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Objective Analysis of environmental DNA (eDNA) is a method that has been used for the detection of various species within water bodies. The great crested newt (Triturus cristatus) has a short eDNA survey season (mid-April to June). Here we investigate whether this season could be extended into other months using the current methodology as stipulated by Natural England. Results Here we present data to show that in monthly water samples taken from two ponds (March 2014–February 2015) we were able to detect great crested newt DNA in all months in at least one of the ponds. Similar levels of great crested newt eDNA (i.e. highly positive identification) were detected through the months of March–August, suggesting it may be possible to extend the current survey window. In order to determine how applicable these observations are for ponds throughout the rest of the UK, further work in multiple other ponds over multiple seasons is suggested. Nevertheless, the current work clearly demonstrates, in two ponds, the efficacy and reproducibility of eDNA detection for determining the presence of great crested newts.

Prions are an enigma amongst infectious disease agents as they lack a genome yet confer specific pathologies thought to be dictated mainly, if not solely, by the conformation of the disease form of the prion protein (PrPSc). Prion diseases affect humans and animals, the latter including the food-producing ruminant species cattle, sheep, goats and deer. Importantly, it has been shown that the disease agent of bovine spongiform encephalopathy (BSE) is zoonotic, causing variant Creutzfeldt Jakob disease (vCJD) in humans. Current diagnostic tests can distinguish different prion types and in food- producing animals these focus on the differentiation of BSE from the non-zoonotic agents. Whilst BSE cases are now rare, atypical forms of both scrapie and BSE have been reported, as well as two types of chronic wasting disease (CWD) in cervids. Typing of animal prion isolates remains an important aspect of prion diagnosis and is now becoming more focused on identifying the range of prion types that are present in food-producing animals and also developing tests that can screen for emerging, novel prion diseases. Here, we review prion typing methodologies in light of current and emerging prion types in food-producing animals.

Current ecological surveys for great crested newts are time-consuming and expensive and can only be carried out within a short survey window. Additional survey methods which would facilitate the detection of rare or protected species such as the great crested newt (Triturus cristatus) would be extremely advanta- geous. Environmental DNA (eDNA) analysis has been utilized for the detection of great crested newts in Denmark. Here, the same methodology has been applied to water samples taken from UK ponds concurrently with conventional field surveying techniques. Our eDNA analysis exhibited an 84% success rate with a kappa coefficient of agreement between field and eDNA surveys of 0.86. One pond determined to be negative for great crested newt by field survey was positive by eDNA analysis, revealing the potential for improved detection rates using this methodology. Analysis of water samples collected in late summer indicates that eDNA analysis could be used to detect great crested newt after the optimal survey window for current field techniques had passed. Conse- quently, eDNA analysis could augment currently stipulated techniques for great crested newt surveying as a relatively quick and inexpensive tool for collecting great crested newt presence and distribution data within the UK instead of or prior to full field surveys.

1. Knowledge of species distribution is critical to ecological management and conservation biology. Effective management requires the detection of populations, which can sometimes be at low densities and is usually based on visual detection and counting. 2. Recently, there has been considerable interest in the detection of short species-specific environmental DNA (eDNA) fragments to allow aquatic species monitoring within different environments due to the potential of greater sensitivity over traditional survey methods which can be time-consuming and costly. 3. Environmental DNA analysis is increasingly being used in the detection of rare or invasive species and has also been applied to eDNA persistence studies and estimations of species biomass and distribution. When combined with next-generation sequencing methods, it has been demonstrated that entire faunas can be identified. 4. Different environments require different sampling methodologies, but there remain areas where laboratory methodologies could be standardized to allow results to be compared across studies. 5. Synthesis and applications. We review recently published studies that use eDNA to moni- tor aquatic populations, discuss the methodologies used and the application of eDNA analysis as a survey tool in ecology. We include innovative ideas for how eDNA can be used for conservation and management citing test cases, for instance, the potential for on-site analyses, including the application of eDNA analysis to carbon nanotube platforms or laser transmission spectroscopy to facilitate rapid on-site detections. The use of eDNA monitoring is already being adopted in the UK for ecological surveys.

