scholarly journals A DNA metabarcoding approach for recovering plankton communities from archived samples fixed in formalin

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0245936
Author(s):  
Takuhei Shiozaki ◽  
Fumihiro Itoh ◽  
Yuu Hirose ◽  
Jonaotaro Onodera ◽  
Akira Kuwata ◽  
...  
Keyword(s):  
Community Structure ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Formalin Fixation ◽  
Plankton Sample ◽  
Rrna Gene ◽  
Dna Metabarcoding ◽  
Plankton Diversity ◽  
The Impact ◽  
Formalin Fixed

Plankton samples have been routinely collected and preserved in formalin in many laboratories and museums for more than 100 years. Recently, attention has turned to use DNA information from formalin-fixed samples to examine changes in plankton diversity over time. However, no molecular ecological studies have evaluated the impact of formalin fixation on the genetic composition of the plankton community structure. Here, we developed a method for extracting DNA from archived formalin-preserved plankton samples to determine their community structure by a DNA metabarcoding approach. We found that a lysis solution consisting of borate-NaOH buffer (pH 11) with SDS and proteinase K effectively cleaved the cross-link formed by formalin fixation. DNA was extracted from samples preserved for decades in formalin, and the diatom community of the extracted DNA was in good agreement with the microscopy analysis. Furthermore, we stored a plankton sample for 1.5 years and demonstrated that 18S rRNA gene community structures did not change significantly from non-formalin-fixed, time-zero samples. These results indicate that our method can be used to describe the original community structure of plankton archived in formalin for years. Our approach will be useful for examining the long-term variation of plankton diversity by metabarcoding analysis of 18S rRNA gene community structure.

scholarly journals 16S rRNA gene and 18S rRNA gene diversity in microbial mat communities in meltwater ponds on the McMurdo Ice Shelf, Antarctica

Polar Biology ◽  
2021 ◽  
Author(s):  
Eleanor E. Jackson ◽  
Ian Hawes ◽  
Anne D. Jungblut
Keyword(s):  
16S Rrna ◽  
18S Rrna ◽  
High Throughput Sequencing ◽  
Microbial Mats ◽  
Gene Diversity ◽  
Salinity Gradient ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Ice Shelf ◽  
The Impact

AbstractThe undulating ice of the McMurdo Ice Shelf, Southern Victoria Land, supports one of the largest networks of ice-based, multiyear meltwater pond habitats in Antarctica, where microbial mats are abundant and contribute most of the biomass and biodiversity. We used 16S rRNA and 18S rRNA gene high-throughput sequencing to compare variance of the community structure in microbial mats within and between ponds with different salinities and pH. Proteobacteria and Cyanobacteria were the most abundant phyla, and composition at OTU level was highly specific for the meltwater ponds with strong community sorting along the salinity gradient. Our study provides the first detailed evaluation of eukaryote communities for the McMurdo Ice Shelf using the 18S rRNA gene. They were dominated by Ochrophyta, Chlorophyta and Ciliophora, consistent with previous microscopic analyses, but many OTUs belonging to less well-described heterotrophic protists from Antarctic ice shelves were also identified including Amoebozoa, Rhizaria and Labyrinthulea. Comparison of 16S and 18S rRNA gene communities showed that the Eukaryotes had lower richness and greater similarity between ponds in comparison with Bacteria and Archaea communities on the McMurdo Ice shelf. While there was a weak correlation between community dissimilarity and geographic distance, the congruity of microbial assemblages within ponds, especially for Bacteria and Archaea, implies strong habitat filtering in ice shelf meltwater pond ecosystems, especially due to salinity. These findings help to understand processes that are important in sustaining biodiversity and the impact of climate change on ice-based aquatic habitats in Antarctica.

scholarly journals Phylogeny of Intestinal Ciliates, Including Charonina ventriculi, and Comparison of Microscopy and 18S rRNA Gene Pyrosequencing for Rumen Ciliate Community Structure Analysis

2015 ◽  
Vol 81 (7) ◽  
pp. 2433-2444 ◽  
Author(s):  
Sandra Kittelmann ◽  
Savannah R. Devente ◽  
Michelle R. Kirk ◽  
Henning Seedorf ◽  
Burk A. Dehority ◽  
...  
Keyword(s):  
Community Structure ◽  
High Throughput ◽  
18S Rrna ◽  
Gene Sequence ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Content Type ◽  
Advantages And Disadvantages ◽  
Ciliate Community

