scholarly journals Comparing sediment DNA extraction methods for assessing organic enrichment associated with marine aquaculture

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10231
Author(s):  
John K. Pearman ◽  
Nigel B. Keeley ◽  
Susanna A. Wood ◽  
Olivier Laroche ◽  
Anastasija Zaiko ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Community Composition ◽  
Oncorhynchus Tshawytscha ◽  
Environmental Dna ◽  
Illumina Miseq ◽  
Ecological Impacts ◽  
Biotic Index ◽  
Soil Dna ◽  
Additional Advantage ◽  
Marine Aquaculture

Marine sediments contain a high diversity of micro- and macro-organisms which are important in the functioning of biogeochemical cycles. Traditionally, anthropogenic perturbation has been investigated by identifying macro-organism responses along gradients. Environmental DNA (eDNA) analyses have recently been advocated as a rapid and cost-effective approach to measuring ecological impacts and efforts are underway to incorporate eDNA tools into monitoring. Before these methods can replace or complement existing methods, robustness and repeatability of each analytical step has to be demonstrated. One area that requires further investigation is the selection of sediment DNA extraction method. Environmental DNA sediment samples were obtained along a disturbance gradient adjacent to a Chinook (Oncorhynchus tshawytscha) salmon farm in Otanerau Bay, New Zealand. DNA was extracted using four extraction kits (Qiagen DNeasy PowerSoil, Qiagen DNeasy PowerSoil Pro, Qiagen RNeasy PowerSoil Total RNA/DNA extraction/elution and Favorgen FavorPrep Soil DNA Isolation Midi Kit) and three sediment volumes (0.25, 2, and 5 g). Prokaryotic and eukaryotic communities were amplified using primers targeting the 16S and 18S ribosomal RNA genes, respectively, and were sequenced on an Illumina MiSeq. Diversity and community composition estimates were obtained from each extraction kit, as well as their relative performance in established metabarcoding biotic indices. Differences were observed in the quality and quantity of the extracted DNA amongst kits with the two Qiagen DNeasy PowerSoil kits performing best. Significant differences were observed in both prokaryotes and eukaryotes (p < 0.001) richness among kits. A small proportion of amplicon sequence variants (ASVs) were shared amongst the kits (~3%) although these shared ASVs accounted for the majority of sequence reads (prokaryotes: 59.9%, eukaryotes: 67.2%). Differences were observed in the richness and relative abundance of taxonomic classes revealed with each kit. Multivariate analysis showed that there was a significant interaction between “distance” from the farm and “kit” in explaining the composition of the communities, with the distance from the farm being a stronger determinant of community composition. Comparison of the kits against the bacterial and eukaryotic metabarcoding biotic index suggested that all kits showed similar patterns along the environmental gradient. Overall, we advocate for the use of Qiagen DNeasy PowerSoil kits for use when characterizing prokaryotic and eukaryotic eDNA from marine farm sediments. We base this conclusion on the higher DNA quality values and richness achieved with these kits compared to the other kits/amounts investigated in this study. The additional advantage of the PowerSoil Kits is that DNA extractions can be performed using an extractor robot, offering additional standardization and reproducibility of results.

scholarly journals Optimizing an eDNA protocol for monitoring endangered Chinook Salmon in the San Francisco Estuary: balancing sensitivity, cost and time

2019 ◽  
Author(s):  
Thiago M. Sanches ◽  
Andrea M. Schreier
Keyword(s):  
Dna Extraction ◽  
San Francisco ◽  
Chinook Salmon ◽  
Magnetic Beads ◽  
Oncorhynchus Tshawytscha ◽  
Environmental Dna ◽  
San Francisco Estuary ◽  
Time Cost ◽  
Dna Yield ◽  
Pcr Inhibitor

AbstractEnvironmental DNA (eDNA) analysis has gained traction as a precise and cost effective method for species and waterways management. To date, publications on eDNA protocol optimization have focused primarily on DNA yield. Therefore, it has not been possible to evaluate the cost and speed of specific components of the eDNA protocol, such as water filtration and DNA extraction method when designing or choosing an eDNA pipeline. At the same time, these two parameters are essential for the experimental design of a project. Here we evaluate and rank different eDNA protocols in the context of Chinook salmon (Oncorhynchus tshawytscha) eDNA detection in an aquatic environment, the San Francisco Estuary. We present a comprehensive evaluation of multiple eDNA protocol parameters, balancing time, cost and DNA yield. For estuarine waters, which are challenging for eDNA studies due to high turbidity, variable salinity, and the presence of PCR inhibitors, we find that a protocol combining glass filters and magnetic beads, along with an extra step for PCR inhibitor removal, is the method that best balances time, cost, and yield. In addition, we provide a generalized decision tree for determining the optimal eDNA protocol for other studies on aquatic systems. Our findings should be applicable to most aquatic environments and provide a clear guide for determining which eDNA pipeline should be used for a given environmental condition.Author SummaryThe use of environmental DNA (eDNA) analysis for monitoring wildlife has steadily grown in recent years. Though, due to differences in the ecology of the environment studied and the novelty of the technique, eDNA currently shows a lack of standards compared to other fields. Here we take a deep look into each step of an eDNA assay, looking at common protocols and comparing their efficiencies in terms of time to process the samples, cost and how much DNA is recovered. We then analyze the data to provide a concise interpretation of best practices given different project constraints. For the conditions of the San Francisco Estuary we suggest the use of glass fiber filtration, the use of paramagnetic beads for DNA extraction and the use of a secondary inhibitor removal. We expect our findings to provide better support for managers to decide their standards ahead of project submission not only for estuarine conditions but for other waterine conditions alike.