1. In Rees et al. (2014b), we reviewed the current status of environmental DNA (eDN A) tomonitor aquatic populations. Our aim was to focus on discus sion of methodologies used,application of eDNA analysis as a survey tool in ecology, and to include some innovativeideas for using eDNA in conservation and management. 2. Roussel et al. (2015) claim that analysis of Rees et al. (2014b) and other publicationshighlights the downsides of the method, and they suggest that some conclusions should betoned down. Many of their arguments were covered in our original paper (Rees et al. ,2014b); however, they make the point that modelling approaches should be encouraged, andwe fully agree with this suggestion. 3. Roussel et al. (2015) also claim that we neglected to recognize that there are two sourcesof imperfect detection (at the field level and at the laboratory level). We feel that our reviewpaper implies this point. 4. Synthesis and applications. Roussel et al. (2015) reiterate many of the points made in theoriginal paper but do cover some additional areas that improve the debate on the use of envi-ronmental DNA (eDNA). Both the comment (Roussel et al., 2015) and our rebuttal clearlyhighlight that detailed laboratory protocols and rigorous field sampling design are crucial fac-tors which require sufficient reporting in the literature to allow for experimenta l comparisonand replication. Any development of a new method for eDNA detection should be compareddirectly with established ‘gold standard’ methods for the detection of the species or habitatunder investigation. None of the issues raised in Roussel et al. (2015) would alter our mainconclusions.

Recently the analytical power of the latest high throughput next generation DNA sequencing platforms has been used to analyse phage that have been selected from the panning of large combinatorial libraries displaying either peptide or antibody ligands. This process, commonly referred to as next generation phage display (NGPD), allows the researcher to determine the identity of specific phage that are being enriched against an antigen target by analysis of the DNA sequence encoding the displayed ligand. This method bypasses several steps in conventional phage panning that include laborious colony picking and functional ligand screening. A downside of this approach is that the only output from such experiments is the DNA sequence information of such enriched phage particles. In the case of peptides, the peptide sequence can be synthesised directly and used for further screening; however this is more difficult with larger antibody fragments such as ScFvs. In the case of ScFvs, their coding sequence would have to be fully elucidated, synthesised and re-cloned before expression. We describe here the application of an inverse PCR-ligation methodology that enables the specific recovery of ScFvs of interest from enriched sub-libraries of phage clones. Phagemid particles are recovered using sequence information derived from their unique heavy chain CDR3/FR4 domains and specific clones can be recovered irrespective of CDR3 size and at levels of abundance that would be refractory to their discovery during conventional phage panning and screening.

Objective Analysis of environmental DNA (eDNA) is a method that has been used for the detection of various species within water bodies. The great crested newt (Triturus cristatus) has a short eDNA survey season (mid-April to June). Here we investigate whether this season could be extended into other months using the current methodology as stipulated by Natural England. Results Here we present data to show that in monthly water samples taken from two ponds (March 2014–February 2015) we were able to detect great crested newt DNA in all months in at least one of the ponds. Similar levels of great crested newt eDNA (i.e. highly positive identification) were detected through the months of March–August, suggesting it may be possible to extend the current survey window. In order to determine how applicable these observations are for ponds throughout the rest of the UK, further work in multiple other ponds over multiple seasons is suggested. Nevertheless, the current work clearly demonstrates, in two ponds, the efficacy and reproducibility of eDNA detection for determining the presence of great crested newts.

Prions are an enigma amongst infectious disease agents as they lack a genome yet confer specific pathologies thought to be dictated mainly, if not solely, by the conformation of the disease form of the prion protein (PrPSc). Prion diseases affect humans and animals, the latter including the food-producing ruminant species cattle, sheep, goats and deer. Importantly, it has been shown that the disease agent of bovine spongiform encephalopathy (BSE) is zoonotic, causing variant Creutzfeldt Jakob disease (vCJD) in humans. Current diagnostic tests can distinguish different prion types and in food- producing animals these focus on the differentiation of BSE from the non-zoonotic agents. Whilst BSE cases are now rare, atypical forms of both scrapie and BSE have been reported, as well as two types of chronic wasting disease (CWD) in cervids. Typing of animal prion isolates remains an important aspect of prion diagnosis and is now becoming more focused on identifying the range of prion types that are present in food-producing animals and also developing tests that can screen for emerging, novel prion diseases. Here, we review prion typing methodologies in light of current and emerging prion types in food-producing animals.

