Sequencing Degenerate Sequences

[tttaaa]

Hello!

My research right now involves sequencing DNA sequences that were amplified by degenerate primers in PCR. I have been informed by Laragen that it's not possible to sequence my PCR amplicons because of the degeneracy at the beginning and end of each sequence. So I have had to use TOPO clone libraries to get M13 sequences incorporated at the very beginning and ends of my amplicons. Using this approach, I have gotten sequence results, but I am wondering if I am overlooking a possibly easier protocol.

Thanks in advance for your replies.

[Andrew_Slatter]

Can't you put M13 or NGS primer sequences in a 5' tail to the 3' degeneracy ? That way your amplicons would be directly sequencable with no extra steps

[krobison]

How many do you want to sequence & how long are your amplicons? Deep sequencing on a NGS platform would give you a lot of data without cloning, but if you don't want/need lots of sequence the cost might be an issue.

[[email protected]]

Agree with krobison, PCR amplicons produced using denegerate primers can be sequenced using MiSeq. The cost is dependent upon the number of amplicons/samples (multiplexing) and amplicon length (different library construction methods).

[tttaaa]

Thank you for your replies!
We tried using M13 as part of our primers, but the PCR was not successful. And at the end, if it does work, it's not guaranteed that there won't be a variation of the same gene within our samples, which is why we're using degenerate primers anyway. It's worth trying to optimize further, or maybe just increase the concentration of degenerate primers when sending the primers for sequencing.

The miseq method sounds good though it's more pricey than I'd like it to be. I have 8 different pcr reactions for every sample (not sure about number of amplicons, it very much varies), and I have around 20 samples.

I haven't done a miseq experiment before. It seems like you can run everything on one lane with multiplexing. And every lane is 2000 bucks or so + library prep?

[tttaaa]

I forgot to answer the question about amplicon length: it varies between 300-400 bp for these 8 sets of primers

[[email protected]]

The amplicons are within the sequencing length so it is possible to contruct sequencing libraries directly using secondary PCRs, which could significantly reduce the library construction costs. A single MiSeq run should produce about 75,000 pair end reads per primer set, assuming that you would run 20 samples in the single run and it costs less than $2000 per run. If sequencing adaptors and barcodes can be added using secondary PCRs, library construction costs should be manageable. Otherwise, you can always add sequencing adaptor and index using library construction kits, which will cost more.