Step 3: Isolating and sequencing DNA
Isolating DNA from pollen is relatively straightforward. First, the pollen is lysed, usually through bead-beating. There are off-the-shelf kits that work well, along with cheaper extraction methods based on phenol and chloroform; acetone-based DNA isolation has also been effective (Gouker, Guo, & Pooler, 2020). Some studies have shown that it is possible to extract and sequence DNA from pollen without a lysing step (Swenson & Gemeinholzer, 2021). The high-throughput capabilities of DNA metabarcoding are reliant on the addition of sample-specific nucleotides which allow the identification of hundreds of individual samples following pooling into a single sample sent for sequencing (multiplexing). Three multiplexing methods are commonly used, and the most appropriate method should be chosen based on an assessment of the risk of cross-contamination, PCR efficiency and overall cost (reviewed in Bohmann et al., in press). For sequencing, most amplicon-based studies use the Illumina MiSeq platform with v3 chemistry (300 paired-end sequencing, 600 cycle) to sequence 300-550 base pair reads. New long-read sequencing technologies could add improved taxonomic resolution. These methods are discussed further in section 5.