An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

Champlot, Sophie; Berthelot, Camille; Pruvost, Mélanie; Bennett, E. Andrew; Grange, Thierry; Geigl, Eva-María

doi:10.1371/journal.pone.0013042

Cited by 247 publications

(204 citation statements)

References 79 publications

(116 reference statements)

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“…The source of these two spurious taxa in the mock communities may be contamination from DNA extraction and/or PCR reagents during production in the manufacturer's laboratories, or laboratory processing (Champlot et al . 2010). The low‐frequency noise filter removed 26 of 99 taxa identified by blast (Fig.…”

Section: Resultsmentioning

confidence: 99%

Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA

et al. 2015

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Preserving biodiversity is a global challenge requiring data on species’ distribution and abundance over large geographic and temporal scales. However, traditional methods to survey mobile species’ distribution and abundance in marine environments are often inefficient, environmentally destructive, or resource‐intensive. Metabarcoding of environmental DNA (eDNA) offers a new means to assess biodiversity and on much larger scales, but adoption of this approach for surveying whole animal communities in large, dynamic aquatic systems has been slowed by significant unknowns surrounding error rates of detection and relevant spatial resolution of eDNA surveys. Here, we report the results of a 2.5 km eDNA transect surveying the vertebrate fauna present along a gradation of diverse marine habitats associated with a kelp forest ecosystem. Using PCR primers that target the mitochondrial 12S rRNA gene of marine fishes and mammals, we generated eDNA sequence data and compared it to simultaneous visual dive surveys. We find spatial concordance between individual species’ eDNA and visual survey trends, and that eDNA is able to distinguish vertebrate community assemblages from habitats separated by as little as ~60 m. eDNA reliably detected vertebrates with low false‐negative error rates (1/12 taxa) when compared to the surveys, and revealed cryptic species known to occupy the habitats but overlooked by visual methods. This study also presents an explicit accounting of false negatives and positives in metabarcoding data, which illustrate the influence of gene marker selection, replication, contamination, biases impacting eDNA count data and ecology of target species on eDNA detection rates in an open ecosystem.

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Section: Resultsmentioning

confidence: 99%

Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA

et al. 2015

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“…Standard laboratory hygiene practices should prevent contamination in typical circumstances. For more sensitive applications, methods commonly used in ancient DNA and forensics could also be applied to pollen DNA barcoding to eliminate contamination from the laboratory, reagents, and cross-contamination between samples (e.g., Champlot et al 2010). Pollen sample decontamination methods could take advantage of the chemical resistance of the pollen exine (Southworth 1974;Kearns and Inouye 1993).…”

Section: Component 1: the Pollen Dna Templatementioning

confidence: 99%

Pollen DNA barcoding: current applications and future prospects

Bell

Vere

Keller

et al. 2016

Genome

183

166

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Identification of the species origin of pollen has many applications, including assessment of plant-pollinator networks, reconstruction of ancient plant communities, product authentication, allergen monitoring, and forensics. Such applications, however, have previously been limited by microscopy-based identification of pollen, which is slow, has low taxonomic resolution, and has few expert practitioners. One alternative is pollen DNA barcoding, which could overcome these issues. Recent studies demonstrate that both chloroplast and nuclear barcoding markers can be amplified from pollen. These recent validations of pollen metabarcoding indicate that now is the time for researchers in various fields to consider applying these methods to their research programs. In this paper, we review the nascent field of pollen DNA barcoding and discuss potential new applications of this technology, highlighting existing limitations and future research developments that will improve its utility in a wide range of applications.Key words: DNA metabarcoding, metagenomics, pollen, palynology, high-throughput sequencing, next-generation sequencing.Résumé : L'identification de l'espèce à l'origine d'un pollen se prête à de nombreuses applications dont la description des réseaux plante-pollinisateur, la reconstruction de communautés de plantes anciennes, l'authentification de produits, la surveillance des allergènes et les enquêtes médicolégales. Cependant, ces applications ont précédemment été limitées à l'identification du pollen par examen microscopique, un processus lent, à faible résolution taxonomique et qui compte peu de praticiens experts. Une alternative est l'identification du pollen par le recours aux codes à barres de l'ADN, une avenue qui permettrait de surmonter plusieurs de ces limitations. De récentes études ont montré qu'il était possible d'amplifier les marqueurs de codage tant chloroplastiques que nucléaires à partir du pollen. Ces récentes validations du métacodage à barres chez le pollen indiquent qu'il est maintenant opportun pour les chercheurs dans divers domaines de considérer l'emploi de ces méthodes dans leurs programmes de recherche. Dans cet article, les auteurs passent en revue le domaine naissant du codage à barres du pollen et discutent des nouvelles applications potentielles de cette technologie en mettant en lumière les limitations existantes ainsi que de futurs développements qui pourraient accroître son utilité dans un grand nombre d'applications. [Traduit par la Rédaction] Mots-clés : métacodage à barres, métagénomique, pollen, palynologie, séquençage à haut débit, séquençage de nouvelle génération.

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“…Primers applied in this study were specific for the genus Sus and showed no amplification with DNA of human or other animals either. All extractions were independently amplified and sequenced twice at least for each fragment, to rule out no misincorporated bases (Champlot et al, 2010).…”

Section: Authenticity Criteria For the Ancient Dna Datamentioning

confidence: 99%

Like a pig out of water: seaborne spread of domestic pigs in Southern Italy and Sardinia during the Bronze and Iron Ages

et al. 2016

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Southern Italy has a long history of human occupation and passage of different cultures since the Early Holocene. Repeated, ancient introductions of pigs in several geographic areas in Europe make it difficult to understand pig translocation and domestication in Italy. The archeozoological record may provide fundamental information on this, hence shedding light on peopling and on trading among different ancient cultures in the Mediterranean. Yet, because of the scanty nature of the fossil record, ancient remains from human-associated animals are somewhat rare. Fortunately, ancient DNA analysis as applied to domestic species proved to be a powerful tool in revealing human migrations. Herein, we analyzed 80-bp fragment of mitochondrial DNA control region from 27 Sus scrofa ancient samples retrieved from Southern Italian and Sardinian archeological sites, spanning in age from the Mesolithic to the Roman period. Our results surprisingly indicate the presence of the Near Eastern haplotype Y1 on both Italy's major islands (Sardinia and Sicily) during the Bronze Age, suggesting the seaborne transportation of domestic pigs by humans at least during 1600-1300 BC. The presence of the Italian E2 clade in domestic contexts shows that the indigenous wild boar was effectively domesticated or incorporated into domestic stocks in Southern Italy during the Bronze Age, although the E2 haplotype has never been found in modern domestic breeds. Pigs belonging to the endemic E2 clade were thus traded between the Peninsula and Sardinia by the end of the second millennium BC and this genetic signature is still detected in Sardinian feral pigs.

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An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

Cited by 247 publications

References 79 publications

Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA

Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA

Pollen DNA barcoding: current applications and future prospects

Like a pig out of water: seaborne spread of domestic pigs in Southern Italy and Sardinia during the Bronze and Iron Ages

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