Hey Everyone. I am trying to DNA metabarcode (COI) bulk samples of aquatic insects.I would like to use fusion primers consisting of an Illumina MiSeq flow cell bind, sequencing primer binding regions, unique tag (base shift), and my amplicon of interest as done by Elbrecht and Leese here:
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130324
The problem is choosing an appropriate taq. After contacting the authors, I learned that the specific taq used in this study is currently not available and several others have failed to get amplification with this type of primer.
What type of taq should I use? The literature suggests that some of the TaKaRa Biosystems taqs might work (Hot start or Ex-taq), at least they did for metabarcoding using pyrosequencing. Any suggestions. Thanks!
Hi Brian,
We performed a lot of metabarcoding sequencing runs in our lab and we used the Multiplex PCR Kit from Qiagen (e.g. in Galan M, Pagès M, Cosson JF. 2012. Next-generation sequencing for rodent barcoding: species identification from fresh, degraded and environmental samples. PLoSOne 7(11): e48374. doi:10.1371/journal.pone.0048374).
I compared it with others polymerases (e.g. Taq Gold from Life Tech, or Molzym and Sigma MTP for 16SrRNA amplicon seq) and the Multiplex PCR Kit is definitively the best (in sensitivity, repeatability and efficiency) when I used fusion primers.
All the best
Max
Thanks Maxime! Indeed, I can verify that the Qiagen Multiplex PCR kit works for one step metabarcoding library prep with fusion primers.
Hi Brian,
If you need an alternative to that, Ive found that Kapa have some great taqs, especially if you are having any sort of issues with inhibition.
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