Current ecological surveys for great crested newts are time-consuming and expensive and can only be carried out within a short survey window. Additional survey methods which would facilitate the detection of rare or protected species such as the great crested newt (Triturus cristatus) would be extremely advanta- geous. Environmental DNA (eDNA) analysis has been utilized for the detection of great crested newts in Denmark. Here, the same methodology has been applied to water samples taken from UK ponds concurrently with conventional field surveying techniques. Our eDNA analysis exhibited an 84% success rate with a kappa coefficient of agreement between field and eDNA surveys of 0.86. One pond determined to be negative for great crested newt by field survey was positive by eDNA analysis, revealing the potential for improved detection rates using this methodology. Analysis of water samples collected in late summer indicates that eDNA analysis could be used to detect great crested newt after the optimal survey window for current field techniques had passed. Conse- quently, eDNA analysis could augment currently stipulated techniques for great crested newt surveying as a relatively quick and inexpensive tool for collecting great crested newt presence and distribution data within the UK instead of or prior to full field surveys.

1. In Rees et al. (2014b), we reviewed the current status of environmental DNA (eDN A) tomonitor aquatic populations. Our aim was to focus on discus sion of methodologies used,application of eDNA analysis as a survey tool in ecology, and to include some innovativeideas for using eDNA in conservation and management. 2. Roussel et al. (2015) claim that analysis of Rees et al. (2014b) and other publicationshighlights the downsides of the method, and they suggest that some conclusions should betoned down. Many of their arguments were covered in our original paper (Rees et al. ,2014b); however, they make the point that modelling approaches should be encouraged, andwe fully agree with this suggestion. 3. Roussel et al. (2015) also claim that we neglected to recognize that there are two sourcesof imperfect detection (at the field level and at the laboratory level). We feel that our reviewpaper implies this point. 4. Synthesis and applications. Roussel et al. (2015) reiterate many of the points made in theoriginal paper but do cover some additional areas that improve the debate on the use of envi-ronmental DNA (eDNA). Both the comment (Roussel et al., 2015) and our rebuttal clearlyhighlight that detailed laboratory protocols and rigorous field sampling design are crucial fac-tors which require sufficient reporting in the literature to allow for experimenta l comparisonand replication. Any development of a new method for eDNA detection should be compareddirectly with established ‘gold standard’ methods for the detection of the species or habitatunder investigation. None of the issues raised in Roussel et al. (2015) would alter our mainconclusions.

1. Knowledge of species distribution is critical to ecological management and conservation biology. Effective management requires the detection of populations, which can sometimes be at low densities and is usually based on visual detection and counting. 2. Recently, there has been considerable interest in the detection of short species-specific environmental DNA (eDNA) fragments to allow aquatic species monitoring within different environments due to the potential of greater sensitivity over traditional survey methods which can be time-consuming and costly. 3. Environmental DNA analysis is increasingly being used in the detection of rare or invasive species and has also been applied to eDNA persistence studies and estimations of species biomass and distribution. When combined with next-generation sequencing methods, it has been demonstrated that entire faunas can be identified. 4. Different environments require different sampling methodologies, but there remain areas where laboratory methodologies could be standardized to allow results to be compared across studies. 5. Synthesis and applications. We review recently published studies that use eDNA to moni- tor aquatic populations, discuss the methodologies used and the application of eDNA analysis as a survey tool in ecology. We include innovative ideas for how eDNA can be used for conservation and management citing test cases, for instance, the potential for on-site analyses, including the application of eDNA analysis to carbon nanotube platforms or laser transmission spectroscopy to facilitate rapid on-site detections. The use of eDNA monitoring is already being adopted in the UK for ecological surveys.

Recently the analytical power of the latest high throughput next generation DNA sequencing platforms has been used to analyse phage that have been selected from the panning of large combinatorial libraries displaying either peptide or antibody ligands. This process, commonly referred to as next generation phage display (NGPD), allows the researcher to determine the identity of specific phage that are being enriched against an antigen target by analysis of the DNA sequence encoding the displayed ligand. This method bypasses several steps in conventional phage panning that include laborious colony picking and functional ligand screening. A downside of this approach is that the only output from such experiments is the DNA sequence information of such enriched phage particles. In the case of peptides, the peptide sequence can be synthesised directly and used for further screening; however this is more difficult with larger antibody fragments such as ScFvs. In the case of ScFvs, their coding sequence would have to be fully elucidated, synthesised and re-cloned before expression. We describe here the application of an inverse PCR-ligation methodology that enables the specific recovery of ScFvs of interest from enriched sub-libraries of phage clones. Phagemid particles are recovered using sequence information derived from their unique heavy chain CDR3/FR4 domains and specific clones can be recovered irrespective of CDR3 size and at levels of abundance that would be refractory to their discovery during conventional phage panning and screening.

