Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa

Similar Threads
Thread Thread Starter Forum Replies Last Post
MiSeq Custom Sequencing Primer Tm? pmiguel Illumina/Solexa 43 11-12-2015 11:52 AM
Miseq Custom primer for 16s V1V3 region lmma Illumina/Solexa 12 02-26-2015 01:13 PM
Primer and adapter design for small RNA sequencing in HiSeq2000 and MiSeq windhorse8 Illumina/Solexa 3 10-09-2013 06:39 AM
Validation of Custom Sequencing Primer Akira Illumina/Solexa 1 11-05-2012 09:22 AM
50+% of my HiSEQ reads are 3' primer (custom primer used) ZAAB Sample Prep / Library Generation 3 02-06-2012 11:00 AM

Thread Tools
Old 02-17-2014, 05:19 PM   #1
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 08:14 PM.
bunce is offline   Reply With Quote
Old 02-24-2014, 01:14 AM   #2
Location: London

Join Date: Nov 2012
Posts: 96

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.
JamieHeather is offline   Reply With Quote

custom primers, fusion primers, lna, miseq

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off

All times are GMT -8. The time now is 01:41 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO