Sensitivity and accuracy of high-throughput metabarcoding methods for early detection of invasive fish species

Abstract

High-throughput DNA metabarcoding has gained recognition as a potentially powerful tool for biomonitoring, including early detection of aquatic invasive species (AIS). DNA based techniques are advancing, but our understanding of the limits to detection for metabarcoding complex samples is inadequate. For detecting AIS at an early stage of invasion when the species is rare, accuracy at low detection limits is key. To evaluate the utility of metabarcoding in future fish community monitoring programs, we conducted several experiments to determine the sensitivity and accuracy of routine metabarcoding methods. Experimental mixes used larval fish tissue from multiple “common” species spiked with varying proportions of tissue from an additional “rare” species. Pyrosequencing of genetic marker, COI (cytochrome c oxidase subunit I) and subsequent sequence data analysis provided experimental evidence of low-level detection of the target “rare” species at biomass percentages as low as 0.02% of total sample biomass. Limits to detection varied interspecifically and were susceptible to amplification bias. Moreover, results showed some data processing methods can skew sequence-based biodiversity measurements from corresponding relative biomass abundances and increase false absences. We suggest caution in interpreting presence/absence and relative abundance in larval fish assemblages until metabarcoding methods are optimized for accuracy and precision.

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

“…Numerous studies have highlighted variations in biological communities identified when different genes are targeted in high throughput sequencing (HTS) studies. This variation is commonly attributed to: lack of primer universality or specificity, preferential amplification of certain organisms (e.g., taxa with short sequences), incomplete or inaccurate reference databases, and differences in the levels of taxonomy that can be resolved by target genes (e.g., De Barba et al, ; Deagle et al, ; Hatzenbuhler, Kelly, Martinson, Okum, & Pilgrim, ; Wilcox et al, ). Although not a specific aim of this study, we observed marked differences in taxonomic composition of the communities when targeting the COI and 18S rRNA gene.…”

Optimization and performance testing of a sequence processing pipeline applied to detection of nonindigenous species

“…With respect to marker choice, we used 18S in our study but COI has shown higher sequence variability and improved taxonomic assignment (Hatzenbuhler et al, 2017;Tang et al, 2012;Zhan, Bailey et al, 2014). This variability can be a double-edged sword; as it is apparent even in primer binding sites, COI can have issues with primer generality (Deagle et al, 2014;Ficetola et al, 2010;Hatzenbuhler et al, 2017;Zhan, Bailey et al, 2014). Consequently, false-negative errors may be more likely to occur because of inconsistent amplification which would be particularly detrimental to early detection of AIS.…”

“…Primers for this marker effectively amplify a broad range of zooplankton taxa, making 18S a suitable marker for zooplankton metabarcoding studies (Zhan, Bailey, Heath, & MacIsaac, 2014). Conversely, the COI marker is highly variable for these taxa (sometimes, even in the primer binding sites) which may make it more suitable for studies taking the targeted approach than for metabarcoding highly divergent communities (Deagle, Jarman, Coissac, Pompanon, & Taberlet, 2014;Hatzenbuhler, Kelly, Martinson, Okum, & Pilgrim, 2017;Zhan, Bailey et al, 2014). The drawback of 18S is that due to lower variability, it may be more difficult to assign identity at the species level.…”

Gaps in DNA-Based Biomonitoring Across the Globe

“…whereas sediment/benthos or soil samples are termed "bulktissue DNA" (Hatzenbuhler et al, 2017;Hajibabaei et al, 2019;Harper et al, 2019), despite these types of sample all referring to DNA which is isolated from an environmental sample (Dickie et al, 2018). This lack of consistency is particularly challenging when attempting to amalgamate literature and compare studies from different research groups and for effectively communicating results of DNA-based studies to non-specialists.…”