Extracellular DNA in natural environments: features, relevance and applications

Nagler, Magdalena; Insam, Heribert; Pietramellara, Giacomo; Ascher‐Jenull, Judith

doi:10.1007/s00253-018-9120-4

Cited by 171 publications

(156 citation statements)

References 170 publications

(253 reference statements)

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“…Over the last decade, environmental DNA (eDNA) studies of lake sediments have added a new dimension to traditional biological proxies (e.g., pollen and plant macrofossil analyses) often used to investigate and reconstruct past vegetation changes and palaeoenvironments (Parducci et al, 2017). We know that in sediments eDNA binds to mineral and organic components (extracellular DNA) and that it is also present within cells in plant and animal remains that become embedded in the sediments (for a review see Nagler et al, 2018). Indeed, PCR (polymerase chain reaction) amplification of short fragments of chloroplast DNA from sediments (metabarcoding) has been successfully used to demonstrate the presence of animals and plants in different palaeoenvironmental settings (e.g.…”

Section: Introductionmentioning

confidence: 99%

Shotgun Environmental DNA, Pollen, and Macrofossil Analysis of Lateglacial Lake Sediments From Southern Sweden

et al. 2019

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The lake sediments of Hässeldala Port in south-east Sweden provide an archive of local and regional environmental conditions ∼14.5-9.5 ka BP (thousand years before present) and allow testing DNA sequencing techniques to reconstruct past vegetation changes. We combined shotgun sequencing with plant micro-and macrofossil analyses to investigate sediments dating to the Allerød (14.1-12.7 ka BP), Younger Dryas (12.7-11.7 ka BP), and Preboreal (<11.7 ka BP). Number of reads and taxa were not associated with sample age or organic content. This suggests that, beyond the initial rapid degradation, DNA is still present. The proportion of recovered plant DNA was low, but allowed identifying an important number of plant taxa, thus adding valid information on the composition of the local vegetation. Importantly, DNA provides a stronger signal of plant community changes than plant micro-and plant macrofossil analyses alone, since a larger number of new taxa were recorded in Younger Dryas samples. A comparison between the three proxies highlights differences and similarities and supports earlier findings that plants growing close to or within a lake are recorded by DNA. Plant macrofossil remains moreover show that tree birch was present close to the ancient lake since the Allerød; together with the DNA results, this indicates that boreal to subarctic climatic conditions also prevailed during the cold Younger Dryas interval. Increasing DNA reference libraries and enrichment strategies prior to sequencing are necessary to improve the potential and accuracy of plant identification using the shotgun metagenomic approach.

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Section: Introductionmentioning

confidence: 99%

Shotgun Environmental DNA, Pollen, and Macrofossil Analysis of Lateglacial Lake Sediments From Southern Sweden

et al. 2019

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“…Exposure to direct solar radiation can result in DNA degradation by denaturation in exposed samples (Schuch et al, 2017). Similarly, high temperatures generated by direct radiation can drive a high degradation of DNA in contrast to low temperatures (Nagler et al, 2018). In contrast to cold environments where nucleic acids can be stored for long-term due to the decrease of reaction rates by an order of magnitude for every degree drop in temperature (Smith et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Experimental evaluation of genomic DNA degradation rates for the pathogen Pseudogymnoascus destructans (Pd) in bat guano

Urbina

Chestnut

Schwalm

et al. 2020

PeerJ

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Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome in bats (WNS), has led to dramatic declines of bat populations in eastern North America. In the spring of 2016, WNS was first detected at several locations in Washington State, USA, which has prompted the need for large scale surveillance efforts to monitor the spread of Pd. Pd is typically detected in bats using invasive methods requiring capturing and swabbing individual bats. However, Pd can also be detected in guano, which may provide an efficient, affordable, and noninvasive means to monitor Pd in bats across North America. The widespread implementation of Pd surveillance in guano is hindered by substantial uncertainty about the probability of detecting Pd when present, and how this probability is influenced by the time since defecation, local environmental conditions, the amount of guano sampled, and the original concentration of DNA shed in the guano. In addition, the expected degradation rate of Pd DNA depends on whether the Pd DNA found in guano represents extracellular DNA fragments, intracellular DNA from dead Pd fungal cells, or from intracellular and viable Pd cells. While this is currently unknown, it has been posited that most environmental DNA, such as Pd found in guano long after defecation, is fragmented extracellular DNA. Using non-viable isolated DNA at precise quantities, we experimentally characterized the degradation rates of Pd DNA in guano samples. We spiked 450 guano samples with Pd gDNA in a 10-fold dilution series from 1 million to 1,000 fg and placed them in variable environmental conditions at five sites at Mount Rainier National Park in Washington State, which is a priority location for Pd surveillance. We evaluated DNA degradation over 70 days by quantifying the amount of DNA in samples collected every 14 days using real-time quantitative PCR (qPCR). Our sampling period was from July 10th to September 17th 2018 which overlaps with bat movement between summer roosts as well as movement from maternity colonies fall swarms. We detected Pd DNA in guano 56 and 70 days after inoculation with 1 million and 100,000 fg respectively, while the lowest quantity (1,000 fg) was detected until 42 days. Detection probability was variable among sites and lower where samples were left exposed without overhead cover. If Pd is shed as extracellular DNA in guano at quantities above 1,000 fg, then guano collection is likely to provide an effective tool for environmental screening of Pd that can be employed in an early detection and rapid response framework throughout Washington and other regions where this disease is rapidly emerging.

