EMB ITS primers explained

[Dilliplaine33]

Does anyone have experience using ITS primers with the Illumina platforms (specifically MiSeq)?

I do not understand why the ITS primers require specific considerations on the Earth Microbiome website. http://www.earthmicrobiome.org/proto...standards/its/

Users at my university have used ITS Truseq fusion primers for quadruple indexing and sequenced successfully while ignoring this melting temperature issue. Does the addition of the itru and internal barcode segments render this issue null and void?

[thermophile]

I don't use the EMP ITS, I use the original ITS3 and ITS4 from White (same reverse, different forward). you have to adjust the pad to increase the melting temp because ITS3/ITS4 have a theoretical TM of 48, the MiSeq annealing is 65. If you are using truseq fusion you use truseq sequencing primers which are already at 65, but you will waste cycles sequencing your target primers.

[Dilliplaine33]

Thank you!