DNA Metabarcoding allows for simultaneous identification of many taxa within the same sample. This allows for more rapid sample processing and data yield. Metabarcoding is helpful to understand species composition at the community level using mixed bulk samples. Previous barcoding practices focus on individual organisms, making it inefficient for processing bulk samples. Traditional morphological arthropod identification is time consuming, requires expertise, and is not practical for community-level analysis. The development of DNA metabarcoding methods and technologies has been revolutionary for entomological ecology research.
How Does it Work?
DNA metabarcoding involves similar steps to general barcoding:
Metabarcoding is possible due to the variance of barcode regions across taxonomic groups. A barcode region is a short variable gene region flanked by highly conserved gene regions. Commonly used barcode regions for arthropods include portions of cytochrome oxidase 1 (COI), ADP Ribosylation Factor 1 (ARF1), and multicopper oxidase (MCO). These target genes are chosen because they are highly conserved among diverse arthropods, meaning almost all arthropod species possess this gene but with some unique taxa-specific variation. Primers are designed based on the highly conserved gene regions for the PCR amplification step. To reduce PCR amplification success bias, we use degenerate primers, which are mixtures of similar primers to complement the degenerate variation of DNA itself.
Compared to general barcoding, metabarcoding requires more complex bioinformatics due to the sample containing a heterogenous mixture from the community sampled. The five main steps of the bioinformatics workflow for DNA metabarcoding are: demultiplexing, merging pair-end reads, quality filtering, OTU curation, and taxonomic identification. The first step of demultiplexing refers to “undoing” the multiplexing from previous molecular steps. This separates the mixed sequencing data based on species-specific barcodes.
Our Application of DNA Metabarcoding
The EDIAs project uses DNA metabarcoding to develop a rapid monitoring system for community assemblages on Pacific islands. In particular, we are interested in identifying introduced and nuisance arthropod species. Current traditional sampling and monitoring is time- and resource-consuming.
We begin with sampling understory on Pacific islands using traditional arthropod sampling methods (vegetative beat samples, malaise traps, and leaf little sifting) and novel eDNA sampling methods (air and water filtration, spider webs, and surface rolling of tree bark). Environmental DNA sampling techniques have promising applications to increase sampling efficiency and capture more history of species frequenting the field sites. This entails week long collection trips to our designated field sites. Once collected and labeled, the samples are shipped to our lab at UC Berkeley where they are sorted, extracted, amplified, and sequenced. Next, the genetic data is analyzed using bioinformatics to understand the arthropod communities we sampled.
References
- Arribas, Paula, et al. “Toward global integration of biodiversity big data generation module for terrestrial arthropods.” GigaScience 11 (2022)
- Compson, Zacchaeus G., et al. “Metabarcoding from microbes to mammals: comprehensive bioassessment on a global scale.” Frontiers in Ecology and Evolution 8 (2020): 581835.
- Deiner, Kristy, et al. “Environmental DNA metabarcoding: Transforming how we survey animal and plant communities.” Molecular ecology 26.21 (2017): 5872-5895.
- Liu, Mingxin, et al. “A practical guide to DNA metabarcoding for entomological ecologists.” Ecological entomology 45.3 (2020): 373-385.