Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types

Sellers, Graham S.; Muri, Cristina Di; Gómez, África; Hänfling, Bernd

doi:10.3897/mbmg.2.24556

Cited by 68 publications

(77 citation statements)

References 36 publications

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“…Dilution of eDNA samples (and inhibitory substances present) can release inhibition, but also reduce detection probability (Piggott, ) and induce false negatives (Buxton, Groombridge, & Griffiths, ). We used TaqMan ® Environmental Master Mix 2.0 (Life Technologies, CA, USA) in qPCR reactions to counter inhibition (Jane et al., ), but it may be advisable to use DNA extraction kits that perform inhibitor removal (Sellers, Di Muri, Gómez, & Hänfling, ) or include bovine‐serum albumin in qPCR reactions (Jane et al., ). Alternatively, ddPCR may handle inhibitors better than qPCR and provide more accurate abundance or biomass estimates (Nathan et al., ).…”

Section: Discussionmentioning

confidence: 99%

Development and application of environmental DNA surveillance for the threatened crucian carp (Carassius carassius)

et al. 2018

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The crucian carp (Carassius carassius) is one of few fish species associated with small ponds in the UK. These populations contain genetic diversity not found in Europe and are important to conservation efforts for the species which has declined across its range in Europe. Detection and monitoring of extant crucian carp populations are crucial for conservation success. Environmental DNA (eDNA) analysis could be very useful in this respect as a rapid, cost‐efficient monitoring tool. We developed a species‐specific quantitative polymerase chain reaction (qPCR) assay for eDNA surveillance of crucian carp to enable non‐invasive, large‐scale distribution monitoring. We compared fyke netting and eDNA analysis at ponds with (n = 10) and without (n = 10) crucian carp for presence–absence detection. We examined biotic (crucian carp density represented by catch‐per‐unit‐effort [CPUE] estimate) and abiotic influences on eDNA detection probability using a hierarchical occupancy model, and eDNA quantification using a mixed‐effects model. eDNA analysis achieved 90% detection for crucian carp (n = 10), failing in only one pond where presence was known. CPUE estimate and conductivity had positive and negative influences on eDNA detection probability in qPCR replicates respectively. Similarly, conductivity had a negative effect on DNA copy number, whereas copy number increased with CPUE estimate. Our results demonstrate that eDNA analysis could enable detection of crucian carp populations in ponds and benefit ongoing conservation efforts, but imperfect species detection in relation to biotic and abiotic factors and eDNA workflow requires further investigation. Nonetheless, we have established an eDNA framework for the crucian carp as well as sources of imperfect detection which future investigations can build upon.

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Section: Discussionmentioning

confidence: 99%

Development and application of environmental DNA surveillance for the threatened crucian carp (Carassius carassius)

et al. 2018

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“…DNA was extracted within 2 weeks of filtration at the UoH eDNA facility using the mu-DNA water protocol (Sellers, Di Muri, Gómez, & Hänfling, 2018). Duplicate filters from samples in Experiment 1 were lysed independently and the lysate from each loaded onto one spin column.…”

Section: Methodsmentioning

confidence: 99%

Environmental DNA (eDNA) metabarcoding of pond water as a tool to survey conservation and management priority mammals

Harper

Handley

Carpenter

et al. 2019

Preprint

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23Environmental DNA (eDNA) metabarcoding can identify terrestrial taxa utilising aquatic habitats 24 alongside aquatic communities, but terrestrial species' eDNA dynamics are understudied. We 25 evaluated eDNA metabarcoding for monitoring semi-aquatic and terrestrial mammals, 26 specifically nine species of conservation or management concern, and examined 27 spatiotemporal variation in mammal eDNA signals. We hypothesised eDNA signals would be 28 stronger for semi-aquatic than terrestrial mammals, and at sites where individuals exhibited 29 behaviours. In captivity, we sampled waterbodies at points where behaviours were observed 30 ('directed' sampling) and at equidistant intervals along the shoreline ('stratified' sampling). We 31 surveyed natural ponds (N = 6) where focal species were present using stratified water 32 sampling, camera traps, and field signs. eDNA samples were metabarcoded using vertebrate-33 specific primers. All focal species were detected in captivity. eDNA signal strength did not differ 34 between directed and stratified samples across or within species, between semi-aquatic or 35 terrestrial species, or according to behaviours. eDNA was evenly distributed in artificial 36 waterbodies, but unevenly distributed in natural ponds. Survey methods deployed at natural 37 ponds shared three species detections. Metabarcoding missed badger and red fox recorded by 38 cameras and field signs, but detected small mammals these tools overlooked, e.g. water vole. 39Terrestrial mammal eDNA signals were weaker and detected less frequently than semi-aquatic 40 mammal eDNA signals. eDNA metabarcoding could enhance mammal monitoring through 41 large-scale, multi-species distribution assessment for priority and difficult to survey species, and 42 provide early indication of range expansions or contractions. However, eDNA surveys need high 43 3 spatiotemporal resolution and metabarcoding biases require further investigation before 44 routine implementation. 45 46

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“…Some DNA extraction kits contain specific inhibitor removal steps that can be adapted for use with difficult (e.g. turbid, high algal content) pond eDNA samples (Buxton et al, 2018;Sellers et al, 2018), while stand-alone clean-up kits (e.g. Zymo Ò or Qiagen Ò ) can be effective when applied to inhibited samples after DNA extraction (McKee et al, 2015;Williams et al, 2016;Niemillar et al, 2017;Mosher et al, 2018).…”

Section: Inhibitionmentioning

confidence: 99%

Prospects and challenges of environmental DNA (eDNA) monitoring in freshwater ponds

et al. 2018

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Environmental DNA (eDNA) analysis is a rapid, non-invasive, cost-efficient biodiversity monitoring tool with enormous potential to inform aquatic conservation and management. Development is ongoing, with strong commercial interest, and new uses are continually being discovered. General applications of eDNA and guidelines for best practice in freshwater systems have been established, but habitat-specific assessments are lacking. Ponds are highly diverse, yet understudied systems that could benefit from eDNA monitoring. However, eDNA applications in ponds and methodological constraints specific to these environments remain unaddressed. Following a stakeholder workshop in 2017, researchers combined knowledge and expertise to review these applications and challenges that must be addressed for the future and consistency of eDNA monitoring in ponds. The greatest challenges for pond eDNA surveys are representative sampling, eDNA capture, and potential PCR inhibition. We provide recommendations for sampling, eDNA capture, inhibition testing, and laboratory practice, which should aid new and ongoing eDNA projects in ponds. If implemented, these recommendations will contribute towards an eventual broad standardisation of eDNA research and practice, with room to tailor workflows for optimal analysis and

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Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types

Cited by 68 publications

References 36 publications

Development and application of environmental DNA surveillance for the threatened crucian carp (Carassius carassius)

Development and application of environmental DNA surveillance for the threatened crucian carp (Carassius carassius)

Environmental DNA (eDNA) metabarcoding of pond water as a tool to survey conservation and management priority mammals

Prospects and challenges of environmental DNA (eDNA) monitoring in freshwater ponds

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