scholarly journals Quantitative and qualitative evaluation of the impact of the G2 enhancer, bead sizes and lysing tubes on the bacterial community composition during DNA extraction from recalcitrant soil core samples based on community sequencing and qPCR

2018 ◽  
Author(s):  
Alex Gobbi ◽  
Rui G. Santini ◽  
Elisa Filippi ◽  
Lea Ellegaard- Jensen ◽  
Carsten S. Jacobsen ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Dna Extraction ◽  
Community Composition ◽  
Bacterial Community Composition ◽  
Microbial Community Composition ◽  
Soil Core ◽  
Soil Matrix ◽  
Soil Dna ◽  
Dna Recovery ◽  
The Impact

AbstractSoil DNA extraction encounters numerous challenges that can affect both yield and purity of the recovered DNA. Clay particles lead to reduced DNA extraction efficiency, and PCR inhibitors from the soil matrix can negatively affect downstream analyses when applying DNA sequencing. Further, these effects impede molecular analysis of bacterial community compositions in lower biomass samples, as often observed in deeper soil layers. Many studies avoid these complications by using indirect DNA extraction with prior separation of the cells from the matrix, but such methods introduce other biases that influence the resulting microbial community composition.To address these issues, a direct DNA extraction method was applied in combination with the use of a commercial product, the G2 DNA/RNA Enhancer®, marketed as being capable of improving the amount of DNA recovered after the lysis step. The results showed that application of G2 increased DNA yields from the studied clayey soils from layers between 1.00 and 2.20 m below ground level.Importantly, the use of G2 did not introduce bias, as it did not result in any significant differences in the biodiversity of the bacterial community measured in terms of alpha and beta diversity and taxonomical composition.Finally, this study considered a set of customised lysing tubes for evaluating possible influences on the DNA yield. Tubes customization included different bead sizes and amounts, along with lysing tubes coming from two suppliers. Results showed that the lysing tubes with mixed beads allowed greater DNA recovery compared to the use of either 0.1 or 1.4 mm beads, irrespective of the tube supplier.These outcomes may help to improve commercial products in DNA/RNA extraction kits, besides raising awareness about the optimal choice of additives, offering opportunities for acquiring a better understanding of topics such as vertical microbial characterisation and environmental DNA recovery in low biomass samples.

scholarly journals A large-scale ecological assessment of Swiss rivers using environmental DNA for the monitoring of macroinvertebrates 

2021 ◽  
Vol 4 ◽  
Author(s):  
Jeanine Brantschen ◽  
Rosetta Blackman ◽  
Jean-Claude Walser ◽  
Florian Altermatt
Keyword(s):  
Random Forest ◽  
Community Composition ◽  
Large Scale ◽  
Anthropogenic Activities ◽  
Ecological Status ◽  
Environmental Dna ◽  
Biotic Index ◽  
Reference Database ◽  
Macroinvertebrate Communities ◽  
Monitoring Methods

Anthropogenic activities are changing the state of ecosystems worldwide, affecting community composition and often resulting in loss of biodiversity. Riverine ecosystems are among the most impacted ecosystems. Recording their current state with regular biomonitoring is important to assess the future trajectory of biodiversity. However, traditional monitoring methods for ecological assessments are costly and time-intense. Here, we compare environmental DNA (eDNA) to traditional kick-net sampling in a standardized framework of surface water quality assessment. We use surveys of macroinvertebrate communities to assess biodiversity and the biological state of riverine systems. Both methods were employed to monitor aquatic macroinvertebrate indicator groups at 92 sites across major Swiss river catchments. The eDNA data were taxonomically assigned using a customised reference database. All zero-radius Operational Taxonomic Units (zOTUs) mapping to one of the 142 traditionally used indicator taxon levels were used for subsequent diversity analyses (n = 205). At the site level, eDNA detected less indicator taxa than the kick-net method and alpha diversity correlated only weakly between the methods. However, the methods showed a strong congruence in the overall community composition (gamma diversity), as the same indicator groups were commonly detected. In order to set the community composition in relation to the biotic index, the ecological states of the sampling sites were predicted by a random forest approach. Using all zOTUs mapping to macroinvertebrate indicator groups (n = 693) as predictive features, the random forest models successfully predicted the ecological status of the sampled sites. The majority of the predictions (71%) resulted in the same classification like the kick-net based scores. Thus, the sampling of eDNA enabled the detection of indicator communities and provided valuable classifications of the ecological state, when combined with machine learning. Overall, eDNA based sampling has the potential to complement traditional surveys of macroinvertebrate communities in routine large-scale assessments in a non-invasive and scalable approach.

