MiSeq indexing amplicons and custom primer Tm

[melcar]

I am planning to sequence 3 mtDNA amplicons per sample using the Nextera XT kit. First I want to see if it is OK for the Nextera indexing be done once after the 3 amplicons are pooled in equal molar ratios?

Second many of my custom template primers have Tms below the 60-65C range outlined by illumnia. Just wondered if anyone has tried adding a CG tail on the 5' end of the primer (positioned between the primer and the adapter) to increase the Tm of the Custom template primer? My custom primers are AT rich and placed in universal regions, so increasing the length by adding sequence would be difficult.

Any advice most welcome ...

[bunce]

Hi

To address your second question - you could use LNA's to boost the Tm's of your custom primer. Fluidigm uses LNA's on the first, third and 6th base of from the 5' end.

If there are just a few degrees off you could (at the p5 end if you are doing single end reads) add the bases ACAC to the 5' end of your sequencing primer..... this 'borrows' a bit of P5 but is still outside of where the strand is cleaved. I would avoid encroaching further into P5 (we have had issues).

(edit; I should point out that this ACAC addition will only work if your indexes are not situated between p5 and the sequencing primer)

Hope this helps?

[melcar]

Just curious what type of issues you had with playing wit the 5' end? Was it to do with PCR of the amplicons or the illumina sequencing? Still getting my head around the MiSeq technology. I am more accustom to 454 pyrosequening.

[bunce]

the issues we had was trying to sequence illumina libraries like they were "454 & Ion Torrent" - i.e. using the adapters themselves as sequencing primers. When we tried this (and even play around with tweaking recipes and melt temps) we got clustering but no sequencing. We ended up using a short LNA custom primer - which is working very well.

I should state that the reason we are doing this is to generate sequenceable indexed amplicons in a single step (and to do this the PCR efficiency goes down when you are using really long primers).

[melcar]

Thanks for your response. Where did you order you LNA primers from?

Also regarding you edit. My primers are the custom primer followed by the illumina adaptor. So if i did modify the 5' end of the primer it would have the adaptor on the other side not an index. I am planning to use the Nextra kit to index the samples and do a 300bp paired end run

[bunce]

In Australia you need to order them through GeneWorks - they licence them from https://www.exiqon.com/oligo-tools

The whole illumina "separate index" architecture frustrates me a bit - it is really a hang-up from times when sequencing reads were so short that it was a waste to sequence indexes "in line" with the rest of your library. We have modified this in our amplicon sequencing workflows. LNA's may be the only way to go? - although the primers are expensive (a few hundred $) so you would want to get more than a single experiment out of them.

One other option is to drop the annealing temperature in your run recipe - I have never done this so it would be risky without getting some advice first. As always it somewhat depends on how much money you want to throw at your options to explore them!