scholarly journals A quest for the biological sources of the ubiquitous long chain alkyl diols in the marine realm

2018 ◽  
Author(s):  
Sergio Balzano ◽  
Julie Lattaud ◽  
Laura Villanueva ◽  
Sebastiaan Rampen ◽  
Corina P. D. Brussaard ◽  
...  
Keyword(s):  
Water Column ◽  
18S Rrna ◽  
Amplicon Sequencing ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Phylogenetic Groups ◽  
Suspended Particulate ◽  
Marine Realm ◽  
Biological Sources ◽  
Long Chain

Abstract. Long chain alkyl diols (LCDs) are widespread in the marine water column and sediments but their biological sources are mostly unknown. Here we combine lipid analyses with 18S rRNA gene amplicon sequencing on suspended particulate matter (SPM) collected in the photic zone of the tropical North Atlantic at 24 stations to infer relationships between LCDs and potential LCD-producers. The C30 1,15-diol was detected in all SPM samples and accounted for > 95 % of the total LCDs, while minor proportions of C28 and C30 1,13-diols, C28 and C30 1,14-diols as well as C32 1,15-diol were found. The concentration of the C30 and C32 diols was higher in the mixed layer of the water column compared to the deep chlorophyll maximum (DCM), whereas concentrations of C28 diols were comparable. Sequencing analyses revealed extremely low contributions (≈ 0.1 % of the 18S rRNA gene reads) of known LCD-producers but the contributions from two taxonomic classes to which known producers are affiliated, i.e. Dictyochophyceae and Chrysophyceae, followed a trend similar to that of the concentrations of C30 and C32 diols. Statistical analyses indicated that the abundance of 4 operational taxonomic units (OTUs) of the Chrysophyceae and Dictyochophyceae, along with 23 OTUs falling in other phylogenetic groups, were significantly correlated with C30 diol concentrations. However, it is not clear whether some of these OTUs might indeed correspond to LCD-producers or whether these correlations are just indirect. Furthermore, based on the average LCD-content measured in cultivated LCD-producing algae, the detected concentrations of LCDs in SPM are too high to be explained by the abundances of the suspected LCD-producing OTUs. This is likely explained by the slower degradation of LCDs compared to DNA in the oxic water column and suggests that some of the LCDs found here were likely to be associated to suspended debris, while the DNA from the related LCD-producers had been already fully degraded. This suggests that care should be taken in constraining biological sources of relatively stable biomarker lipids by quantitative comparisons of DNA and lipid abundances.

scholarly journals A quest for the biological sources of long chain alkyl diols in the western tropical North Atlantic Ocean

2018 ◽  
Vol 15 (19) ◽  
pp. 5951-5968 ◽  
Author(s):  
Sergio Balzano ◽  
Julie Lattaud ◽  
Laura Villanueva ◽  
Sebastiaan W. Rampen ◽  
Corina P. D. Brussaard ◽  
...  
Keyword(s):  
Atlantic Ocean ◽  
Water Column ◽  
North Atlantic ◽  
18S Rrna ◽  
North Atlantic Ocean ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Tropical North Atlantic ◽  
Biological Sources ◽  
Long Chain

