I have previously worked with 454, but fairly new to Miseq platform. Using Nextera XT sample preparation kit, I am going to study soil and root fungal communities using fungal specific primers with TMs of 56.4 °C and 52.7 °C (Illumina overhanger not included). Illumina 16 S protocol recommends using primers with TM of 60-65 °C. I have seen papers that have used primers with TMs less than 60 °C, but I am told using primers with lower TM may cause low sequencing yield and poor sequence quality. One solution would be to add few nucleotides to the primers, but I am concerned adding nucleotides may negatively affect amplification of some fungal taxa.
In another matter, Illumina 16 S protocol suggests using an equal concentration of metagenomic DNA (5 ng/μl) for Amplicon PCR reactions. I am wondering if this is a strict requirement, I guess this concentration is too low for a successful PCR on environmental samples.
Hi Ahmad,
Is your goal to characterize fungal diversity in your samples? If so, you might consider targeting the ITS rather than 18S. 18S does not distinguish fungal species. With respect to ITS, primer choice is critical, and there are pros and cons to all of the currently published primers. My lab has designed fungal primers targeting ITS2 that work well on the MiSeq: https://portal.lternet.edu/nis/mapbrowse?packageid=knb-lter-bnz.579.2
You can run these primers at 55-58C.
Cheers,
Lee
Dear,
I'll suggest you to read protocols in the site of enterprises which give services for it such as BGI, Macrogen, FASTERIS or other...You can contact them as well. Best,
I used primers developed by Caporaso (attached) with the annealing temperature of 50°C for bacterial 16S and we got good clusters. I do not know if it works for fungal ...
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