denaturation step prior to cluster generation

[dvh]

Hi,

Has anybody made clusters on a GAII flowcell using different protocol to Illumina's?

We have a low conc library to sequence. Doing more PCR has lead to clonal reads (~50% of reads duplicate as determined by outer co-ordinates of aligned read-pairs).

The Illumina cluster protocol is quite wasteful of DNA: (1-19ul DNA, to 1ul 2N NaOH in 20ul volume), take 1-8ul max of this in total 1ml hyb buffer. Then you only need 120ul of this per lane of flowcell.

We'd like to use lots of input DNA, and put all this into 120ul to load on the cluster station to sequence only one lane for this sample.

Anyone tried different methods? Especially different hyb buffers that can buffer more NaOH? I think the Illumina hyb buffer HT1 is SSC/SDS but am not sure. Or different denaturation - heating, formamide etc.

Of course we could experiment. But costly at > $1500 per flowcell lane. Just wondered if anyone had already tried alternatives.

regards

david

[elaney_k]

There was a protocol in Nature Methods by the guys in the sanger centre last year "A large genome center's improvements to the Illumina sequencing system" Nature Methods 5, 1005 - 1010 (supplementary protocol 12) but I think it may be only applicable if your using v2 sequencing kits.. worth a look though