8.8.3. Amplicon-based or shotgun sequencing

Given the low costs of sequencing, it is feasible now to simply survey all nucleic acids in a sample and then assign them to taxa in various kingdoms via searches of local or online databases. Nevertheless, targeted deep sequencing of specific taxonomic groups can benefit from a selection of specific regions via PCR-based amplification prior to generating the sequencing libraries. This has been done most frequently with the 454 sequencing platform since relatively long read lengths on this platform (> 400 bp) enable the capture of sequence data for a substantial section of the targeted species. Several studies have now used amplicon-based sequencing to describe bacterial populations carried by honey bees. As with any PCR protocol, this approach will under sample taxa with mismatches to the initial primer sequences since no PCR primers are truly ‘universal’ to a targeted group. Nevertheless, there are many examples of primers that amplify broadly across all of the major bacterial taxonomic groups, and amplicon-based 454 sequencing has appears to provide a consistent and accurate view of bacterial communities in bees. The software environment Qiime (http://qiime.org/) is widely used to match amplicon-based sequences to microbial databases in order to identify and quantify taxa.