Complementary strand in cluster generation

Question

[KirGen]

Hello! I don't understand,when a library is denatured before loading in the ngs instrument, I'll have the two complementary strands separately; so they will form two different clusters, the system recognizes them as duplicates because they come from the same molecule and give the same informations?Or if not how the system could recognize that they come from the same molecule but the are one the opposite strand of the other and so not duplicates? Thank you!

[finswimmer]

Interesting question.

As ATpoint notice you have different adapters on the p5 and p7 ends. For both there are complement sequences attached to the flow cell surface. I guess one complementary sequence on the surface is initially blocked, so only one strand can bind. After this strand is amplified, the new double strand is denatured and the original strand is washed away. At the same time the second strand, that could not bind because the adapter on the surface is blocked, is washed away as well. Finally the blocked surface adapters gets unblocked and bridge amplification can start.

Answer 1

[ATpoint]

No because the adapters on each side of the fragments are different and only one fits the specific oligo on the flow cell surface during initial hybridization. The other one is washed away, see the video at about 1:05min.

[KirGen]

Yes, but the two complementary strands have both p5 and p7 to the opposite ends. So when they are denatured and in single strand they behave as two different fragment with p5 at one end and p7 at the other end..so they will form separate clusters is correct? And from this cluster I'll have the same information.. Sorry but I continue to don't understand.

[ATpoint]

Yes but not the same sequence, the second strand has the complement which does not fit the oligos on the flowcell.

[KirGen]

You mean that the system enable the hybridization of only one oligo first?because on the other hand the opposite strand will hybridize with the other adapter. For example adapter p5 is attack to the flowcell in direction 5'-3' ,so it will be complementary to the reverse strand p5 adapter sequence while p7 on the flowcell will hybridize with 3'p7 sequence on the forward;if is it possible the hybridization of both oligos togheter,both strands will hybridize at the same time

[ATpoint]

From how I understand this only the adapter regions of one of the two strands are complementary to the flow cell oligos so one strand forms the cluster. This is an important basis for strand specific kits to work and retain strand information.

[Marvin]

I have the same question and I don't think the question was really answered. You have a double-stranded fragment, Adapter1 on one side and Adapter2 on the other side. Now you dehybridize. Let's say on the flow cell there is ONLY the complement to Adapter1. But BOTH single-stranded fragments have Adapter1!! I think that is the main point that @KirGen was trying to make. I'm not 100% sure about this but I THINK the answer must be that on single-stranded-fragment 1 the Adapter1 ends with a 5' base whereas on single-stranded-fragment 2 the Adapter1 Ends with a 3' base. That might explain why only 1 of the 2 single-stranded fragments can bind. BUT now I have another question. We were assuming that on the flow cell there is ONLY the complement to Adapter1. But in the videos you can clearly see that there are 2 types of Adapters on the flow cell!! So if the FIRST single-stranded fragment attaches to the Adapter1-complement on the flow cell (because it is the 5' end), I would expect the SECOND single-stranded fragment to attach to the Adapter2-complement on the flow cell (because this time, fragment 2 has the 5' end!). So once again I would expect both single-stranded fragments to form separate clusters on the flow cell. Why is this not the case?