Environmental DNA (eDNA) analysis is a rapid, cost-effective, non-invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species-specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and ‘metabarcoding’ have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real-time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using High-Throughput Sequencing technology. With qPCR and a detection threshold of 1/12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4/12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can

Objective Analysis of environmental DNA (eDNA) is a method that has been used for the detection of various species within water bodies. The great crested newt (Triturus cristatus) has a short eDNA survey season (mid-April to June). Here we investigate whether this season could be extended into other months using the current methodology as stipulated by Natural England. Results Here we present data to show that in monthly water samples taken from two ponds (March 2014–February 2015) we were able to detect great crested newt DNA in all months in at least one of the ponds. Similar levels of great crested newt eDNA (i.e. highly positive identification) were detected through the months of March–August, suggesting it may be possible to extend the current survey window. In order to determine how applicable these observations are for ponds throughout the rest of the UK, further work in multiple other ponds over multiple seasons is suggested. Nevertheless, the current work clearly demonstrates, in two ponds, the efficacy and reproducibility of eDNA detection for determining the presence of great crested newts.

Prions are an enigma amongst infectious disease agents as they lack a genome yet confer specific pathologies thought to be dictated mainly, if not solely, by the conformation of the disease form of the prion protein (PrPSc). Prion diseases affect humans and animals, the latter including the food-producing ruminant species cattle, sheep, goats and deer. Importantly, it has been shown that the disease agent of bovine spongiform encephalopathy (BSE) is zoonotic, causing variant Creutzfeldt Jakob disease (vCJD) in humans. Current diagnostic tests can distinguish different prion types and in food- producing animals these focus on the differentiation of BSE from the non-zoonotic agents. Whilst BSE cases are now rare, atypical forms of both scrapie and BSE have been reported, as well as two types of chronic wasting disease (CWD) in cervids. Typing of animal prion isolates remains an important aspect of prion diagnosis and is now becoming more focused on identifying the range of prion types that are present in food-producing animals and also developing tests that can screen for emerging, novel prion diseases. Here, we review prion typing methodologies in light of current and emerging prion types in food-producing animals.

Environmental DNA (eDNA) analysis is a rapid, cost-effective, non-invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species-specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and ‘metabarcoding’ have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real-time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using High-Throughput Sequencing technology. With qPCR and a detection threshold of 1/12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4/12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can

Objective Analysis of environmental DNA (eDNA) is a method that has been used for the detection of various species within water bodies. The great crested newt (Triturus cristatus) has a short eDNA survey season (mid-April to June). Here we investigate whether this season could be extended into other months using the current methodology as stipulated by Natural England. Results Here we present data to show that in monthly water samples taken from two ponds (March 2014–February 2015) we were able to detect great crested newt DNA in all months in at least one of the ponds. Similar levels of great crested newt eDNA (i.e. highly positive identification) were detected through the months of March–August, suggesting it may be possible to extend the current survey window. In order to determine how applicable these observations are for ponds throughout the rest of the UK, further work in multiple other ponds over multiple seasons is suggested. Nevertheless, the current work clearly demonstrates, in two ponds, the efficacy and reproducibility of eDNA detection for determining the presence of great crested newts.

