16S amplicon sequencing of Daphnia magna gut microbiota: experimental population given 100mcg OTC at day 23 replicate 1
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.