The V4 region of the 16S-rRNA encoding gene was amplified from each sample using the Dual-indexing sequencing strategy reported in Kozich et al. (2013) (Reference #1).  Accuprime High Fidelity Taq (Life Cat # 12346094 ) was used instead of Accuprime Pfx supermix.Libraries are prepared according to Illumina’s protocol for Preparing Libraries for Sequencing on the MiSeq (part# 15039740 Rev. D) for 2nM or 4nM libraries. If the library concentration is below 1nM an alternative method is used for denaturation. (Reference # 2).  The final load concentration is 4pM with a 10% PhiX spike to add diversity.    Reference #1: Kozich JJ et al. (2013)  Appl. Environ. Microbiol. 2013, 79(17):5112.   Reference #2: Quail et al, Nature Methods 5,1005-1010(2008), supplemental protocol 12
MiSeqIllumina