Does extra sequence preceeding the adapter interfere with efficient sequencing

[clueheart]

Hi,

using a HiSeq device I plan to do amplicon sequencing on a fragment isolated from a plasmid library following restriction digestion with a homing endonuclease. As such, the fragments to be deep-sequenced contain a 11/15 base-long sequence at its 3' end, preceding the adapter sequence. Would that cause any problems at any steps during sequencing, specifically, in attaching them efficiently to the flow cell?

[nucacidhunter]

It should not affect hybridization to flow cell, unless it is highly similar to flow cell reverse complement. If those sequences are at 3' end of fragments they will not affect Read1, but they will result in low diversity region at the start of Read2 if you are doing paired end sequencing.

[clueheart]

Thanks, Nucacidhunter, for your reply.

I am sorry, I might not have put my question correctly. Typically, adapters containing the flow cell binding site are flanking the library fragment to be sequenced. But in our case there is an extra sequence at the 5' or at the 3' very end of the fragment.

Using a HiSeq device I plan to do amplicon sequencing on a fragment isolated from a plasmid library following restriction digestion with a homing endonuclease. As such, the fragment to be deep-sequenced contain a 11/15 base-long sequence at its end, terminal to the adapter sequence. Would that cause any problems at any steps during sequencing, specifically, in attaching them efficiently to the flow cell?

Here is the order of the relevant sequence units:
EXTRA sequence - adapter 1(P5) - sequence of interest - adapter 2(P7)
or
adapter 1(P5) - sequence of interest - adapter 2(P7) - EXTRA sequence

I wonder if anyone has used such sequences.

[microgirl123]

So you are saying that you have extra sequence on the outside of the adapters? I don't think this will work - the ends of the adapters are what bind to the flow cell. I envision excess DNA getting in the way and not letting flow cell binding happen efficiently. Even if it bends out of the way to allow binding to happen, it seems like it would interfere with the binding of neighboring clusters.

[nucacidhunter]

This is a difficult call and I am not sure. In theory the extra sequences should not interfere with clustering. After hybridisation and 1st extension the original template is denatured and washed away. However, the extra sequence in its 5’ end will be copied to 3’ end of newly synthesized complementary strand which will form bridge and extended at consequent cycles of extension carrying those sequences. This strand will be cleaved before read 1 sequencing primer hybridisation and the complementary strand which has normal adapter sequences will be used as template for sequencing. Here is the video link to reaction on flow cell: https://www.youtube.com/watch?v=HMyCqWhwB8E