Loading a flow cell with ssDNA (one strand only)

[Yepler]

Hi, all-

I'm wondering if anyone has experience with loading just one strand of a ds duplex onto a flow cell. In this case, it would be the antisense (3'--5') strand, so that elongation would occur during the first round of amplification on the flow cell. Would this work and form productive clusters?

I'd be happy with comments about the logic of the idea, even if you haven't tried it before.

Thanks much!

Deb

[GW_OK]

Don't we already load flowcells with ssDNA? With the 5 minute NaOH incubation right before diluting to XpM with hyb buffer 1?

[Yepler]

Yep, we do! But it's normally denatured dsDNA and has both strands present. I want to know if I can load only one of those two strands and still get productive clusters - for simplicity's sake, assume that I have one strand biotin-labeled and captured on an avidin bead, and I denature off only the other strand.

Logically, I think it will work, but I'd like to hear other opinions (my pool of sequencing people to talk to live is pretty limited!).

ty-
Deb

[kmcarr]

Deb,

I'm having trouble understanding why you would need (want) to do this. You refer to one strand being antisense, which only makes sense in the context of the coding sequence for a gene. If your intention is, for example, directional sequencing of mRNA that is accomplished by directional library preparation protocols. If the library was not prepared in a directional manner it won't matter which strand you originally load into the flow cell.

[HESmith]

Why do you doubt that it would work? As long as the end sequences are correct, either strand is capable of annealing to the flowcell.

[Yepler]

Thanks for the responses...I contacted someone from Illumina who agreed that it should be fine (although she was nervous about degradation).