Abstract. Long chain alkyl diols (LCDs) are widespread in the marine water column and sediments, but their biological sources are mostly unknown. Here we combine lipid analyses with 18S rRNA gene amplicon sequencing on suspended particulate matter (SPM) collected in the photic zone of the western tropical North Atlantic Ocean at 24 stations to infer relationships between LCDs and potential LCD producers. The C30 1,15-diol was detected in all SPM samples and accounted for >95 % of the total LCDs, while minor proportions of C28 and C30 1,13-diols, C28 and C30 1,14-diols, as well as C32 1,15-diol were found. The concentration of the C30 and C32 diols was higher in the mixed layer of the water column compared to the deep chlorophyll maximum (DCM), whereas concentrations of C28 diols were comparable. Sequencing analyses revealed extremely low contributions (≈0.1 % of the 18S rRNA gene reads) of known LCD producers, but the contributions from two taxonomic classes with which known producers are affiliated, i.e. Dictyochophyceae and Chrysophyceae, followed a trend similar to that of the concentrations of C30 and C32 diols. Statistical analyses indicated that the abundance of 4 operational taxonomic units (OTUs) of the Chrysophyceae and Dictyochophyceae, along with 23 OTUs falling into other phylogenetic groups, were weakly (r≤0.6) but significantly (p value <0.01) correlated with C30 diol concentrations. It is not clear whether some of these OTUs might indeed correspond to C28−32 diol producers or whether these correlations are just indirect and the occurrence of C30 diols and specific OTUs in the same samples might be driven by other environmental conditions. Moreover, primer mismatches were unlikely, but cannot be excluded, and the variable number of rRNA gene copies within eukaryotes might have affected the analyses leading to LCD producers being undetected or undersampled. Furthermore, based on the average LCD content measured in cultivated LCD-producing algae, the detected concentrations of LCDs in SPM are too high to be explained by the abundances of the suspected LCD-producing OTUs. This is likely explained by the slower degradation of LCDs compared to DNA in the oxic water column and suggests that some of the LCDs found here were likely to be associated with suspended debris, while the DNA from the related LCD producers had been already fully degraded. This suggests that care should be taken in constraining biological sources of relatively stable biomarker lipids by quantitative comparisons of DNA and lipid abundances.

scholarly journals Long-chain diols in rivers: distribution and potential biological sources

2018 ◽  
Author(s):  
Julie Lattaud ◽  
Frédérique Kirkels ◽  
Francien Peterse ◽  
Chantal V. Freymond ◽  
Timothy I. Eglinton ◽  
...  
Keyword(s):  
River Flow ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
River Systems ◽  
Eukaryotic Diversity ◽  
Suspended Particulate ◽  
Fractional Abundance ◽  
Biological Sources ◽  
Rrna Gene Sequencing ◽  
Long Chain

Abstract. Long chain diols (LCDs) occur widespread in marine environments and also in lakes and rivers. Transport of LCDs from rivers may impact the distribution of LCDs in coastal environments, however relatively little is known about the distribution and biological sources of LCDs in river systems. In this study, we investigated the distribution of LCDs in suspended particulate matter (SPM) of three river systems (Godavari, Danube, and Rhine) in relation with season, precipitation, temperature, and source catchments. The dominant long-chain diol is the C32 1,15-diol followed by the C30 1,15-diol in all studied river systems. In regions influenced by marine waters, such as delta systems, the fractional abundance of the C30 1,15-diol is substantially higher than in the river itself, suggesting different LCD producers in marine and freshwater environments. A change in the LCD distribution along the downstream transects of the rivers studied was not observed. However, an effect of river flow is observed, i.e. the concentration of the C32 1,15-diol is higher in stagnant waters, such as reservoirs and during seasons with river low stands. A seasonal change in the LCD distribution was observed in the Rhine, likely due to a change in the producers. Eukaryotic diversity analysis by 18S rRNA gene sequencing of SPM from the Rhine showed extremely low abundances of sequences (i.e.

scholarly journals Long-chain diols in rivers: distribution and potential biological sources

2018 ◽  
Vol 15 (13) ◽  
pp. 4147-4161 ◽  
Author(s):  
Julie Lattaud ◽  
Frédérique Kirkels ◽  
Francien Peterse ◽  
Chantal V. Freymond ◽  
Timothy I. Eglinton ◽  
...  
Keyword(s):  
River Flow ◽  
River System ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
River Systems ◽  
Eukaryotic Diversity ◽  
Fractional Abundance ◽  
Biological Sources ◽  
Rrna Gene Sequencing ◽  
Long Chain