Recently the analytical power of the latest high throughput next generation DNA sequencing platforms has been used to analyse phage that have been selected from the panning of large combinatorial libraries displaying either peptide or antibody ligands. This process, commonly referred to as next generation phage display (NGPD), allows the researcher to determine the identity of specific phage that are being enriched against an antigen target by analysis of the DNA sequence encoding the displayed ligand. This method bypasses several steps in conventional phage panning that include laborious colony picking and functional ligand screening. A downside of this approach is that the only output from such experiments is the DNA sequence information of such enriched phage particles. In the case of peptides, the peptide sequence can be synthesised directly and used for further screening; however this is more difficult with larger antibody fragments such as ScFvs. In the case of ScFvs, their coding sequence would have to be fully elucidated, synthesised and re-cloned before expression. We describe here the application of an inverse PCR-ligation methodology that enables the specific recovery of ScFvs of interest from enriched sub-libraries of phage clones. Phagemid particles are recovered using sequence information derived from their unique heavy chain CDR3/FR4 domains and specific clones can be recovered irrespective of CDR3 size and at levels of abundance that would be refractory to their discovery during conventional phage panning and screening.

Current ecological surveys for great crested newts are time-consuming and expensive and can only be carried out within a short survey window. Additional survey methods which would facilitate the detection of rare or protected species such as the great crested newt (Triturus cristatus) would be extremely advanta- geous. Environmental DNA (eDNA) analysis has been utilized for the detection of great crested newts in Denmark. Here, the same methodology has been applied to water samples taken from UK ponds concurrently with conventional field surveying techniques. Our eDNA analysis exhibited an 84% success rate with a kappa coefficient of agreement between field and eDNA surveys of 0.86. One pond determined to be negative for great crested newt by field survey was positive by eDNA analysis, revealing the potential for improved detection rates using this methodology. Analysis of water samples collected in late summer indicates that eDNA analysis could be used to detect great crested newt after the optimal survey window for current field techniques had passed. Conse- quently, eDNA analysis could augment currently stipulated techniques for great crested newt surveying as a relatively quick and inexpensive tool for collecting great crested newt presence and distribution data within the UK instead of or prior to full field surveys.

1. Knowledge of species distribution is critical to ecological management and conservation biology. Effective management requires the detection of populations, which can sometimes be at low densities and is usually based on visual detection and counting. 2. Recently, there has been considerable interest in the detection of short species-specific environmental DNA (eDNA) fragments to allow aquatic species monitoring within different environments due to the potential of greater sensitivity over traditional survey methods which can be time-consuming and costly. 3. Environmental DNA analysis is increasingly being used in the detection of rare or invasive species and has also been applied to eDNA persistence studies and estimations of species biomass and distribution. When combined with next-generation sequencing methods, it has been demonstrated that entire faunas can be identified. 4. Different environments require different sampling methodologies, but there remain areas where laboratory methodologies could be standardized to allow results to be compared across studies. 5. Synthesis and applications. We review recently published studies that use eDNA to moni- tor aquatic populations, discuss the methodologies used and the application of eDNA analysis as a survey tool in ecology. We include innovative ideas for how eDNA can be used for conservation and management citing test cases, for instance, the potential for on-site analyses, including the application of eDNA analysis to carbon nanotube platforms or laser transmission spectroscopy to facilitate rapid on-site detections. The use of eDNA monitoring is already being adopted in the UK for ecological surveys.

1. In Rees et al. (2014b), we reviewed the current status of environmental DNA (eDN A) tomonitor aquatic populations. Our aim was to focus on discus sion of methodologies used,application of eDNA analysis as a survey tool in ecology, and to include some innovativeideas for using eDNA in conservation and management. 2. Roussel et al. (2015) claim that analysis of Rees et al. (2014b) and other publicationshighlights the downsides of the method, and they suggest that some conclusions should betoned down. Many of their arguments were covered in our original paper (Rees et al. ,2014b); however, they make the point that modelling approaches should be encouraged, andwe fully agree with this suggestion. 3. Roussel et al. (2015) also claim that we neglected to recognize that there are two sourcesof imperfect detection (at the field level and at the laboratory level). We feel that our reviewpaper implies this point. 4. Synthesis and applications. Roussel et al. (2015) reiterate many of the points made in theoriginal paper but do cover some additional areas that improve the debate on the use of envi-ronmental DNA (eDNA). Both the comment (Roussel et al., 2015) and our rebuttal clearlyhighlight that detailed laboratory protocols and rigorous field sampling design are crucial fac-tors which require sufficient reporting in the literature to allow for experimenta l comparisonand replication. Any development of a new method for eDNA detection should be compareddirectly with established ‘gold standard’ methods for the detection of the species or habitatunder investigation. None of the issues raised in Roussel et al. (2015) would alter our mainconclusions.