ABSTRACTThe development of high-throughput methods, such as the construction of 18S rRNA gene clone or pyrosequencing libraries, has allowed evaluation of ciliate community composition in hundreds of samples from the rumen and other intestinal habitats. However, several genera of mammalian intestinal ciliates have been described based only on morphological features and, to date, have not been identified using molecular methods. Here, we isolated single cells of one of the smallest but widely distributed intestinal ciliates,Charonina ventriculi, and sequenced its 18S rRNA gene. We verified the sequence in a full-cycle rRNA approach using fluorescencein situhybridization and thereby assigned an 18S rRNA gene sequence to this species previously known only by its morphology. Based on its full-length 18S rRNA gene sequence,Charonina ventriculiwas positioned within the phylogeny of intestinal ciliates in the subclass Trichostomatia. The taxonomic framework derived from this phylogeny was used for taxonomic assignment of trichostome ciliate 18S rRNA gene sequence data stemming from high-throughput amplicon pyrosequencing of rumen-derived DNA samples. The 18S rRNA gene-based ciliate community structure was compared to that obtained from microscopic counts using the same samples. Both methods allowed identification of dominant members of the ciliate communities and classification of the rumen ciliate community into one of the types first described by Eadie in 1962. Notably, each method is associated with advantages and disadvantages. Microscopy is a highly accurate method for evaluation of total numbers or relative abundances of different ciliate genera in a sample, while 18S rRNA gene pyrosequencing represents a valuable alternative for comparison of ciliate community structure in a large number of samples from different animals or treatment groups.

scholarly journals Preliminary analysis of New Zealand scampi (Metanephrops challengeri) diet using metabarcoding

2018 ◽  
Author(s):  
Aimee L van der Reis ◽  
Olivier Laroche ◽  
Andrew G Jeffs ◽  
Shane D Lavery
Keyword(s):  
New Zealand ◽  
Preliminary Analysis ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Coi Gene ◽  
Taxonomic Resolution ◽  
Gut Contents ◽  
Rrna Gene ◽  
Diverse Range ◽  
Dna Metabarcoding

Deep sea lobsters are highly valued for seafood and provide the basis of important commercial fisheries in many parts of the world. Despite their economic significance, relatively little is known about their natural diets. Microscopic analyses of foregut content in some species have suffered from low taxonomic resolution, with many of the dietary items difficult to reliably identify as their tissue is easily digested. DNA metabarcoding has the potential to provide greater taxonomic resolution of the diet of the New Zealand scampi (Metanephrops challengeri) through the identification of gut contents, but a number of methodological concerns need to be overcome first to ensure optimum DNA metabarcoding results. In this study, a range of methodological parameters were tested to determine the optimum protocols for DNA metabarcoding, and provide a first view of M. challengeri diet. Several PCR protocols were tested, using two universal primer pairs targeting the 18S rRNA and COI genes, on DNA extracted from both frozen and ethanol preserved samples for both foregut and hindgut digesta. The selection of appropriate DNA polymerases, buffers and methods for reducing PCR inhibitors (including the use of BSA) were found to be critical. Amplification from frozen or ethanol preserved gut contents appeared similarly dependable, but metabarcoding outcomes indicated that the ethanol samples produced better results from the COI gene. The COI gene was found to be more effective than 18S rRNA gene for identifying large eukaryotic taxa from the digesta, however, it was less successfully amplified. The 18S rRNA gene was more easily amplified, but identified mostly smaller marine organisms such as plankton and parasites. This preliminary analysis of the diet of M. challengeri identified a range of species (13,541 reads identified as diet), which included the ghost shark (Hydrolagus novaezealandiae), silver warehou (Seriolella punctate), tall sea pen (Funiculina quadrangularis) and the salp (Ihlea racovitza), suggesting that they have a varied diet, with a high reliance on scavenging a diverse range of pelagic and benthic species from the seafloor.

scholarly journals New Insights Into Nematode DNA-metabarcoding as Revealed by the Characterization of Artificial and Spiked Nematode Communities

