I would like to create custom primers for MiSeq that will have a higher and more specific coverage of archaea. I have 2 questions pertaining to this:
1. What is the best hypervariable region for archaea using MiSeq? I have read the Klindworth et al, 2012 paper on the best primers for NGS and it suggests that the best archaeal primers for Illumina are in the V3 region, however, I believe that the region may be too short for complete overlap on MiSeq 2x250 (they may have used different Illumina chemistry, or HiSeq?). I have also come across the V4-V5 region for Archaea on MiSeq using 517F and 958R. Does anyone have any knowledge/experience with the best primers to use for archaea with MiSeq?
2. How exactly to design these primers for MiSeq? I would like to use dual barcoding with these primers. I already have dual barcoded primers designed for the v4 region for bacteria (basically the same set up as Pat Schloss uses for MiSeq). Would I use the same adapters, pad, and linker sequences for these archaea primers and only change the 16S primers (F/R) based on what region I use (As long as the entire oligo sequences have a Tm of at least 65 deg.)?
I apologize if these are elementary questions, I am relatively new to NGS and MiSeq. Any insight to these questions would be greatly appreciated, thanks!
1. What is the best hypervariable region for archaea using MiSeq? I have read the Klindworth et al, 2012 paper on the best primers for NGS and it suggests that the best archaeal primers for Illumina are in the V3 region, however, I believe that the region may be too short for complete overlap on MiSeq 2x250 (they may have used different Illumina chemistry, or HiSeq?). I have also come across the V4-V5 region for Archaea on MiSeq using 517F and 958R. Does anyone have any knowledge/experience with the best primers to use for archaea with MiSeq?
2. How exactly to design these primers for MiSeq? I would like to use dual barcoding with these primers. I already have dual barcoded primers designed for the v4 region for bacteria (basically the same set up as Pat Schloss uses for MiSeq). Would I use the same adapters, pad, and linker sequences for these archaea primers and only change the 16S primers (F/R) based on what region I use (As long as the entire oligo sequences have a Tm of at least 65 deg.)?
I apologize if these are elementary questions, I am relatively new to NGS and MiSeq. Any insight to these questions would be greatly appreciated, thanks!