scholarly journals Assessment of Passive Traps Combined with High-Throughput Sequencing To Study Airborne Fungal Communities

2018 ◽  
Vol 84 (11) ◽  
Author(s):  
Jaime Aguayo ◽  
Céline Fourrier-Jeandel ◽  
Claude Husson ◽  
Renaud Ioos
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Linear Models ◽  
Environmental Dna ◽  
Illumina Miseq ◽  
Fungal Communities ◽  
Correct Identification ◽  
Spore Trap

ABSTRACTTechniques based on high-throughput sequencing (HTS) of environmental DNA have provided a new way of studying fungal diversity. However, these techniques suffer from a number of methodological biases which may appear at any of the steps involved in a metabarcoding study. Air is one of the most important environments where fungi can be found, because it is the primary medium of dispersal for many species. Looking ahead to future developments, it was decided to test 20 protocols, including different passive spore traps, spore recovery procedures, DNA extraction kits, and barcode loci. HTS was performed with the Illumina MiSeq platform targeting two subloci of the fungal internal transcribed spacer. Multivariate analysis and generalized linear models showed that the type of passive spore trap, the spore recovery procedure, and the barcode all impact the description of fungal communities in terms of richness and diversity when assessed by HTS metabarcoding. In contrast, DNA extraction kits did not significantly impact these results. Although passive traps may be used to describe airborne fungal communities, a study using specific real-time PCR and a mock community showed that these kinds of traps are affected by environmental conditions that may induce losses of biological material, impacting diversity and community composition results.IMPORTANCEThe advent of high-throughput sequencing (HTS) methods, such as those offered by next-generation sequencing (NGS) techniques, has opened a new era in the study of fungal diversity in different environmental substrates. In this study, we show that an assessment of the diversity of airborne fungal communities can reliably be achieved by the use of simple and robust passive spore traps. However, a comparison of sample processing protocols showed that several methodological biases may impact the results of fungal diversity when assessed by metabarcoding. Our data suggest that identifying these biases is of paramount importance to enable a correct identification and relative quantification of community members.

scholarly journals Environmental DNA gives comparable results to morphology-based indices of macroinvertebrates in a large-scale ecological assessment

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257510
Author(s):  
Jeanine Brantschen ◽  
Rosetta C. Blackman ◽  
Jean-Claude Walser ◽  
Florian Altermatt
Keyword(s):  
Machine Learning ◽  
Random Forest ◽  
Community Composition ◽  
Large Scale ◽  
Anthropogenic Activities ◽  
Environmental Dna ◽  
Biotic Index ◽  
Ecological Classification ◽  
Indicator Taxa

Anthropogenic activities are changing the state of ecosystems worldwide, affecting community composition and often resulting in loss of biodiversity. Rivers are among the most impacted ecosystems. Recording their current state with regular biomonitoring is important to assess the future trajectory of biodiversity. Traditional monitoring methods for ecological assessments are costly and time-intensive. Here, we compared monitoring of macroinvertebrates based on environmental DNA (eDNA) sampling with monitoring based on traditional kick-net sampling to assess biodiversity patterns at 92 river sites covering all major Swiss river catchments. From the kick-net community data, a biotic index (IBCH) based on 145 indicator taxa had been established. The index was matched by the taxonomically annotated eDNA data by using a machine learning approach. Our comparison of diversity patterns only uses the zero-radius Operational Taxonomic Units assigned to the indicator taxa. Overall, we found a strong congruence between both methods for the assessment of the total indicator community composition (gamma diversity). However, when assessing biodiversity at the site level (alpha diversity), the methods were less consistent and gave complementary data on composition. Specifically, environmental DNA retrieved significantly fewer indicator taxa per site than the kick-net approach. Importantly, however, the subsequent ecological classification of rivers based on the detected indicators resulted in similar biotic index scores for the kick-net and the eDNA data that was classified using a random forest approach. The majority of the predictions (72%) from the random forest classification resulted in the same river status categories as the kick-net approach. Thus, environmental DNA validly detected indicator communities and, combined with machine learning, provided reliable classifications of the ecological state of rivers. Overall, while environmental DNA gives complementary data on the macroinvertebrate community composition compared to the kick-net approach, the subsequently calculated indices for the ecological classification of river sites are nevertheless directly comparable and consistent.

