Biology subjects

Leese, F.

Publications and source records attributed to Leese, F..

3 recordsLinked to original sources

bioRxiv2018

DNA metabarcoding from sample fixative as a quick and voucher preserving biodiversity assessment method

Metabarcoding is a powerful tool for biodiversity assessment and has become increasingly popular in recent years. Although its reliability and applicability have been proven in numerous scientific studies, metabarcoding still suffers from some drawbacks. One is the usually mandatory destruction of specimens before DNA extraction, which is problematic because it does not allow a later taxonomic evaluation of the results. Additionally, metabarcoding often implements a time-consuming step, where specimens need to be separated from substrate or sorted in different size classes. A non-destructive protocol, excluding any sorting step, where the extraction of DNA is conducted from a samples fixative (ethanol) could serve as an alternative. We test an innovative protocol, where the sample preserving ethanol is filtered and DNA extracted from the filter for subsequent DNA metabarcoding. We first tested the general functionality of this approach on 15 mock communities comprising one individual of eight different macroinvertebrate taxa each and tried to increase DNA yield through different treatments (ultrasonic irradiation, shaking, freezing). Application of the method was successful for most of the samples and taxa, but showed weaknesses in detecting mollusc taxa. In a second step, the community composition detected in DNA from ethanol was compared to conventional bulk sample metabarcoding of complex environmental samples. We found that especially taxa with pronounced exoskeleton or shells (Coleoptera, Isopoda) and small taxa (Trombidiformes) were underrepresented in ethanol samples regarding taxa diversity and read numbers. However, read numbers of Diptera (mainly chironomids) and Haplotaxida were higher in ethanol derived DNA samples, which might indicate the detection of stomach content, which would be an additional advantage of this approach. Concerning EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa which are decisive for the determination of ecological statuses, both methods had 46 OTUs in common with 4 unique to the ethanol samples and 10 to the bulk samples. Results indicate that fixative-based metabarcoding is a non-destructive, time-saving alternative for biodiversity assessments focussing on taxa used for ecological status determination. For a comprehensive picture on total biodiversity, the method might however not be sufficient and conventional bulk sample metabarcoding should be applied.

molecular biology↗

bioRxiv2017

Environmental DNA metabarcoding of rivers: Not all eDNA is everywhere, and not all the time

Environmental DNA metabarcoding has become a popular tool for the assessment of freshwater biodiversity, but it is largely unclear how sampling time and location influence the assessment of communities. Abiotic factors in rivers can change on small spatial and temporal scale and might greatly influence eDNA metabarcoding results. In this study, we sampled three German rivers at four locations per sampling site: 1. Left river side, surface water 2. Right river side, surface water, 3. Left side, close to the riverbed, 4. Right side, close to the riverbed. For the rivers Ruhr and Mohne, sampling was conducted three times in spring, each sampling one week apart. The Ruhr was again sampled in autumn and the Gillbach was sampled in winter. Sequencing on an Illumina MiSeq with COI primers Bf2/BR2 revealed diverse communities (6493 Operational taxonomic units, OTUs), which largely differed between rivers. Communities changed significantly over time in the Ruhr, but not in the Mohne. Sampling location influenced recovered communities in the Mohne and in the Ruhr in autumn. Our results have important implications for future eDNA studies, which should take into account that not all eDNA in rivers is everywhere and not at all times.

molecular biology↗

bioRxiv2017

A simple centrifugation protocol leads to a 55-fold mitochondrial DNA enrichment and paves the way for future mitogenomic research

DNA (meta)barcoding is used to study biodiversity and is available for standardised assessments. However, it suffers from PCR bias, which can lead to the loss of specific taxa. PCR-free techniques such as shotgun metagenomics are therefore thought to be more suited for biodiversity assessments, but are currently limited by incomplete reference libraries.\n\nThe technique of mitogenome-skimming or mitogenomics, in which complete mitochondrial genomes are sequenced, is ideal to bridge the techniques of (meta)barcoding and metagenomics. However, without the enrichment of mitochondria, roughly 99 % of all sequencing reads are of non-mitochondrial origin and mostly useless for common applications, e.g. species identification.\n\nHere, we present a simple centrifugation protocol that leads to an average 140-fold enrichment of mitochondrial DNA. By sequencing six mock- communities - comprising the freshwater taxa Corbicula fluminea, Gammarus roeselii and Hydropsyche exocellata each - we recovered whole mitochondrial genomes of these species and the acanthocephalan endoparasite Pomphorhynchus laevis.\n\nThe presented protocol will greatly speed up building reference libraries for whole mitochondrial genomes, as dozens of species could be sequenced on a single MiSeq run. Subsequently, it will also allow biodiversity assessments using mitogenomics at greatly reduced costs in comparison to mitogenomic approaches without prior enrichment for mitochondria.

molecular biology↗