SR Adapters on a PE flowcell?

[andibody]

Does this work? Of course only for the first read...
Or do you think colony formation will not work at all?

Thanks

[ETHANol]

Use the search function and look through recent posts before asking a question. Thanks.

[andibody]

Thanks for your comment but I haven't found the exact answer I'm looking for.
I know single read adapters can not be used for paired end sequencing, the question is if I get only one read or if colony formation is totally inhibited.

[ein_io]

Indeed andibody

I just asked this question yesterday.

I also called our Seq. rep. She said that she could make a single read from a library made with PE adapters on a single read flow cell. But of course she cannot do PE seq on a single read flow cell.

hope that helps

let me know what you think

ein_io

[Robby]

You can sequence the first read, but in the second read are of course just N's (we tested that). Furthermore you might have problems to recognize the Index-sequence => so it might be better to add an index at the beginning of the first read

[pmiguel]

Originally Posted by Robby

Furthermore you might have problems to recognize the Index-sequence => so it might be better to add an index at the beginning of the first read

Not sure what you mean here. TruSeq Index reads are possible (they do work) on SR or PE flow cells. Do you mean there is another non-inline indexing scheme that might not work?

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Phillip

[andibody]

We had to fill up a PE flowcell and took therefore this SR library (small RNA adapters no Truseq).
It didn't really work, very few, very small clusters. I was expecting to get the first read only for that sample as Robby mentioned...

[Robby]

As far as I understand the adapter-sequences, the difference between the small RNA adapter and the pe-adapter is just the P7-part. Because of that the bridge amplification can't be performed in the correct way, which causes small clusters. Also due to the different P7-part, just the P5-part can bind to the flowcell and the sequencing for the second read can't start. Nevertheless the sequencing primer and indexing primer can still bind.

In theory I think the sequencing of a single end library with a paired end kit should work very bad, but in practice we observed that the sequencing of the forward reads works quite good (many reads in good quality), although the quality of the index/barcode read was very bad and the sorting was impossible.

[andibody]

Great, thanks a lot, that's exactly what I wanted to know.

Best Wishes
Andi

[TonyBrooks]

As I understand it, the oligos on the SR flowcell are similar as on the PE flowcell, only shorter (at the 3' end). This means PE libraries can bind to SR flowcells and amplify, but SR libraries can't bind to PE flowcells (as the 3' end of the flowcell oligo is unbound to the library and therefore can't extend - or does so very poorly).
Read 1 sequencing oligo is the same for all applications (except small RNA).

[protist]

Tony is correct re the length differences - they can be seen in the original customer letter re adapters from 2008-2009. Also the linearisation reaction is different for PE versus SR flowcells. The single read P5 undergoes a periodate mediated linearisation whereas the PE P5 is enzymatic based. So when clustering the LMX mix from a PE cluster kit cannot be substituted for the mix in SR kit. I have attached a screen-shot from a very nice BROAD/Illumina presentation (http://www.broadinstitute.org/files/...PrepSlides.pdf). PE libraries for PE/SR flowcells, SR libraries for SR flowcells.

[TonyBrooks]

So theoretically, it may be possible to do PE sequencing on a SR flowcell, as long as you use the PE cluster gen reagents? Or maybe the ribose residue (I'm assuming this is how the cleavage site is marked) used to mark the cleavage site in the SR flowcell would interfere with the enzymatic linearisation in the PE kit?

[ECO]

Originally Posted by TonyBrooks

So theoretically, it may be possible to do PE sequencing on a SR flowcell, as long as you use the PE cluster gen reagents? Or maybe the ribose residue (I'm assuming this is how the cleavage site is marked) used to mark the cleavage site in the SR flowcell would interfere with the enzymatic linearisation in the PE kit?

Nope. The magic of the PE chemistry is a P5 primer that can be cleaved (via a uracil) and leave behind a cluster compatible primer for the paired end turnaround, in addition to the periodate-labile other primer. For PE seq you need to be able to linearize twice...at first cluster gen, then after the PE-turnaround.

Please correct me if I'm wrong.

[HESmith]

Originally Posted by ECO

Please correct me if I'm wrong.

You are 100% correct.

[DJ Flowcell]

If I accidentally clustered PE libraries with a PE cluster kit on the SR flowcell, would the reads be good or bad? Let's forget about index and turnaround and second read, what would the first few cycles look like? Weird question but I'm troubleshooting a weird problem.

Thanks!

[pmiguel]

My guess is that they would be fine. But your really just want to phone Illumina's tech support. They are generally excellent, but this is the sort of issue they know backwards and forwards.

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Phillip