16S amplicon sequencing of Daphnia magna gut microbiota: experimental population given no OTC at day 23
DNA was extracted from three pooled adult Daphnia following Huang et al. (40). Because of initially low bacterial DNA concentrations in some samples, we applied a nested PCR to increase specificity and amplicon yield . First, the full length 16S rRNA gene was amplified with primers 27F and 1492R on 10 ng of template (94°C - 30s; 50°C - 45s; 68°C - 90s; 30 cycles) using a high-fidelity Pfx polymerase (Life technologies). PCR products were purified using the QIAquick PCR purification kit (Qiagen). To obtain dual-index amplicons of the V4 region, a second amplification was performed on 5 µL of PCR product using primers 515F (42) and a slightly modified version of primer 806R to increase detection of SAR11 bacterioplankton for 30 cycles (94°C - 30s; 55°C - 30s; 68°C - 60s). Both primers contained an Illumina adapter and an 8-nt barcode at the 5'-end. For each sample, PCRs were performed in triplicate, pooled and gel purified using the QIAquick gel extraction kit (Qiagen). An equimolar library was prepared by normalizing amplicon concentrations with a SequalPrep Normalization Plate (Applied Biosystems) and subsequent pooling. Amplicons were sequenced using a v2 PE500 kit with custom primers (42) on the Illumina Miseq platform, producing 2 x 250-nt paired-end reads.