16S rRNA Amplicon Sequencing of Bacteria

Bacterial colonies were subcultured on the same media as used for primary growth. Crude extract of bacterial DNA was prepared as previously described (Scholz et al., 2014 (link)). 16S rRNA gene amplification was performed using the B4 and B5 primers that amplify the V1–V3 region (Mikkelsen et al., 2000 (link)). A PCR reaction mixture of 25 μl containing 8 μl sterile PCR grade water, 2 μl primer mix (5 μmol each of B4 and B5 primers), 5 μl of the 1:100 diluted DNA, and 10 μl of 5Prime Hotmaster mix (Quanta Bio) was prepared. The following thermocycling scheme was used: 94°C for 5 min (1 cycle), 94°C for 1 min, 60°C for 1 min (30 cycles), 72°C for 2 min, and 72°C for 8 min (1 cycle). The PCR products were verified on agarose gels. Sequencing of the PCR products using the B4 and B5 primers was done at Eurofins Genomics (Ebersberg, Germany), and sequence comparison with the NR database at NCBI was done using blastn. A sequence identity >99% of the amplicon sequence with a database entry led to species assignment.