| 1. |
- Iwaszkiewicz-Eggebrecht, Elzbieta, et al.
(författare)
-
FAVIS : Fast and versatile protocol for nondestructive metabarcoding of bulk insect samples
- 2023
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 18:7, s. e0286272-
-
Tidskriftsartikel (refereegranskat)abstract
- Insects are diverse and sustain essential ecosystem functions, yet remain understudied. Recent reports about declines in insect abundance and diversity have highlighted a pressing need for comprehensive large-scale monitoring. Metabarcoding (high-throughput bulk sequencing of marker gene amplicons) offers a cost-effective and relatively fast method for characterizing insect community samples. However, the methodology applied varies greatly among studies, thus complicating the design of large-scale and repeatable monitoring schemes. Here we describe a non-destructive metabarcoding protocol that is optimized for high-throughput processing of Malaise trap samples and other bulk insect samples. The protocol details the process from obtaining bulk samples up to submitting libraries for sequencing. It is divided into four sections: 1) Laboratory workspace preparation; 2) Sample processing-decanting ethanol, measuring the wet-weight biomass and the concentration of the preservative ethanol, performing non-destructive lysis and preserving the insect material for future work; 3) DNA extraction and purification; and 4) Library preparation and sequencing. The protocol relies on readily available reagents and materials. For steps that require expensive infrastructure, such as the DNA purification robots, we suggest alternative low-cost solutions. The use of this protocol yields a comprehensive assessment of the number of species present in a given sample, their relative read abundances and the overall insect biomass. To date, we have successfully applied the protocol to more than 7000 Malaise trap samples obtained from Sweden and Madagascar. We demonstrate the data yield from the protocol using a small subset of these samples.
|
|
| 2. |
- Iwaszkiewicz-Eggebrecht, Elzbieta, et al.
(författare)
-
Optimizing insect metabarcoding using replicated mock communities
- 2023
-
Ingår i: Methods in Ecology and Evolution. - : Wiley. - 2041-210X. ; 14:4, s. 1130-1146
-
Tidskriftsartikel (refereegranskat)abstract
- Metabarcoding (high-throughput sequencing of marker gene amplicons) has emerged as a promising and cost-effective method for characterizing insect community samples. Yet, the methodology varies greatly among studies and its performance has not been systematically evaluated to date. In particular, it is unclear how accurately metabarcoding can resolve species communities in terms of presence-absence, abundance and biomass. Here we use mock community experiments and a simple probabilistic model to evaluate the effect of different DNA extraction protocols on metabarcoding performance. Specifically, we ask four questions: (Q1) How consistent are the recovered community profiles across replicate mock communities?; (Q2) How does the choice of lysis buffer affect the recovery of the original community?; (Q3) How are community estimates affected by differing lysis times and homogenization? and (Q4) Is it possible to obtain adequate species abundance estimates through the use of biological spike-ins? We show that estimates are quite variable across community replicates. In general, a mild lysis protocol is better at reconstructing species lists and approximate counts, while homogenization is better at retrieving biomass composition. Small insects are more likely to be detected in lysates, while some tough species require homogenization to be detected. Results are less consistent across biological replicates for lysates than for homogenates. Some species are associated with strong PCR amplification bias, which complicates the reconstruction of species counts. Yet, with adequate spike-in data, species abundance can be determined with roughly 40% standard error for homogenates, and with roughly 50% standard error for lysates, under ideal conditions. In the latter case, however, this often requires species-specific reference data, while spike-in data generalize better across species for homogenates. We conclude that a non-destructive, mild lysis approach shows the highest promise for the presence/absence description of the community, while also allowing future morphological or molecular work on the material. However, homogenization protocols perform better for characterizing community composition, in particular in terms of biomass.
|
|
| 3. |
|
|
| 4. |
- Vinuesa, Ricardo, et al.
(författare)
-
Convergence of numerical simulations of turbulent wall-bounded flows and mean cross-flow structure of rectangular ducts
- 2016
-
Ingår i: Meccanica (Milano. Print). - : Springer Netherlands. - 0025-6455 .- 1572-9648. ; 51:12, s. 3025-3042
-
Tidskriftsartikel (refereegranskat)abstract
- Convergence criteria for direct numerical simulations of turbulent channel and duct flows are proposed. The convergence indicator for channels is defined as the deviation of the nondimensional total shear-stress profile with respect to a linear profile, whereas the one for the duct is based on a nondimensional streamwise momentum balance at the duct centerplane. We identify the starting () and averaging times () necessary to obtain sufficiently converged statistics, and also find that optimum convergence rates are achieved when the spacing in time between individual realizations is below . The in-plane structure of the flow in turbulent ducts is also assessed by analyzing square ducts at and 360 and rectangular ducts with aspect ratios 3 and 10 at . Identification of coherent vortices shows that near-wall streaks are located in all the duct cases at a wall-normal distance of as in Pinelli et al. (J Fluid Mech 644:107-122, 2010). We also find that large-scale motions play a crucial role in the streamline pattern of the secondary flow, whereas near-wall structures highly influence the streamwise vorticity pattern. These conclusions extend the findings by Pinelli et al. to other kinds of large-scale motions in the flow through the consideration of wider ducts. They also highlight the complex and multiscale nature of the secondary flow of second kind in turbulent duct flows.
|
|
