Illumina Sequencing Technology

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This video provides an overview of the DNA sequencing workflow on an Illumina sequencer. The process begins with Nextera sample preparation, followed by cluster generation on a system flow cell, sequencing with Illumina’s proprietary sequencing by synthesis technology, and culminating with data analysis.

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  1. 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?

  2. 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 … ?!

  3. 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. 

  4. 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?

  5. 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?

  6. 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?

  7. 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?

  8. I admit I came here for more illuminati bologna, ended up watching the entire video. I was captivated despite being completely lost. these are some smart mutherf*ckerz. unlike the illuminati freaks.

  9. 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 .

  10. 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? …

  11. 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!

  12. 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.

  13. 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'?

  14. 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

  15. 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? 😅

  16. Sample Prep, Cluster Generation, Sequencing and Data Analysis. Sample prep. begins with extracted and purified DNA. First step of sample prep is tagmentation. During tagmentation, transposomes simultanetously fragment and tag the input DNA with adapters. Once adapters are ligated, reduced cycle amplification adds more motifs such as sequencing primer bind sites, indices and regions which are the same as flow cell oligo or complimentary to flow cell oligo. Clustering/cluster generation is where each fragment is isothermally amplified. The flowcell is a glass slide with lanes. Each lane is a channel coated with 2 types of oligos. Hybridization is enabled by the first type of oligo on the surface. This oligo is complementary to the adapter region on one of the fragement strands. A polymerase creates a complement of the hybridized fragment. The double stranded molecule is denatured and the original template is washed away. The strands are clonally amplified to bridge amplification. In this process, the strand folds over and the adapter region hybridizes to the second type of oligo on the flowcell. Polymerases generate the complementary strand forming a double stranded bridge. This bridge is denatured resulting in two single stranded copies of the molecule that are tethered to the flowcell. The process is then repeated and occurs simultaneously for millions of clusters resulting in clonal amplification of all the fragments. After bridge amplification the reverse strands are cleaved and washed off leaving only the forward strands. The 3' ends are blocked to prevent unwanted priming. Sequencing begins with the extension of the first sequencing primer to produce the first read. With each cycle 4 fluorescently tagged nucleotides compete for addition to the growing chain. Only one is incorporated based on the sequence of the template. After the addition of each nucleotide, the clusters are excited by a light source and a characteristic fluorescent signal is emitted. This propertiary process is called sequencing by synthesis. The number of cycles determines the length of the read. The emission wavelength along with the signal intensity to determine the base call. For a given cluster all identical strands are read simultaneously. Hundreds of millions of clusters are sequenced in a massively parallel process. After the completion of the first read, the read product is washed away. In this step the Index 1 read primer is introduced and hybridized to the template. The read is generated similar to the first read. After completion of the index read, the rad product is washed off and the 3' end of the template is deprotected. Index 2; The template now bends over and binds the second oligo on the flowcell. Index 2 is read in the same manner as Index 1. Index 2 read product is washed off at the completion of this step. Paired-end sequencing – polymerases extend the second flowcell oligo forming a double stranded bridge. This double stranded DNA is then linearized and 3' ends blocked. The forward strand is cleaved off and washed away leaving the reverse strand. Read 2 begins with the introduction of the read 2 sequencing primer. As with Read 1, the sequencing steps are repeated until the desired read length is acheived. The Read 2 product is washed away. This entire process generates billions of reads representing all the fragments. Sequences from pooled sample libraries are separated based on the unique Indices introduced during sample preparation. Local sequencing clustering; for each sample, reads with similar stretches of basecalls are locally clustered. Forward and reverse reads are paired creating contingous sequences. These contigous sequences are aligned back to the reference genome for variant identification. The paired in information is used to resolve ambiguous alignments. (Transcribed from video so you can read as you watch!)

  17. 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.

  18. 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.

  19. 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?

  20. 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.

  21. What is a reference strand? Is it the original DNA sequence which was fractured? Or is it simply a DNA with lot of known different sequence on it?


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