problems with M13 universal tailed fusion PCR for 454 pyrosequencing

[melcar]

I am having great difficulty getting the 2nd round PCRs (where I add the 454 fusion tailed primers to my template specific primers using an M13 primer tail) working effectively. I generally get a very low amount of PCR product that is often a smear. I have been gel purifying the 1st round add trying a variety of PCR protocols and profiles (adding 150ng 1st round template and PCRing for 4 cycles and also just adding 1ul and PCRing for 15 cycles), but nothing seems to work. Does anyone have a protocol that works for this step?
I am also concerned that the overhanging "A" added by the taq polymerase may be a issue for annealing of fusion tailed primers. Has anybody found the type polymerase has an effect? I have tried Bio-act short mix and Roche's FastStart.

Any advice would be most welcome!

[neophyte]

Maybe a "suppressive PCR" effect ?
see Evrogen...

[ParkerKB]

I am just about to embark on this so don't have much to offer...have you seen Bybee et al. 2011 and Hajibabaei et al. 2011? Bybee uses a different tail and Hajibabaei doesn't specify, but I am assuming it is also M13 as I know they use M13 tails for standard sanger sequencing. Daigle et al. 2011 also has some details. I also recently came across a blog of someone who is troubleshooting this, I haven't read it thoroughly but it may have some ideas.

http://enggen-nau.blogspot.ca/2012/03/our-lab-has-been-sending-samples-off-to.html

[melcar]

Thanks for the tips. I ended up having alot of trouble using an m13 tail with my COI primers. I ended up developing my own tail, which was G-C rich. This meant I could use a high Tm and avoid problems with secondary structure in the 2nd round PCR's. Although I haven't got the data from the run yet to see how well it has worked

It is hard to find detailed information on the success of different universal tails

[lotijeo]

universal tail

Hi Melcar,
Did you have any success using customized universal tails? I wanted to try the M13 sequences but maybe you had a better luck with your customized GC rich tail?

[melcar]

The GC rich Tail work really well, I got heaps of product in the secondary PCR and it sequenced well using 454. I would definitely use these types of primers over M13. I have submitted the work for publication, so finger crossed it will be out soon

[alezhe]

melcar

we would like to know details of your success. could you share with the public the sequences of universal tails you have developed.

[melcar]

Universal tail

The work in now published. See below

Title: Environmental monitoring using next generation sequencing: rapid identification of macroinvertebrate bioindicator species

Authors: Carew E Melissa, Pettigrove J Vincent, Metzeling Leon, Hoffmann A Ary,

DOI: 10.1186/10.1186/1742-9994-10-45

URL: http://www.frontiersinzoology.com/content/10/1/45

You will find the sequences for the universal tails in the methods

Cheers

[alezhe]

Thank you a lot!