16S UniCPR amplicon seq of droplet enrichment of groundwater PNK148 with Anammox necromass and soil extract, day 30, replicate 3
PCR amplification of partial bacterial 16S genes of CPR was performed in a 25 L reaction. Illumina sequencing adapters TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG and GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG were attached at the 5 ends of the forward and reverse primers, respectively. PCR was conducted using UniCPR primer 684F (GTA GKR RTR AAA TSC GTT) and universal bacterial 16S primer 907R (Lane et al., Evolution (N Y). 1985;82(October):69559.). The cycling conditions for 16S rRNA with an initial denaturation at 98C for 3 min, 40 cycles of denaturation at 98C for 30 s, annealing at 51C for 40s, elongation at 72C for 1 min, and final extension at 72C for 5 min. In a second PCR, Illumina sequencing adapters were introduced using primer combination IAf/IAr (AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGT/CAAGCAGAAGACGGCATACGAGATGTCTCGTGGGCTCGG). The second PCR was done for 6 cycles using 1 L product of the first PCR as template, with the following cycling conditions: 15 min 95C pre-denaturation; 94C for 45 s, 55C for 60 s, 72C for 60 s; 10 min final elongation at 72C. PCR products were purified after each PCR with the NucleoSpin Gel and PCR clean-up kit according to the manufacturers instructions (Macherey-Nagel GmBH, Dren, Germany). Amplicon sequencing was then carried out in-house on a MiSeq Illumina platform (Illumina, Eindhoven, The Netherlands) with v3 chemistry (2 x 300 bp paired-end).