Diversity ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 52 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nancy de Sutter ◽  
Nicole Viaene ◽  
Annelies Haegeman
Keyword(s):  
Species Richness ◽  
18S Rrna ◽  
Nematode Species ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Soil Nematode ◽  
Mock Community ◽  
Nematode Communities ◽  
Dna Metabarcoding ◽  
Soil Nematode Communities

Nematodes are ideal biological indicators to monitor soil biodiversity and ecosystem functioning. For this reason, they have been receiving increasing attention from a broad range of scientists. The main method to characterize soil nematode communities until at least genus level is still based on microscopic observations of nematode morphology. Such an approach is time-consuming, labor-intensive, and requires specialized personnel. The first studies on the potential use of DNA-metabarcoding to characterize nematode communities showed some shortcomings: under- or overestimation of species richness caused by failure to detect a number of nematode species or caused by intraspecific sequence variants increasing the number of OTUs (operational taxonomic units) or ‘molecular’ species, and flaws in quantification. We set up experiments to optimize this metabarcoding approach. Our results provided new insights such as the drastic effect of different DNA-extraction methods on nematode species richness due to variation in lysis efficacy. Our newly designed primer set (18S rRNA gene, V4-V5 region) showed in silico an improved taxonomic coverage compared with a published primer set (18S rRNA gene, V6-V8 region). However, results of DNA-metabarcoding with the new primer set showed less taxonomic coverage, and more non-nematode reads. Thus, the new primer set might be more suitable for whole soil faunal analysis. Species-specific correction factors calculated from a mock community with equal amounts of different nematode species were applied on another mock community with different amounts of the same nematode species and on a biological sample spiked with four selected nematode species. Results showed an improved molecular quantification. In conclusion, DNA-metabarcoding of soil nematode communities is useful for monitoring shifts in nematode composition but the technique still needs further optimization to enhance its precision.

scholarly journals Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities

2016 ◽  
Vol 82 (19) ◽  
pp. 5878-5891 ◽  
Author(s):  
Ian M. Bradley ◽  
Ameet J. Pinto ◽  
Jeremy S. Guest
Keyword(s):  
Community Structure ◽  
18S Rrna ◽  
Target Genes ◽  
Hypervariable Region ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Sequencing Bias ◽  
Primer Sets

ABSTRACTThe use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3′ end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest.IMPORTANCEThe quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential amplification efficiencies result in inaccurate estimates of community structure. The extent to which amplification bias affects community representation and the accuracy with which different gene targets represent community structure are not known. As a result, there is no consensus on which region provides the most suitable representation of diversity for eukaryotes. This study determined the accuracy with which commonly used 18S rRNA gene primer sets represent community structure and identified particular biases related to PCR amplification and Illumina MiSeq sequencing in order to more accurately study eukaryotic microbial communities.

scholarly journals Seasonal Diversity of Planktonic Protists in Southwestern Alberta Rivers over a 1-Year Period as Revealed by Terminal Restriction Fragment Length Polymorphism and 18S rRNA Gene Library Analyses

2012 ◽  
Vol 78 (16) ◽  
pp. 5653-5660 ◽  
Author(s):  
Matthew C. Thomas ◽  
L. Brent Selinger ◽  
G. Douglas Inglis
Keyword(s):  
Community Structure ◽  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Fragment Length ◽  
18S Rrna ◽  
Fragment Length Polymorphism ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Seasonal Diversity ◽  
Restriction Fragment Length

ABSTRACTThe temporal dynamics of planktonic protists in river water have received limited attention despite their ecological significance and recent studies linking phagotrophic protists to the persistence of human-pathogenic bacteria. Using molecular-based techniques targeting the 18S rRNA gene, we studied the seasonal diversity of planktonic protists in Southwestern Alberta rivers (Oldman River Basin) over a 1-year period. Nonmetric multidimensional scaling analysis of terminal restriction fragment length polymorphism (T-RFLP) data revealed distinct shifts in protistan community profiles that corresponded to season rather than geographical location. Community structures were examined by using clone library analysis; HaeIII restriction profiles of 18S rRNA gene amplicons were used to remove prevalent solanaceous plant clones prior to sequencing. Sanger sequencing of the V1-to-V3 region of the 18S rRNA gene libraries from spring, summer, fall, and winter supported the T-RFLP results and showed marked seasonal differences in the protistan community structure. The spring library was dominated by Chloroplastidae (29.8%), Centrohelida (28.1%), and Alveolata (25.5%), while the summer and fall libraries contained primarily fungal clones (83.0% and 88.0%, respectively). Alveolata (35.6%), Euglenozoa (24.4%), Chloroplastida (15.6%), and Fungi (15.6%) dominated the winter library. These data demonstrate that planktonic protists, including protozoa, are abundant in river water in Southwestern Alberta and that conspicuous seasonal shifts occur in the community structure.

