Does anyone have experience modifying the MiSeq to run at an annealing temperature that is less than 65 C? I am following the Kozich et al. 2013 protocol to sequence 18S rRNA amplicons and my custom sequencing primers fall short of a Tm of 65. Had I realized this issue sooner, I might have attempted to change the pad to increase the Tm, but now >300 libraries have been made. It would be expensive and time consuming to redo all that work.
I have the option of extending the length of my sequencing primers into the beginning of my amplicon to increase the Tm, but this will result in losing some diversity that I wanted to detect.
Alternatively, an Illumina technician said that they could change the MiSeq's annealing temperature for my run, but they had no data on whether that might be successful or not.
The latter option would be ideal, but I'm hesitant to try given the cost of each run. Has anyone out there attempted something like this before with or without success?
Thank you
I have the option of extending the length of my sequencing primers into the beginning of my amplicon to increase the Tm, but this will result in losing some diversity that I wanted to detect.
Alternatively, an Illumina technician said that they could change the MiSeq's annealing temperature for my run, but they had no data on whether that might be successful or not.
The latter option would be ideal, but I'm hesitant to try given the cost of each run. Has anyone out there attempted something like this before with or without success?
Thank you
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