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Old 02-17-2014, 06:19 PM   #1
bunce
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Location: Perth

Join Date: Sep 2012
Posts: 55
Default 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.

Last edited by bunce; 02-17-2014 at 09:14 PM.
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Old 02-24-2014, 02:14 AM   #2
JamieHeather
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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.
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