Evaluation of non-destructive DNA extraction protocols for insect metabarcoding: gentler and shorter is better

Abstract

DNA metabarcoding can accelerate research on insect diversity, as it is cheap and fast compared to manual sorting and identification. Most metabarcoding protocols require homogenisation of the sample, preventing further work on the specimens. Mild digestion of the tissue by incubation in a lysis buffer has been proposed as an alternative, and, although some mild lysis protocols have already been presented, they have so far not been evaluated against each other. Here, we analyse how two mild lysis buffers (one more aggressive, one gentler in terms of tissue degradation), two different incubation times, and two DNA purification methods (a manual precipitation and an automated protocol) affect the accuracy of retrieving the true composition of mock communities using two mitochondrial markers (COI and 16S). We found that protocol-specific variation in concentration and purity of the DNA extracts produced had little effect on the recovery of species. However, the two lysis treatments differed in quantification of species abundances. Digestion in the gentler buffer and for a shorter time yielded better representation of original sample composition. Digestion in a more aggressive buffer or longer incubation time yielded lower alpha diversity values and increased differences between metabarcoding results and the true species-abundance distribution. We conclude that the details of non-destructive protocols can have a significant effect on metabarcoding performance. A short and mild lysis treatment appears the best choice for recovering the true composition of the sample. This not only improves accuracy, but also comes with a faster processing time than the other treatments.

Optimizing insect metabarcoding using replicated mock communities

“…Overall, our findings are in line with the results of Marquina et al (2022), who found that small and soft species were more difficult to detect after treatment with a chemically more aggressive buffer (higher SDS, DTT, and proteinase K concentrations) than after lysis with the milder buffer corresponding to Buffer 1 in the current study. They also agree with the results of Marquina et al (2019), who showed that hard-bodied and large insects are more easily detected in homogenates, while analysis of preservative ethanol – similar to extremely mild lysis – is better for the detection of small and soft-bodied insects.…”

Development of a cost-effective, morphology-preserving method for DNA isolation from bulk invertebrate trap catches: Tephritid fruit flies as an exemplar

“…Our method is rapid, cost-effective, suitable for bulk samples and is gentle on specimens. Crucially, we were able to isolate sufficient DNA for many downstream molecular applications without addition of proteinase K or chemical lysis reagents which are common components in many published non-destructive methods [ 22 , 33 , 34 ] and are cost-prohibitive for large scale bulk sample processing. Another consideration that we felt crucial was to reduce physical damage to specimens by decreasing the processing time and minimising sample handling.…”

Depicting environmental gradients from Malaise trap samples: Is ethanol‐basedDNAmetabarcoding enough?

“…A striking subject of today's (and the future's) research concerns the advancing methodology of non‐destructive DNA extractions. Numerous studies dedicated to the development of non‐destructive methodologies for sequencing are emerging, showing that it is possible to extract DNA (although in smaller quantities) from specimens while keeping their structural integrity intact (Batovska et al, 2021; Carew et al, 2018; Kirse et al, 2022; Marquina et al, 2022; Martins et al, 2019; Martoni et al, 2022; Nielsen et al, 2019). Such protocols roughly consist of leaching DNA from whole individuals by temporarily submerging them in a digestive buffer (Castalanelli et al, 2010; Krosch & Cranston, 2012; Nielsen et al, 2019; Porco et al, 2010; Wong et al, 2014).…”

“…Such protocols roughly consist of leaching DNA from whole individuals by temporarily submerging them in a digestive buffer (Castalanelli et al, 2010; Krosch & Cranston, 2012; Nielsen et al, 2019; Porco et al, 2010; Wong et al, 2014). While various studies have tested non‐destructive DNA extractions on single arthropod specimens or samples of mock communities (see Castalanelli et al, 2010; Marquina et al, 2022; Nielsen et al, 2019), we only found one study that did so on real‐life bulk samples of terrestrial arthropods from Malaise traps (see Kirse et al, 2022). Malaise traps are especially challenging to process as they can contain hundreds to thousands of individuals (Geiger et al, 2016), each displaying various degrees of sclerotisation, which require different incubation times for adequate non‐destructive DNA extraction (Elbrecht et al, 2017).…”

“…Malaise traps are especially challenging to process as they can contain hundreds to thousands of individuals (Geiger et al, 2016), each displaying various degrees of sclerotisation, which require different incubation times for adequate non‐destructive DNA extraction (Elbrecht et al, 2017). Moreover, there are many options in which non‐destructive DNA extractions can be performed, ranging from an optional step of sample sorting, to the choice of lysis buffer, to incubation times of specimen in the fluid, to the protocol used for extraction (Kirse et al, 2022; Marquina et al, 2022; Martoni et al, 2022). With so many factors, numerous researchers are in the process of testing these different options in determining which combination is most effective.…”