Abstract. Long-chain diols (LCDs) occur widespread in marine environments and also in lakes and rivers. Transport of LCDs from rivers may impact the distribution of LCDs in coastal environments, however relatively little is known about the distribution and biological sources of LCDs in river systems. In this study, we investigated the distribution of LCDs in suspended particulate matter (SPM) of three river systems (Godavari, Danube, and Rhine) in relation with precipitation, temperature, and source catchments. The dominant long-chain diol is the C32 1,15-diol followed by the C30 1,15-diol in all studied river systems. In regions influenced by marine waters, such as delta systems, the fractional abundance of the C30 1,15-diol is substantially higher than in the river itself, suggesting different LCD producers in marine and freshwater environments. A change in the LCD distribution along the downstream transects of the rivers studied was not observed. However, an effect of river flow is observed; i.e., the concentration of the C32 1,15-diol is higher in stagnant waters such as reservoirs and during seasons with river low stands. A seasonal change in the LCD distribution was observed in the Rhine, likely due to a change in the producers. Eukaryotic diversity analysis by 18S rRNA gene sequencing of SPM from the Rhine showed extremely low abundances of sequences (i.e., < 0.32 % of total reads) related to known algal LCD producers. Furthermore, incubation of the river water with 13C-labeled bicarbonate did not result in 13C incorporation into LCDs. This indicates that the LCDs present are mainly of fossil origin in the fast-flowing part of the Rhine. Overall, our results suggest that the LCD producers in rivers predominantly reside in lakes or side ponds that are part of the river system.

scholarly journals Revealing the Composition of the Eukaryotic Microbiome of Oyster Spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS)

2021 ◽  
Author(s):  
Kevin Xu Zhong ◽  
Anna Cho ◽  
Christophe M. Deeg ◽  
Amy M. Chan ◽  
Curtis A. Suttle
Keyword(s):  
18S Rrna ◽  
Amplicon Sequencing ◽  
18S Rrna Gene ◽  
Model Organisms ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Oyster Spat ◽  
Guide Rna ◽  
16S Rna ◽  
Eukaryotic Microbes

Abstract BackgroundThe microbiome affects the health of plants and animals, including humans, and has many biological, ecological and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbes this approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped by contaminating host rRNA gene sequences. ResultsTo overcome this problem, we developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS), a high-resolution and efficient approach for characterizing eukaryotic microbiomes. CCSAS uses taxon-specific single-guide RNA (sgRNA) to direct Cas9 to cut 18S rRNA gene sequences of the host, while leaving protistan and fungal sequences intact. We validated the specificity of the sgRNA on ten model organisms and an artificially constructed (mock) community of nine protistan and fungal pathogens. The results showed that >96.5% of host rRNA gene amplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used to assess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community of eukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than other methods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that were not detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryotic microbiome in complex communities. To facilitate the application of CCSAS, we designed taxon-specific sgRNA for ~16,000 metazoan and plant taxa, making CCSAS widely available for characterizing eukaryotic microbiomes that have largely been neglected. ConclusionCCSAS provides a high-through-put and cost-effective approach for resolving the eukaryotic microbiome of metazoa and plants with minimal contamination from host 18S rRNA gene sequences. Keywords: Eukaryotic microbiome, 18S rRNA gene, Microeukaryote, CRISPR-Cas, Taxon-specific single-guide RNA, gRNA-target-site, CasOligo, CCSAS

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 Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS)

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Kevin Xu Zhong ◽  
Anna Cho ◽  
Christoph M. Deeg ◽  
Amy M. Chan ◽  
Curtis A. Suttle
Keyword(s):  
Fungal Pathogens ◽  
18S Rrna ◽  
Amplicon Sequencing ◽  
18S Rrna Gene ◽  
Model Organisms ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Oyster Spat ◽  
16S Rna ◽  
Eukaryotic Microbes

Abstract Background The microbiome affects the health of plants and animals, including humans, and has many biological, ecological, and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbes this approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped by contaminating host rRNA gene sequences. Results To overcome this problem, we developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS), a high-resolution and efficient approach for characterizing eukaryotic microbiomes. CCSAS uses taxon-specific single-guide RNA (sgRNA) to direct Cas9 to cut 18S rRNA gene sequences of the host, while leaving protistan and fungal sequences intact. We validated the specificity of the sgRNA on ten model organisms and an artificially constructed (mock) community of nine protistan and fungal pathogens. The results showed that > 96.5% of host rRNA gene amplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used to assess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community of eukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than other methods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that were not detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryotic microbiome in complex communities. To facilitate the application of CCSAS, we designed taxon-specific sgRNA for ~16,000 metazoan and plant taxa, making CCSAS widely available for characterizing eukaryotic microbiomes that have largely been neglected. Conclusion CCSAS provides a high-through-put and cost-effective approach for resolving the eukaryotic microbiome of metazoa and plants with minimal contamination from host 18S rRNA gene sequences.