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“…Their phylogenetic behavior is anomalous to a degree never observed with genomes of real organisms. CPR and Asgard MAGs are binning artefacts, assembled from environments where up to 90% of the DNA is from dead cells [9][10][11][12] . Asgard and CPR MAGs are unnatural constructs, genome-like patchworks of genes that have been stitched together into computer files by binning.…”

Section: Discussionmentioning

confidence: 99%

“…They could easily stem from DNA fragments of 20-30 different genomes that simply occur in the same environment. This problem is exacerbated by the circumstance that up to 90% of sequenceable DNA in some environments such as marine sediment is not packaged in cells but is extracellular DNA (eDNA) from dead cells or biofilms [9][10][11][12] , whereby the proportion of eDNA varies across environments and changes over geological time [13][14] .…”

Section: Discussionmentioning

confidence: 99%

Anomalous phylogenetic behavior of ribosomal proteins in metagenome assembled genomes

Garg

Kapust

Lin

et al. 2019

Preprint

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Metagenomic studies have claimed the existence of novel lineages with unprecedented properties never before observed in prokaryotes. Such lineages include Asgard archaea 1-3 , which are purported to represent archaea with eukaryotic cell complexity, and the Candidate Phyla Radiation (CPR), a novel domain level taxon erected solely on the basis of metagenomic data 4 . However, it has escaped the attention of most biologists that these metagenomic sequences are not assembled into genomes by sequence overlap, as for cultured archaea and bacteria. Instead, short contigs are sorted into computer files by a process called binning in which they receive taxonomic assignment on the basis of sequence properties like GC content, dinucleotide frequencies, and stoichiometric co-occurrence across samples.Consequently, they are not genome sequences as we know them, reflecting the gene content of real organisms. Rather they are metagenome assembled genomes (MAGs). Debates that Asgard data are contaminated with individual eukaryotic sequences 5-7 are overshadowed by the more pressing issue that no evidence exists to indicate that any sequences in binned Asgard MAGs actually stem from the same chromosome, as opposed to simply stemming from the same environment. Here we show that Asgard and CPR MAGs fail spectacularly to meet the most basic phylogenetic criterion 8 fulfilled by genome sequences of all cultured prokaryotes investigated to date: the ribosomal proteins of Asgard and CPR MAGs do not share common evolutionary histories. Their phylogenetic behavior is anomalous to a degree never observed with genomes of real organisms. CPR and Asgard MAGs are binning artefacts, assembled from environments where up to 90% of the DNA is from dead cells 9-12 . Asgard and CPR MAGs are unnatural constructs, genome-like patchworks of genes that have been stitched together into computer files by binning.The sequencing of environmental DNA (metagenomics) has become an essential tool of modern science because it allows microbiologists to uncover the existence of genes and species in environments such as marine sediment or the deep biosphere from which representatives cannot readily be cultured 13,14 . Initially an endeavor involving rRNA sequences 15 , metagenomic investigations have led to the binning-assembly and deposition in databases of MAGs, sequences of similar GC content and stoichiometry across samples. Because rRNA has limited phylogenetic resolution, concatenated sequences of ribosomal proteins (r-proteins) and other universally distributed proteins are commonly used for phylogeny. This practice is well established with over 20 years of tradition, whereby the validity of using concatenated rproteins for phylogeny lies in the reproducible crosschecking result that individual r-proteins 3 from the same sequenced genome give the same or very similar trees [16][17][18][19][20][21] . Based in such precedence, it became common practice to use concatenated r-proteins from sequenced genomes for microbial phylogeny without first crosschecking w...

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Extracellular DNA in natural environments: features, relevance and applications

Cited by 171 publications

References 170 publications

Shotgun Environmental DNA, Pollen, and Macrofossil Analysis of Lateglacial Lake Sediments From Southern Sweden

Shotgun Environmental DNA, Pollen, and Macrofossil Analysis of Lateglacial Lake Sediments From Southern Sweden

Experimental evaluation of genomic DNA degradation rates for the pathogen Pseudogymnoascus destructans (Pd) in bat guano

Anomalous phylogenetic behavior of ribosomal proteins in metagenome assembled genomes

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