Short LNA custom sequencing primer for MiSeq
 

Hi All,

For a couple of reasons we are trying to 'shorten' the custom sequencing primer we use in MiSeq. Primarily it is because we are trying to generate 'complete' fusion-primers in a single step. I have two queries that I was hoping to get comment on.

1) Has anyone used a LNA primer shorter than the 22bp fluidigm primer (A+CA+CTG+ACGACATGGTTCTACA). I am tempted to add a few more LNA's and remove some 3' bases but was hoping someone might have done this already?

2) We tried (very unsuccessfully) to overlap the sequencing primer into the adaptor(s) - has anyone managed to achieve this? The fluidgm LNA primer overlaps by 4bp into the P5 adapter.... but i was hoping that someone in SeqAnswers might have cracked this.

The upshot is that we feel we have lost PCR efficiency (assessed by qPCR) when compared to fusion-tagged 454 and PGM adaptors (which do not have separate sequencing primers). Any thoughts/suggestions would be gratefully appreciated. Best, Mike.

I know that some people on this forum just add some padding nucleotides before the adapter, so you can extend the sequencing primer back into that, bringing your Tm up. I was certainly once told by an Illumina rep that they'd be wary of engineering overlap between the lawn oligos and sequencing primers.