Homopolymeric bases problem

Replies to This Discussion

Permalink Reply by Ursula Eberhardt on February 25, 2011 at 3:21pm

Hi!

I have seen that happening also in other fungi. As long as there are only one of these homopolymeric regions in the ITS1 and ITS2 each, intermediate ITS primers (i.e. ITS2 paired with one of the forward primers; ITS3 paired with one of the reverse primers) are a solution. Even if there are more of such regions, the shorter the fragment, the more likely it can be sequenced completely. But: One of the possible reasons for the 'trailing off' is that there are +/- equally high proportions of several versions of the ITS present in the cells with different repeat numbers. Even if the sequence continues, it will very likely be unreadable. However, even those bad sequences may then give an indication whether there are stretches of sequence missing between the repeats or whether complete ITS sequences can be pieced together from the sequence results of the different primers. 

Hope this will help, Ursula

Permalink Reply by Franck Stefani on February 26, 2011 at 3:56am

Hi there, 

I am facing polymerase slippage with Dermocybe specimens (40% of my ITS sequences are messy...) because of the presence of poly-T or poly-G either in the ITS1 or 2. Until now, I have used the primer sets ITS1-F/ITS4 or ITS1-F/ITS4b to amplify my samples. I am trying next week to amplify shorter ITS fragments with primers ITS1, 2, 3, 4, 5. But I am not certain it would fix the problem.

My clean sequences display these homopolymeric regions, so it looks like the enzyme slippage does not occur systematically. Does anyone have a solution (fitting the barcoding philosophy...) to fix that problem?

Franck

Permalink Reply by karen Hansen on February 26, 2011 at 12:05pm

Hi 

Years ago I had this problem with three groups of Pezizaceae. They possessed sequences with short homopolymere regions of 8 to 12 Ts or As in different positions, which caused sequencing problems. I obtained the majority of these sequences by use of internal primers reading in both directions. To obtain complete sequences from nine of these collections/taxa, however, I had eventually to clone the PCR products (for these I did the PCR using Pfu DNA polymerase).

Karen 

Permalink Reply by Franck Stefani on February 26, 2011 at 4:02am

Some solutions are suggested here: http://pan.stanford.edu/section_html/DNASEQ/analysis.html

Permalink Reply by Ursula Eberhardt on February 26, 2011 at 7:31am

Hi again,

I do not want to go into barcode philosophy ... but barcode philosophy embraces also the idea of minibarcodes when technical problems .

What I forgot yesterday: Obviously, short fragments can be sequenced from much longer PCR products, but results tend to be better from short PCR products, already produced from the same intermediate primers. 

Ursula

Permalink Reply by Franck Stefani on February 27, 2011 at 6:13am

Hi, 

 

I think this paper provides a good set of solutions against polymerase slippage:

http://www.biotechniques.com/multimedia/archive/00087/BTN_A_0001133...

 

Franck

Permalink Reply by Eric Tretter on February 28, 2011 at 1:30pm

Thank you for you help!  I'm a graduate student working in Merlin White's lab.  I'd actually found that paper a while back when researching this issue and we have actually tried the Phusion polymerase.  We have not detected any particular improvement in the homopolymer repeat problem with regard to sequencing, but it does seem particularly robust with regard to the amplification of sequence fragments that contain these repeats. 

 

It may be that we tried the wrong Phusion product (we attempted it with the Phusion Hot-Start II enzyme, perhaps we should have used the normal one) or it may be that the homopolymer repeats are located in regions that also form hairpins. We may try the Herculase II Fusion enzyme as well.  So we haven't completely run through all of the ideas presented by this paper.