scholarly journals A Rapid Bacteriophage DNA Extraction Method

2018 ◽  
Vol 1 (3) ◽  
pp. 27 ◽  
Author(s):  
Džiuginta Jakočiūnė ◽  
Arshnee Moodley
Keyword(s):  
Dna Extraction ◽  
Illumina Miseq ◽  
Extraction Methods ◽  
Resistant Bacteria ◽  
Simple Method ◽  
Antibiotic Resistant ◽  
Phage Dna ◽  
Specialized Equipment ◽  
Miseq Platform ◽  
Dna Extraction Methods

Bacteriophages (phages) are intensely investigated as non-antibiotic alternatives to circumvent antibiotic resistance development as well as last resort therapeutic options against antibiotic resistant bacteria. As part of gaining a better understanding of phages and to determine if phages harbor putative virulence factors, whole genome sequencing is used, for which good quality phage DNA is needed. Traditional phage DNA extraction methods are tedious and time consuming, requiring specialized equipment e.g., an ultra-centrifuge. Here, we describe a quick and simple method (under four hours) to extract DNA from double stranded DNA (dsDNA) phages at titers above 1.0 × 1010 plaque-forming units (PFU)/mL. This DNA was suitable for library preparation using the Nextera XT kit and sequencing on the Illumina MiSeq platform.

DNA extraction from concrete v2

2021 ◽  
Author(s):  
Anders Kiledal ◽  
Julia A Maresca
Keyword(s):  
Dna Extraction ◽  
Dental Plaque ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Metagenomic Sequencing ◽  
Sample Analysis ◽  
Larger Sample ◽  
Laboratory Contamination ◽  
Biomass Sample ◽  
Over Time

This is a protocol for extracting DNA from concrete, based on the protocol developed by L. S. Weyrich, et al. for extraction of DNA from ancient calcified dental plaque. We have scaled it up for larger sample sizes and made some additional modifications for the chemistry of concrete. DNA extracted using this method is suitable for metagenomic sequencing by Illumina MiSeq and NextSeq, as well as amplicon sequencing. This protocol should yield 10 ng to 5 μg DNA per 10 g of concrete, depending on the age and integrity of the sample. Reference: L. S. Weyrich et al., Laboratory contamination over time during low-biomass sample analysis. Mol. Ecol. Resour. 19, 982–996 (2019).

scholarly journals Different Methods of Soil DNA Extraction

BIO-PROTOCOL ◽  
2020 ◽  
Vol 10 (2) ◽  
Author(s):  
Asmita Kamble ◽  
Harinder Singh
Keyword(s):  
Dna Extraction ◽  
Soil Dna ◽  
Soil Dna Extraction

scholarly journals Global Trends of Benthic Bacterial Diversity and Community Composition Along Organic Enrichment Gradients of Salmon Farms

2021 ◽  
Vol 12 ◽  
Author(s):  
Larissa Frühe ◽  
Verena Dully ◽  
Dominik Forster ◽  
Nigel B. Keeley ◽  
Olivier Laroche ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Bacterial Diversity ◽  
Community Composition ◽  
Environmental Dna ◽  
Community Diversity ◽  
Rrna Gene ◽  
Local Conditions ◽  
Global Trends ◽  
Bacterial Taxon ◽  
Metabolic Properties

The analysis of benthic bacterial community structure has emerged as a powerful alternative to traditional microscopy-based taxonomic approaches to monitor aquaculture disturbance in coastal environments. However, local bacterial diversity and community composition vary with season, biogeographic region, hydrology, sediment texture, and aquafarm-specific parameters. Therefore, without an understanding of the inherent variation contained within community complexes, bacterial diversity surveys conducted at individual farms, countries, or specific seasons may not be able to infer global universal pictures of bacterial community diversity and composition at different degrees of aquaculture disturbance. We have analyzed environmental DNA (eDNA) metabarcodes (V3–V4 region of the hypervariable SSU rRNA gene) of 138 samples of different farms located in different major salmon-producing countries. For these samples, we identified universal bacterial core taxa that indicate high, moderate, and low aquaculture impact, regardless of sampling season, sampled country, seafloor substrate type, or local farming and environmental conditions. We also discuss bacterial taxon groups that are specific for individual local conditions. We then link the metabolic properties of the identified bacterial taxon groups to benthic processes, which provides a better understanding of universal benthic ecosystem function(ing) of coastal aquaculture sites. Our results may further guide the continuing development of a practical and generic bacterial eDNA-based environmental monitoring approach.

Sign in / Sign up

Export Citation Format

Share Document