16S rRNA metabarcoding of Apis mellifera: crop content of bees kept in the laboratory
DNA was extracted with the Qiagen DNeasy Blood & Tissue kit. Library preparation and sequencing were outsourced to IGA Technology Services S.R.L. (Italy). Libraries for the V3-V4 region of the 16S rRNA gene were prepared following the Illumina 16S Metagenomic Sequencing Library Preparation protocol (Illumina, 2013). Universal prokaryotic primers, Pro341F and Pro805R (Takahashi et al., 2014), were used in the initial PCR amplification step. The subsequent amplification integrated relevant flow-cell binding domains and unique indices (NexteraXT Index Kit, FC?131?1001/FC?131?1002). Libraries were sequenced on an Aviti instrument (Element Biosciences, San Diego, USA) using a 300-bp paired-end mode. Base calling, demultiplexing, and adapter masking were performed with Bases2fastq software v.1.8 (Element Biosciences).