scholarly journals Microbial communities developing within bulk sediments under fish carcasses on a tidal flat

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247220
Author(s):  
Yasutake Kawamoto ◽  
Hiromi Kato ◽  
Yuji Nagata ◽  
Jotaro Urabe
Keyword(s):  
Tidal Flat ◽  
Indirect Effects ◽  
18S Rrna ◽  
Amplicon Sequencing ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Direct Effects ◽  
Tidal Zone ◽  
Operational Taxonomic Units ◽  
Bacterial Assemblages

Animal carcasses are often brought into tidal flats where they are at the boundary between terrestrial and marine ecosystems. Since these carcasses act as microhabitats with large amounts of energy and nutrients, they likely develop unique bacterial assemblages in the ambient sediment, which in turn may stimulate colonization of other organisms such as protozoans. However, little is known about the microbial assemblages colonized in sediment around animal carcasses in the tidal zone. Herein we examined the bacterial and ciliophoran assemblages developed in association with fish carcasses by incubating the carcasses in the Higashiyachi tidal flat (Sendai, Japan). We collected sediment samples at 2, 9, and 42 days of incubation and analyzed the bacterial and ciliophoran assemblages by 16S and 18S rRNA gene amplicon sequencing. We observed significant differences in the composition and relative abundance of bacterial and ciliophoran operational taxonomic units (OTUs) between the sediments with and without the carcasses. Our analyses suggest that these unique assemblages were created through the direct effects of the carcass and indirect effects through interactions between bacteria and ciliophorans. These results also suggest that animal carcasses developed a temporally unique microbial food web in the sediments close to the carcasses, although it disappeared for several weeks.

scholarly journals Analytical validation of a real-time hydrolysis probe PCR assay for quantifying Plasmodium falciparum parasites in experimentally infected human adults

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Claire Y. T. Wang ◽  
Emma L. Ballard ◽  
Zuleima Pava ◽  
Louise Marquart ◽  
Jane Gaydon ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Plasmodium Falciparum ◽  
18S Rrna ◽  
Drug Efficacy ◽  
18S Rrna Gene ◽  
Molecular Techniques ◽  
Qpcr Assay ◽  
Rrna Gene ◽  
Analytical Sensitivity ◽  
Blood Samples

Abstract Background Volunteer infection studies have become a standard model for evaluating drug efficacy against Plasmodium infections. Molecular techniques such as qPCR are used in these studies due to their ability to provide robust and accurate estimates of parasitaemia at increased sensitivity compared to microscopy. The validity and reliability of assays need to be ensured when used to evaluate the efficacy of candidate drugs in clinical trials. Methods A previously described 18S rRNA gene qPCR assay for quantifying Plasmodium falciparum in blood samples was evaluated. Assay performance characteristics including analytical sensitivity, reportable range, precision, accuracy and specificity were assessed using experimental data and data compiled from phase 1 volunteer infection studies conducted between 2013 and 2019. Guidelines for validation of laboratory-developed molecular assays were followed. Results The reportable range was 1.50 to 6.50 log10 parasites/mL with a limit of detection of 2.045 log10 parasites/mL of whole blood based on a parasite diluted standard series over this range. The assay was highly reproducible with minimal intra-assay (SD = 0.456 quantification cycle (Cq) units [0.137 log10 parasites/mL] over 21 replicates) and inter-assay (SD = 0.604 Cq units [0.182 log10 parasites/mL] over 786 qPCR runs) variability. Through an external quality assurance program, the QIMR assay was shown to generate accurate results (quantitative bias + 0.019 log10 parasites/mL against nominal values). Specificity was 100% after assessing 164 parasite-free human blood samples. Conclusions The 18S rRNA gene qPCR assay is specific and highly reproducible and can provide reliable and accurate parasite quantification. The assay is considered fit for use in evaluating drug efficacy in malaria clinical trials.

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