scholarly journals Preliminary analysis of New Zealand scampi (Metanephrops challengeri) diet using metabarcoding

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5641 ◽  
Author(s):  
Aimee L. van der Reis ◽  
Olivier Laroche ◽  
Andrew G. Jeffs ◽  
Shane D. Lavery
Keyword(s):  
New Zealand ◽  
Preliminary Analysis ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Coi Gene ◽  
Taxonomic Resolution ◽  
Gut Contents ◽  
Rrna Gene ◽  
Diverse Range ◽  
Dna Metabarcoding

Deep sea lobsters are highly valued for seafood and provide the basis of important commercial fisheries in many parts of the world. Despite their economic significance, relatively little is known about their natural diets. Microscopic analyses of foregut content in some species have suffered from low taxonomic resolution, with many of the dietary items difficult to reliably identify as their tissue is easily digested. DNA metabarcoding has the potential to provide greater taxonomic resolution of the diet of the New Zealand scampi (Metanephrops challengeri) through the identification of gut contents, but a number of methodological concerns need to be overcome first to ensure optimum DNA metabarcoding results. In this study, a range of methodological parameters were tested to determine the optimum protocols for DNA metabarcoding, and provide a first view ofM.challengeridiet. Several PCR protocols were tested, using two universal primer pairs targeting the 18S rRNA and COI genes, on DNA extracted from both frozen and ethanol preserved samples for both foregut and hindgut digesta. The selection of appropriate DNA polymerases, buffers and methods for reducing PCR inhibitors (including the use of BSA) were found to be critical. Amplification from frozen or ethanol preserved gut contents appeared similarly dependable. The COI gene was found to be more effective than 18S rRNA gene for identifying large eukaryotic taxa from the digesta; however, it was less successfully amplified. The 18S rRNA gene was more easily amplified, but identified mostly smaller marine organisms such as plankton and parasites. This preliminary analysis of the diet ofM.challengeriidentified a range of species (13,541 reads identified as diet), which included the ghost shark (Hydrolagus novaezealandiae), silver warehou (Seriolella punctata), tall sea pen (Funiculina quadrangularis) and the salp (Ihlea racovitzai), suggesting that they have a varied diet, with a high reliance on scavenging a diverse range of pelagic and benthic species from the seafloor.

scholarly journals 16S and 18S rRNA Gene Metabarcoding Provide Congruent Information on the Responses of Sediment Communities to Eutrophication

2021 ◽  
Vol 8 ◽  
Author(s):  
Jesse P. Harrison ◽  
Panagiota-Myrsini Chronopoulou ◽  
Iines S. Salonen ◽  
Tom Jilbert ◽  
Karoliina A. Koho
Keyword(s):  
Community Structure ◽  
Bottom Water ◽  
18S Rrna ◽  
Ecological Status ◽  
18S Rrna Gene ◽  
Ecological Impacts ◽  
Nutrient Concentrations ◽  
Rrna Gene ◽  
Sediment Depth ◽  
Using Data

Metabarcoding analyses of bacterial and eukaryotic communities have been proposed as efficient tools for environmental impact assessment. It has been unclear, however, to which extent these analyses can provide similar or differing information on the ecological status of the environment. Here, we used 16S and 18S rRNA gene metabarcoding to compare eutrophication-induced shifts in sediment bacterial and eukaryotic community structure in relation to a range of porewater, sediment and bottom-water geochemical variables, using data obtained from six stations near a former rainbow trout farm in the Archipelago Sea (Baltic Sea). Shifts in the structure of both community types were correlated with a shared set of variables, including porewater ammonium concentrations and the sediment depth-integrated oxygen consumption rate. Distance-based redundancy analyses showed that variables typically employed in impact assessments, such as bottom water nutrient concentrations, explained less of the variance in community structure than alternative variables (e.g., porewater NH4+ inventories and sediment depth-integrated O2 consumption rates) selected due to their low collinearity (up to 40 vs. 58% of the variance explained, respectively). In monitoring surveys where analyses of both bacterial and eukaryotic communities may be impossible, either 16S or 18S rRNA gene metabarcoding can serve as reliable indicators of wider ecological impacts of eutrophication.

