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Evaluation of non-destructive extraction protocols for metabarcoding of insects
Stockholm University, Faculty of Science, Department of Zoology.ORCID iD: 0000-0001-5722-058X
Stockholm University, Faculty of Science, Department of Zoology.ORCID iD: 0000-0002-3929-251X
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Researchers in the field of insect diversity can greatly benefit from DNA metabarcoding due to its accuracy, low cost and short processing time compared to manual sorting and identification. But most existing metabarcoding protocols require homogenization of the sample, thus prohibiting any further work on the captured individuals. A mild digestion of the tissue by incubation of the specimens in a lysis buffer has been proposed as an alternative to sample homogenization. Several mild lysis protocols have been presented but they have not been properly evaluated against each other. Here, we analyze the effects of two different mild lysis buffers (one more aggressive than the other), two different incubation times, and two different DNA purification methods (a manual precipitation method and an automated robotic protocol) on metabarcoding performance. Performance was measured as the accuracy of retrieving the true composition of mock insect communities using two different mitochondrial markers (COI and 16S). We found that the variation in concentration and purity of the DNA extracts produced by the different lysis treatments and purification methods had little effect on the recovery of species. However, the two lysis treatments differed significantly in how well they allowed quantification of species abundances. Digestion in the gentler buffer and for a shorter time resulted in metabarcoding results that were more representative of the original sample, while a more aggressive buffer or a longer incubation reduced the values of alpha diversity and increased the differences between metabarcoding results and the true species- abundance distribution. In summary, our results show that the details of non-destructive DNA metabarcoding protocols can have a significant effect on performance. Specifically, our results suggest that a short and mild lysis treatment is the best choice for recovering the true composition of the processed sample. Short and mild lysis protocols not only improve accuracy, they also come with a lower cost and a faster processing time.

National Category
Ecology
Research subject
Systematic Zoology
Identifiers
URN: urn:nbn:se:su:diva-180292OAI: oai:DiVA.org:su-180292DiVA, id: diva2:1417084
Available from: 2020-03-26 Created: 2020-03-26 Last updated: 2022-03-08Bibliographically approved
In thesis
1. Debugging metabarcoding for insect biodiversity studies
Open this publication in new window or tab >>Debugging metabarcoding for insect biodiversity studies
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Insects are one of the most abundant and diverse animal groups, and they include many valuable ecological indicator species, but taxonomic discovery projects and biodiversity surveys targeting this group are often challenging. While mass trapping devices allow the collection of insects in great numbers, the task of identifying the species present is a painstaking and resource-demanding process. Metabarcoding, that is, high throughput sequencing of PCR-amplified species-specific genetic markers in environmental samples, promises to solve this problem. However, metabarcoding is still in its infancy. In this thesis, I optimized metabarcoding methods for inventorying and accelerating species discovery of terrestrial insects. In paper I, we designed new PCR primers for mitochondrial markers and evaluated them against existing ones using in silico methods. We showed that the best marker for metabarcoding of insects is 16S because of its broad taxonomic coverage and low amplification bias. However, there is significantly more reference data for COI, and its taxonomic coverage is reasonable when using sufficiently degenerate primers (mixes of primer sequences). In paper II, we applied 16S and COI metabarcoding to different types of samples of the same insect communities: Malaise trap samples (preservative ethanol or homogenized samples) and soil samples. The results show that the two-marker strategy increases biodiversity detection over single-marker analyses. They also show that 16S is better than COI for metabarcoding of eDNA samples because the less degenerate 16S primers do not amplify as many off-target organisms. Finally, the results show that analyses of tissue homogenate and preservative ethanol yield strikingly different results. Large and heavily sclerotized insects do not leak DNA into preservative ethanol like small and weakly sclerotized ones do, but their DNA tends to swamp the DNA of the latter in homogenized samples.  In paper III we evaluated the performance of various non-destructive mild lysis treatments and DNA purification methods. We subjected mock community samples to incubation in either a milder or a more aggressive digestion buffer for a short or a long period of incubation. The DNA was then extracted using either a manual or an automated purification protocol. We found that the milder digestion buffer and the shorter incubation time preserved the morphology of the insect best while at the same time giving the most accurate DNA metabarcoding results; the purification protocol had little or no effect on metabarcoding results. Finally, in paper IV, we explored the received wisdom that high concentrations of ethanol, although optimal for preservation of the DNA, make insects fragile and difficult to work with from a morphological point of view. We preserved insects in different ethanol concentrations and subjected them to damaging processes, such as shaking or transporting. We verified that high concentrations of ethanol induce brittleness, although the effect is less pronounced in robust insects. Our results also indicate that shipping by mail is safe for samples preserved at intermediate concentrations (70 or 80 %). In summary, this thesis represents a significant step forward in the development of methods for preserving and analyzing samples of terrestrial insects for biodiversity surveys, monitoring programs, and taxonomic research projects.

Place, publisher, year, edition, pages
Stockholm: Department of Zoology, Stockholm University, 2020. p. 40
Keywords
metabarcoding, insects, non-destructive, DNA extraction, Malaise trap, preservative ethanol, environmental DNA
National Category
Ecology Zoology
Research subject
Systematic Zoology
Identifiers
urn:nbn:se:su:diva-180293 (URN)978-91-7911-130-4 (ISBN)978-91-7911-131-1 (ISBN)
Public defence
2020-05-15, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 09:30 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2020-04-22 Created: 2020-03-26 Last updated: 2022-02-26Bibliographically approved

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Marquina, DanielŁukasik, PiotrRonquist, Fredrik

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