people confused on what is the template and non template strand. Also, the color coding you used for forward and reverse strand adapters at the start of the video does not match the rest of the video.
WHAT ARE the unique indices they talk about they introduce during sample prep? is it a particular sequence that allows them to distinguish one fragment from another?
Each kit come with a certain amount of unique index, let's say 12. You can then prepare up to 12 different samples (or patients), each one getting a unique index that identifies it. The 12 samples are then pooled together in 1 unique container, and everything goes through de sequencing process you just saw there. The computer analysis will then use the index reading datas to sort the reads "per sample" and make up a whole exome or genome for each sample :) Hope it makes sens
try giving pauses next time for instructional videos. Let us digest what we hear before shoving more info in the next minute. I know, I did play you at 0.75X speed and nice explanation. So thank you.
Not detailed enough. Has each flow cell different oligos? How does the "denaturing" work? What if around a single and binded strand there's no oligo left and the strand can't build a bridge, do we get a double stranded DNA again? How is the reverse strand washed away? How is the 3' adapter blocked? ...
Each flow cell has the same oligos i think, Strands that fail to have adapters ligated are washed away, and i think they block the 3' adapter by binding with a complementary sequence, I hope I have been a little helpful but i'm not really sure if i'm right.
Maybe I´m just too tired after a long day, but I don´t really get what the index primers/reads are for. Do the index sequences change according to where the tagmentation was carried on or something like that? Otherwise, I don´t really see the point. Could someone explain this to me?
When sample libraries are prepared, unique indices are attached to each fragment. The libraries are then pooled and then run on the machine. The index reads are used afterwards to identify and separate the sample libraries.
katamari30000 - see my reply to AntiPop4Ever at the top of the comments. The indices are used to differentiate between the paired ends of the sequenced read.
Hello, very nice video!, i have only one question, when you run a PE sequencing, as shown in the video, how its posible sintetice a reverse strand (is not the same that complementary), because in 2:20 you can see the sequence of the template strand and in 4:08 you can see the same sequence of the template but in reverse. Is the polymerase able to sintetice the reverse strand?, because the polymerase should sintetice the complementary. Hoping a good reception, Thanks You!
Hello, How do light reading sensors distinguish the strands in the reading phase because there may for example be a shift and therefore the sensor will receive shifted information?
Stupid question but are all the clusters composed of the same fragment of DNA or is each cluster a different fragment? If each one is different, how do you get specific fragments to bind to the oligos in only one place?
Tom Carnwath Each cluster is composed of a different fragment of DNA. When a fragment binds an oligo in the flowcell lane, it undergoes bridge amplification which stays confined to the vicinity of the original fragment.
Carmen Sandoval Yeah, but how to you guarantee "one fragment per cluster"? If you have 2 or more fragments bind near each other, woudln't the bridge amplification yield a mixed set of DNA (arround that same spot)? I'm missing something here I take it.
+rasmasyean from what I understood, you only separate the sequence after synthesis is complete. Listen starting at minute 4:17. You don't have to 'guarantee ' a specific location of any fragment. You only need the sequence and the computer software will sort the sequences out by identity matching, etc.
Nouri Al-Kadhim Let me rephrase the question. OK, each "flash" is from a cluster location of cloned DNA, right? They clone the initial fragment (in each cluster location) into many copies cuz building 1 DNA strand wouldn't make a bright enough flash to be detected by the camera, correct? But in the flowcell, you have to ensure each cluster location has only 1 initial DNA fragment. If you have 2 DNA fragments in one location, it will bridge amplify into dual clones in one spot. Then you can't reliably read flashes from that cluster location. How do you guarantee that there is only 1 initial fragment per cluster. Not 2+.
+Alexander Thomas You don't. And that doesn't happen. There's something called "quality control" after all these. Let me explain: In each cluster, there's the same string with same nucleotides. Let's follow the example from the video. It says that T binds first, and another T after that. But this doesn't always happen at the same time for all the strings in a cluster. For example, when T binds to the second complementary nucleotide, in another string in the same cluster, A binds to the third complementary nucleotide. But when you look at the overall color scheme, you know that T comes the second with high certainty. Sadly, as the sequencing goes, the certainty level drops a lot. And when you come to the, lets say, 100. nucleotide, the certainty drops massively. Paired-end reading can help with dealing with this problem. So, as I said in the beginning, you have to do some quality control after these. Which includes, trimming of low quality base calls, removal of contaminants etc.
Alexander, in short, every base added has a block-cap and a dye. Only one base can be added in each circle. After the reading at the end of each circle, the non-integrated NTs are washed away, then the block and the dye are both cleaved and the circle is repeated. Now the last integrated Nt can receive a new Nt but no longer has a dye. Thus, in each circle only one Nt can be integrated and only the last one can produce light.
At minute 2:51, how can one know that all the identical strands are simultaneously amplified? this should be important, if they are not amplified at the same time there are different fluorescent signals emitted at the same time ... ?!
i think a sufficiently high concentration of dna polymerase, the knowledge of the rate of nucleotide addition of dna pol and sufficient time should solve your problem.
For example, one flow cells can generate 150M reads, but it's too many for one sample. So samples with different indexes can be pooled together and sequenced. All the reads will be assigned to different samples.
This is actually for coupling the index to the sample. It is not related to the sequence of the sample but just to identify to which treatment the sequence belongs to
