A modification of the protocol as described by Caporaso et. al (2011) is used and is available upon request.
- 10-50 ng of DNA are PCR-amplified using V3-V4 primers and 5 PrimeHotMaster Mix.
- PCR samples are purified using AMPure beads.
- 100 ng of each library is pooled, gel-purified, and quantified using a bioanalyser and subsequently with qPCR.
- 12pM of the library mixture, spiked with 20% Phix, is run on the MiSeq.
The V3-V4 primers are used as follows:
~341F (forward primer)
~806R (reverse primer)
Sequences of the primers are in bolded italics
12 N’s (in red) designate barcode sequences
These primers are slightly different from those primers used by Illumina.