mothur

archaea primer set up for MiSeq

I am new to archaea specific amplification and am wondering if anyone has a specific primer set up for archaea to be sequenced using MiSeq with custom dual barcoding?

I have found that the best region with MiSeq may be the v3-v4(517F/958R) region (according to VAMPS) and wanted to know if I could use their suggested 16S archaea specific primers in place of the v4 bacterial primers used in the custom primer method outlined by Kozich and Schloss. In other words, could I use the same adapter-barcode-pad-linker-16Sprimer(F/R) sequences and only change the 16S specific primers (keeping in mind the Tm of course)?

I have also read the paper by Klindworth et al, 2012 suggesting the use of 349F/519R for Illumina sequencing, but I fear that this specific primer set may create sequences too short for MiSeq sequencing. I was unsucessful in finding other published papers that amplified archaea and sequences using MiSeq.

Any insight or suggestions would be greatly appreciated! Thank you.

I guess I would suggest you go more toward the 349/519 primer set because the reads would fully overlap.

Thanks Pat,

I had originally thought though that the 349/519 might be too short for MiSeq considering each read would only be about 185 nt long and the reads for your v4 wet lab SOP were about 290 nt long. Would the reads overlap enough, or would it better to have a longer reads for overlap?

Would you suggest then to use the 349/519 primer set with the exact same set up as with using the v4 region for bacterial primers? so it would be:
adapter->barcode->pad->linker->16Sprimer(ARCH349F)
adapter->barcode->pad-linker->16Sprimer(ARCH519R)

Of course I would make sure to change the pad sequence if needed so that the Tm is at least 65, but I just wanted to make sure that this set up would still work with Archaea specific primers? Thank you for all of your help.

yep, i think that would work well. one thing we’ve recently learned with using the 300PE kit to sequence the v4 region is to limit the number of cycles to 250 instead of 300 (you’ll still get the benefit of the increased number of reads)

Hi Pat,

I am designing my primers to order with the 349/519 primer pair.
I’ve read that this pair produces reads from the V3 region, so do the linkers change? Currently I have them designed with the forward linker GT, and the reverse linker CC.

However, I found on the MiSeq_WetLab_SOP_v3 the following information:
Link:
V4f: GT
V4r: CC
V3f: GG
V5r: GG

So should I be using GG as my forward linker instead?

Thank you in advance!

You may need to adjust the pad and the linker to get your TM to 65

Thanks!

[aadusche], would you be able to share which pad and linker you used for this primer pair?