16S RDNA of bacterioplankton or Daphnia gut microbiome: community after mono- or coculture of two Daphnia species
To characterize the microbial communities of the Daphnia guts, DNA was extracted using a PowerSoil DNA isolation kit (Qiagen). DNA was dissolved in 20 L milliQ water. The total DNA yield was determined using a Qubit dsDNA HS assay (Invitrogen) on 1 L of sample. Because of initially low bacterial DNA concentrations in some samples, a nested PCR was applied to increase specificity and amplicon yield. The full-length 16S rRNA gene was first amplified with EUB8F and 1492R primers on 10 ng of template using a high-fidelity SuperFi polymerase (Life Technologies) for 30 cycles: 98C  10s; 50C  45s; 72C  30s. PCR products were subsequently purified using the QIAquick PCR purification kit (Qiagen). To obtain dual-index amplicons of the V4 region, a second amplification was performed on 5 L (=20-50 ng) of PCR product using 515F and 806R primers for 30 cycles: 98C  10s; 50C  45s; 72C  30s. Both primers contained an Illumina adapter and an 8-nucleotide (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 on the Illumina Miseq platform (KU Leuven Genomics Core), producing 2 x 250-nt paired-end reads.