Fractionating bulk samples for DNA Metabarcoding

Bulk sample analysis

The most common method to investigate species compositions involves sequencing the bulk DNA - or the combined DNA - of all parts of the mixed sample of insects. By its nature, this method is destructive, as the mixed sample needs to be homogenized - in other words pulverized - to be able to obtain an appropriately small, but statistically representative amount of insect powder for DNA extraction. While it may not be able to record every rare and/or very small species, bulk DNA metabarcoding is able to detect a large portion of the biodiversity, and will be sufficient to answer most scientific or ecological questions. At the cost of destroying the samples, it does also enable us to analyze hundreds or thousands of samples on short notice, a unique advantage in cases where we lack the resources and experts to determine these species by manual morphological examination.

Metabarcoding Analysis of ethanol supernatant

Ethanol is commonly used as a fixative that helps preserve mixed insect samples. Amplifying the DNA dissolved in the fixative makes it possible to retrieve genetic information without having to destroy the samples. However, sequencing only the DNA from the fixative records only a small part of the total biodiversity, and it introduces several sources of bias. For example, weakly sclerotized insects with soft bodies will release a lot of DNA into the fixative, while heavily armored species tend not to release any DNA at all. This phenomenon is known, for example, in dragonflies. What is more, large species will release disproportionately more DNA than smaller ones. Thus, fixative-DNA sequencing results in strong biomass effects. For these reasons, while it can be used as a supplement, we do not recommend it as a sole method to estimate biodiversity.

Size fractionation before metacarcoding analysis

The taxonomic resolution of DNA metabarcoding - in other words, the percentage of the total number of species that can be detected in a sample - can be further improved by size fractionation. As the name suggests, this method involves separating the mixed sample by size into two or more fractions. Our laboratory experts have designed a specialized, easy-to-clean and high-throughput system for fractionation that can be customized to fit unique customer needs. In our experience, a mesh size of 6 mm - about the size of the ordinary house fly (Musca domestica) - has shown particularly impressive results. Analyzing small and large fractions separately can overcome the strong biomass effects that are characteristic of fixative-DNA and, to a smaller extent, bulk-DNA metabarcoding. In particular, small and rare species are detected much more readily when compared to regular metabarcoding protocols.

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