scholarly journals Understanding PCR Processes to Draw Meaningful Conclusions from Environmental DNA Studies

2019 ◽  
Author(s):  
Ryan P. Kelly ◽  
Andrew Olaf Shelton ◽  
Ramón Gallego

ABSTRACTAs environmental DNA (eDNA) studies have grown in popularity for use in ecological applications, it has become clear that their results differ in significant ways from those of traditional, non-PCR-based surveys. In general, eDNA studies that rely on amplicon sequencing may detect hundreds of species present in a sampled environment, but the resulting species composition can be idiosyncratic, reflecting species’ true biomass abundances poorly or not at all. Here, we use a set of simulations to develop a mechanistic understanding of the processes leading to the kinds of results common in mixed-template PCR-based (metabarcoding) studies. In particular, we focus on the effects of PCR cycle number and primer amplification efficiency on the results of diversity metrics in sequencing studies. We then show that proportional indices of amplicon reads capture trends in taxon biomass with high accuracy, particularly where amplification efficiency is high (median correlation up to 0.97). Our results explain much of the observed behavior of PCR-based studies, and lead to recommendations for best practices in the field.


Author(s):  
Anita Porath‐Krause ◽  
Alexander T. Strauss ◽  
Jeremiah A. Henning ◽  
Eric W. Seabloom ◽  
Elizabeth T. Borer


2021 ◽  
Vol 4 ◽  
Author(s):  
Tiina Laamanen ◽  
Veera Norros ◽  
Sanna Suikkanen ◽  
Mikko Tolkkinen ◽  
Kristiina Vuorio ◽  
...  

Environmental DNA (eDNA) and other molecular based approaches are revolutionizing the field of biomonitoring. These approaches undergo rapid modifications, and it is crucial to develop the best practices by sharing the newest information and knowledge. In our ongoing project we: assess the state-of-the-art of eDNA methods at Finnish Environment Institute SYKE; identify concrete next steps towards the long-term aim of implementing eDNA methods into environmental and biomonitoring; promote information exchange on eDNA methods and advance future research efforts both within SYKE and with our national and international partners. assess the state-of-the-art of eDNA methods at Finnish Environment Institute SYKE; identify concrete next steps towards the long-term aim of implementing eDNA methods into environmental and biomonitoring; promote information exchange on eDNA methods and advance future research efforts both within SYKE and with our national and international partners. Scientific background Well-functioning and intact natural ecosystems are essential for human well-being, provide a variety of ecosystem services and contain a high diversity of organisms. However, human activities such as eutrophication, pollution, land-use or invasive species, are threatening the state and functioning of ecosystems from local to global scale (e.g. Benateau et al. 2019; Reid et al. 2018; Vörösmarty et al. 2010). New molecular techniques in the field and in the laboratory have enabled sampling and identification of much of terrestrial, marine and freshwater biodiversity. These include environmental DNA (eDNA, e.g. Valentini et al. 2016) and bulk-sample DNA metabarcoding approaches (e.g. Elbrecht et al. 2017) and targeted RNA-based methods (e.g. Mäki and Tiirola 2018). The eDNA technique uses DNA that is released from organisms into their environment, from which a signal of organisms’ presence in the system can be obtained. For example, in aquatic ecosystems, eDNA is typically extracted from sediment or filtered water samples (e.g. Deiner et al. 2016), and this approach is distinguished from bulk DNA metabarcoding, where organisms are directly identified from e.g. complete biological monitoring samples (e.g. Elbrecht et al. 2017). Despite the demonstrated potential of environmental and bulk-sample DNA metabarcoding approaches in recent years, there are still significant bottlenecks to their routine use that need to be addressed (e.g. Pawlowski et al. 2020). Methods and implementati on The project is divided into three work packages: WP1 Gathering existing knowledge, identifying knowledge gaps and proposing best practices, WP2 Roadmap to implementation and WP3 eDNA monitoring pilot. Please see more details in the Fig. 1



2005 ◽  
Vol 51 (5) ◽  
pp. 393-398 ◽  
Author(s):  
Sunny Jiang ◽  
Hojabr Dezfulian ◽  
Weiping Chu

Adenoviruses 40 and 41 have been recognized as important etiological agents of gastroenteritis in children. A real-time PCR method (TaqMan® assay) was developed for rapid quantification of adenovirus 40 (Ad40) by amplifying an 88 bp sequence from the hexon gene. To establish a quantification standard curve, a 1090 bp hexon region of Ad40 was amplified and cloned into the pGEM®-T Vector. A direct correlation was observed between the fluorescence threshold cycle number (Ct) and the starting quantity of Ad40 hexon gene. The quantification was linear over 6-log units and the amplification efficiency averaged greater than 95%. Seeding studies using various environmental matrices (including sterile water, creek water, brackish estuarine water, ocean water, and secondary sewage effluent) suggest that this method is applicable to environmental samples. However, real-time PCR was sensitive to inhibitors present in the environmental samples. Lower efficiency of PCR amplification was found in secondary sewage effluent and creek waters. Application of the method to fecal contaminated waters successfully quantified the presence of Ad40. The sensitivity of the real-time PCR is comparable to the traditional nested PCR assay for environmental samples. In addition, the real-time PCR assay offers the advantage of speed and insensitivity to contamination during PCR set up. The real-time PCR assay developed in this study is suitable for quantitative determination of Ad40 in environmental samples and represents a considerable advancement in pathogen quantification in aquatic environments.Key words: adenovirus, real-time PCR, environmental waters, serotype 40.



