title: 16S sequencing from interstitial water december 2014
design: The 16S rRNA gene V4 variable region PCR primers 515/806 with barcode on the forward primer were used in a 25 cycle PCR (5 cycle used on PCR products) using the HiFi HotStart ReadyMix (KAPA Biosystems®) under the following conditions: 95°C for 3 minutes, followed by 25 cycles of 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds, after which a final elongation step at 72°C for 5 minutes was performed. After amplification, PCR products are checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Products were purified using magnetic beads (Agencourt AMPure XP, Beckman Coulter®) and subjected to a second PCR to incorporate dual indices, as described in the 16S Metagenomic Sequencing Library Preparation Protocol for the Illumina MiSeq System. The size and quality of DNA in the final libraries were verified on a Bioanalyzer 2100 using a Bioanalyzer DNA 1000 chip (Agilent Technologies®). After quantitative PCR with KAPA Library Quantification Kit for Illumina (KAPA Biosystems®) samples were normalized and pooled for sequencing.