scholarly journals Preliminary analysis of New Zealand scampi (Metanephrops challengeri) diet using metabarcoding

2018 ◽  
Author(s):  
Aimee L van der Reis ◽  
Olivier Laroche ◽  
Andrew G Jeffs ◽  
Shane D Lavery
Keyword(s):  
New Zealand ◽  
Preliminary Analysis ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Coi Gene ◽  
Taxonomic Resolution ◽  
Gut Contents ◽  
Rrna Gene ◽  
Diverse Range ◽  
Dna Metabarcoding

Deep sea lobsters are highly valued for seafood and provide the basis of important commercial fisheries in many parts of the world. Despite their economic significance, relatively little is known about their natural diets. Microscopic analyses of foregut content in some species have suffered from low taxonomic resolution, with many of the dietary items difficult to reliably identify as their tissue is easily digested. DNA metabarcoding has the potential to provide greater taxonomic resolution of foregut and hindgut contents of the New Zealand (NZ) scampi (Metanephrops challengeri), but a number of methodological concerns need to first be overcome to ensure optimum DNA metabarcoding results. In this study, a range of methodological parameters were trialled to determine the optimum protocols for DNA metabarcoding, and provide a first view of the NZ scampi diet. Several PCR protocols were trialled, using two universal primer pairs targeting the 18S rRNA and COI genes, on DNA extracted from frozen and ethanol preserved samples of both foregut and hindgut digesta. The selection of appropriate DNA polymerases, buffers and methods for reducing PCR inhibitors (including use of BSA) were found to be critical. Amplification from frozen or ethanol preserved gut contents appeared similarly dependable, but metabarcoding results showed that ethanol samples resulted in better results from the COI gene. The COI gene was found to be more effective than 18S rRNA gene for identifying large eukaryotic taxa from the digesta, however, it was less successfully amplified. The 18S rRNA gene was more easily amplified, but identified mostly smaller marine organisms such as plankton and parasites. This preliminary analysis of the diet of the NZ scampi identified a range of species, which included the ghost shark (Hydrolagus novaezealandiae), silver warehou (Seriolella punctate), tall sea pen (Funiculina quadrangularis ) and salp (Ihlea racovitza), suggesting that they have a varied diet, with a high reliance on scavenging a diverse range of pelagic and benthic species from the seafloor.

scholarly journals Gastrointestinal Segments Influenced Fermentation End-Products, Microbiota and Microbial Abundances in Goats

2021 ◽  
Author(s):  
Tsegay Gebremariam ◽  
Zhiliang Tan
Keyword(s):  
Fatty Acids ◽  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Gene Copies ◽  
End Products ◽  
The Impact

Abstract Purpose: Carbohydrate diets altered fermentation end-products and microbial community in the gastrointestinal tracts (GIT) of goats. Gastrointestinal contents used to determine the impact of carbohydrate feeds on fermentation end-products and microbial community in goats.Methodology: in the study goats were assigned to one of the two treatments corn meal (CM) or Corn gluten (CG) in a randomized block design (400 g/kg DM each). Goats were slaughtered, GIT liquids were used to determine dissolved gasses, fatty acids and microbial community.Results: Goats fed CG increased molar acetate (P < 0.05), lowered butyrate and propionate in the fore and hindgut comparing to those goats received CM. Goats received CM had higher (P < 0.05) dH2 while lowered dH2S in the fore and hindgut than those goats fed with CG treatment. The fore and hindgut had higher (P < 0.01) 16S rRNA gene copies of bacteria, protozoa, methanogens and 18S rRNA gene copies fungi than in the ileum and cecum. Goats fed CG diet had higher (P < 0.05)16S rRNA gene copies of bacteria, protozoa, methanogens, and 18S rRNA gene copies of fungi than those goats fed with CM diet. Conclusion fore and hindguts improved dissolved gasses, fatty acids and microbial community comparing with in the ileum and cecum. Goats fed CM had improved the Methanobacterials order and Methanobrevibacter genus as compared with those goats fed CG. The study suggested that hindgut segments have a reasonable contribution as foregut to methane emissions from goats.

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