2.1.2. Primer choice and 16S rRNA regions.
Several factors influence primer choice. Universality of the primer across the Bacteria is the primary concern, and for this we recommend using the 16S rRNA gene as the target. The region of the 16S rRNA gene that the primers amplify also deserves careful consideration. Different regions can return different results, and therefore, studies that use different regions of the 16S rRNA gene are not directly comparable (Engelbrektson et al., 2010). We recommend two regions that have recently been used successfully for identifying the bee-associated microbiome: V1-V3 (Ahn et al., 2012; Mattila et al., 2012; McFrederick et al., 2012) and V6-V8 (Martinson et al., 2012; Moran et al., 2012). V1 is particularly useful for discriminating Lactobacillus species (McFrederick et al., 2013). Both of these regions provide good taxonomic resolution of bee-associated bacteria, but universal primers associated with these regions can also amplify eukaryotic DNA (McFrederick, unpublished data; Moran et al., 2012). As NGS read lengths increase, obtaining overlapping paired-end reads (i.e. sequencing 250 bases from each end of a 400 bp amplicon) will become feasible, and additional variables will factor into primer choice.
- Suggested V1-V3 region
Forward primer, 28F: 5’– GAGTTTGATCNTGGCTCAG –3’
Reverse primer, 519R: 5’–GTNTTACNGCGGCKGCTG –3’.
- Suggested V6-V8 region
Forward primer, 926F: 5’– AAACTYAAAKGAATTGACGG –3’
Reverse primer, 1392R 5’– ACGGGCGGTGTGTRC –3’