2019 ◽  
Author(s):  
Bryden Fields ◽  
Sara Moeskjær ◽  
Ville-Petri Friman ◽  
Stig U. Andersen ◽  
J. Peter W. Young

AbstractBackgroundSequencing and PCR errors are a major challenge when characterising genetic diversity using high-throughput amplicon sequencing (HTAS).ResultsWe have developed a multiplexed HTAS method, MAUI-seq, which uses unique molecular identifiers (UMIs) to improve error correction by exploiting variation among sequences associated with a single UMI. We show that two main advantages of this approach are efficient elimination of chimeric and other erroneous reads, outperforming DADA2 and UNOISE3, and the ability to confidently recognise genuine alleles that are present at low abundance or resemble chimeras.ConclusionsThe method provides sensitive and flexible profiling of diversity and is readily adaptable to most HTAS applications, including microbial 16S rRNA profiling and metabarcoding of environmental DNA.



2018 ◽  
Author(s):  
Kendell Clement ◽  
Rick Farouni ◽  
Daniel E. Bauer ◽  
Luca Pinello

AbstractMotivationUnique molecular identifiers (UMIs) are added to DNA fragments before PCR amplification to discriminate between alleles arising from the same genomic locus and sequencing reads produced by PCR amplification. While computational methods have been developed to take into account UMI information in genome-wide and single-cell sequencing studies, they are not designed for modern amplicon based sequencing experiments, especially in cases of high allelic diversity. Importantly, no guidelines are provided for the design of optimal UMI length for amplicon-based sequencing experiments.ResultsBased on the total number of DNA fragments and the distribution of allele frequencies, we present a model for the determination of the minimum UMI length required to prevent UMI collisions and reduce allelic distortion. We also introduce a user-friendly software tool called AmpUMI to assist in the design and the analysis of UMI-based amplicon sequencing studies. AmpUMI provides quality control metrics on frequency and quality of UMIs, and trims and deduplicates amplicon sequences with user specified parameters for use in downstream analysis. AmpUMI is open-source and freely available at http://github.com/pinellolab/[email protected]





2013 ◽  
Vol 59 (10) ◽  
pp. 1470-1480 ◽  
Author(s):  
Steve Lefever ◽  
Filip Pattyn ◽  
Jan Hellemans ◽  
Jo Vandesompele

BACKGROUND Genome-sequencing studies have led to an immense increase in the number of known single-nucleotide polymorphisms (SNPs). Designing primers that anneal to regions devoid of SNPs has therefore become challenging. We studied the impact of one or more mismatches in primer-annealing sites on different quantitative PCR (qPCR)-related parameters, such as quantitative cycle (Cq), amplification efficiency, and reproducibility. METHODS We used synthetic templates and primers to assess the effect of mismatches at primer-annealing sites on qPCR assay performance. Reactions were performed with 5 commercially available master mixes. We studied the effects of the number, type, and position of priming mismatches on Cq value, PCR efficiency, reproducibility, and yield. RESULTS The impact of mismatches was most pronounced for the number of mismatched nucleotides and for their distance from the 3′ end of the primer. In addition, having ≥4 mismatches in a single primer or having 3 mismatches in one primer and 2 in the other was required to block a reaction completely. Finally, the degree of the mismatch effect was concentration independent for single mismatches, whereas concentration independence failed at higher template concentrations as the number of mismatches increased. CONCLUSIONS Single mismatches located >5 bp from the 3′ end have a moderate effect on qPCR amplification and can be tolerated. This finding, together with the concentration independence for single mismatches and the complete blocking of the PCR reaction for ≥4 mismatches, can help to chart mismatch behavior in qPCR reactions and increase the rate of successful primer design for sequences with a high SNP density or for homologous regions of sequence.



2011 ◽  
Vol 77 (21) ◽  
pp. 7846-7849 ◽  
Author(s):  
David Berry ◽  
Karim Ben Mahfoudh ◽  
Michael Wagner ◽  
Alexander Loy

ABSTRACT“Barcode-tagged” PCR primers used for multiplex amplicon sequencing generate a thus-far-overlooked amplification bias that produces variable terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing data from the same environmental DNA template. We propose a simple two-step PCR approach that increases reproducibility and consistently recovers higher genetic diversity in pyrosequencing libraries.



2018 ◽  
Author(s):  
Keith Mitchell ◽  
Christopher Dao ◽  
Amanda Freise ◽  
Serghei Mangul ◽  
Jordan Moberg Parker

AbstractMicrobial community profiling and functional inference via 16S rRNA analysis is quickly expanding across various areas of microbiology due to improvements to technology. There are numerous platforms for producing 16S rRNA taxonomic data which often vary in file and sequence formatting, creating a common barrier in microbiome studies. Additionally, many of the methods for analyzing and visualizing this sequencing data each require their own specific formatting. As a result, efficient and reproducible comparative analysis of taxonomic data and corresponding metadata in multiple programs remains a challenge in the investigation of microbial communities. PUMA, the Program for Unifying Microbiome Analysis, alleviates this problem in microbiome studies by allowing users to take advantage of numerous 16S rRNA taxonomic identification platforms and analysis tools in an efficient manner. PUMA accepts sequencing results from several taxonomic identification platforms and then automates configuration of data and file types for analysis and visualization via many popular tools. The protocol accomplishes this by producing a variety of properly configured, annotated, and altered files for both analysis and visualization of taxonomic community profiles and inferred functional profiles. PUMA provides an easy and flexible interface to accommodate for a variety of users to produce all files needed for all-inclusive analysis of targeted amplicon sequencing studies. PUMA is an unprecedented open-source solution for unifying multiple microbiome analysis softwares and uses an adaptable implementation with the potential to improve and consolidate the state of microbiome research.Body/Findings



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