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.