Overview of Illumina Sequencing by Synthesis Workflow

Explore the Illumina next-generation sequencing workflow, including sequencing by synthesis (SBS) technology, in 3-dimensional detail. Go from sample preparation, to cluster generation, to sequencing on a system flow cell with the proprietary SBS process, through to data analysis on the BaseSpace Sequence Hub. #Illumina #sequencingbysynthesis #nextgensequencing

[chrisneumayer]

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

[HaroldBien]

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.

[HenryFarmery]

Thanks, I always wondered how plumbuses are made.

[anuk]

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!

[bratzillla]

is it jus me or is this repetition so heckin confusing to follow .... one thing repeated like 10 times god i hate my degree

[chasecolin]

The loud, repetitive dinging in the background is unfortunately quite distracting.

[dinithividushika]

this is one of the best videos for sequencing by synthesis. thanks a lot

[Romeworld]

In addition, a description providing the video times of each step would be useful. I.e: Paired end joining @ 2:00

[GustavoMazzei]

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!

[timothystark]

I am now understanding how DNA sequencing works clearly!

[MarceloZerillo]

The animation could have mentioned the terminator caps, which are crucial for this technology. Besides that, great explanation.

[stevenkimdmd]

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.

[TheTMSxGaming]

Wait a minute…this isn’t a Minecraft speed run

[greenneko]

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.

[laraince]

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

[dewsdays]

Next Generation Sequencing is really amazing!

[subhashinis]

Need some more explanations on index 1 and index 2

[dsim]

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"?

[reversed_me]

Thank you so much! Visualizing the process while reading about this technique is too difficult, an animated illustration is the perfect solution! ❤️

[Flyingspud]

Did anyone else get the chills at (2:01) or was that just me?