DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms

2016 ◽  
pp. 57-74 ◽  
Author(s):  
Eleanor Jameson ◽  
Martin Taubert ◽  
Sara Coyotzi ◽  
Yin Chen ◽  
Özge Eyice ◽  
...  
Keyword(s):  
Stable Isotope ◽  
High Throughput ◽  
Stable Isotope Probing ◽  
Biomarker Analysis

scholarly journals Identifying the Active Microbiome Associated with Roots and Rhizosphere Soil of Oilseed Rape

2017 ◽  
Vol 83 (22) ◽  
Author(s):  
Konstantia Gkarmiri ◽  
Shahid Mahmood ◽  
Alf Ekblad ◽  
Sadhna Alström ◽  
Nils Högberg ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Stable Isotope ◽  
Oilseed Rape ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Rhizosphere Soil ◽  
Stable Isotope Probing ◽  
Biofuel Production ◽  
Content Type ◽  
Bacteria And Fungi

ABSTRACT RNA stable isotope probing and high-throughput sequencing were used to characterize the active microbiomes of bacteria and fungi colonizing the roots and rhizosphere soil of oilseed rape to identify taxa assimilating plant-derived carbon following 13CO2 labeling. Root- and rhizosphere soil-associated communities of both bacteria and fungi differed from each other, and there were highly significant differences between their DNA- and RNA-based community profiles. Verrucomicrobia, Proteobacteria, Planctomycetes, Acidobacteria, Gemmatimonadetes, Actinobacteria, and Chloroflexi were the most active bacterial phyla in the rhizosphere soil. Bacteroidetes were more active in roots. The most abundant bacterial genera were well represented in both the 13C- and 12C-RNA fractions, while the fungal taxa were more differentiated. Streptomyces, Rhizobium, and Flavobacterium were dominant in roots, whereas Rhodoplanes and Sphingomonas (Kaistobacter) were dominant in rhizosphere soil. “Candidatus Nitrososphaera” was enriched in 13C in rhizosphere soil. Olpidium and Dendryphion were abundant in the 12C-RNA fraction of roots; Clonostachys was abundant in both roots and rhizosphere soil and heavily 13C enriched. Cryptococcus was dominant in rhizosphere soil and less abundant, but was 13C enriched in roots. The patterns of colonization and C acquisition revealed in this study assist in identifying microbial taxa that may be superior competitors for plant-derived carbon in the rhizosphere of Brassica napus. IMPORTANCE This microbiome study characterizes the active bacteria and fungi colonizing the roots and rhizosphere soil of Brassica napus using high-throughput sequencing and RNA-stable isotope probing. It identifies taxa assimilating plant-derived carbon following 13CO2 labeling and compares these with other less active groups not incorporating a plant assimilate. Brassica napus is an economically and globally important oilseed crop, cultivated for edible oil, biofuel production, and phytoextraction of heavy metals; however, it is susceptible to several diseases. The identification of the fungal and bacterial species successfully competing for plant-derived carbon, enabling them to colonize the roots and rhizosphere soil of this plant, should enable the identification of microorganisms that can be evaluated in more detailed functional studies and ultimately be used to improve plant health and productivity in sustainable agriculture.

scholarly journals Novel bacteria capable of degrading phenanthrene in activated sludge revealed by stable-isotope probing coupled with high-throughput sequencing

2017 ◽  
Vol 28 (5-6) ◽  
pp. 423-436 ◽  
Author(s):  
Jibing Li ◽  
Dayi Zhang ◽  
Mengke Song ◽  
Longfei Jiang ◽  
Yujie Wang ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Activated Sludge ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Stable Isotope Probing ◽  
Novel Bacteria

Ultra-high-sensitivity stable-isotope probing of rRNA by high-throughput sequencing of isopycnic centrifugation gradients

2015 ◽  
Vol 7 (2) ◽  
pp. 282-287 ◽  
Author(s):  
Tomo Aoyagi ◽  
Satoshi Hanada ◽  
Hideomi Itoh ◽  
Yuya Sato ◽  
Atsushi Ogata ◽  
...  
Keyword(s):  
Stable Isotope ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
High Sensitivity ◽  
Stable Isotope Probing ◽  
Isopycnic Centrifugation

Assessing fungal contributions to cellulose degradation in soil by using high-throughput stable isotope probing

2019 ◽  
Vol 130 ◽  
pp. 150-158 ◽  
Author(s):  
Chantal Koechli ◽  
Ashley N. Campbell ◽  
Charles Pepe-Ranney ◽  
Daniel H. Buckley
Keyword(s):  
Stable Isotope ◽  
High Throughput ◽  
Cellulose Degradation ◽  
Stable Isotope Probing ◽  
Degradation In Soil

scholarly journals Evaluating the accuracy of DNA stable isotope probing

2017 ◽  
Author(s):  
Nicholas D. Youngblut ◽  
Daniel H. Buckley
Keyword(s):  
Stable Isotope ◽  
Dna Sequencing ◽  
High Throughput ◽  
False Positives ◽  
Stable Isotope Probing ◽  
Detection Sensitivity ◽  
False Negatives ◽  
Isotope Incorporation ◽  
High Throughput Dna Sequencing

Originality-Significance StatementBy combining DNA Stable Isotope Probing (DNA-SIP) with multiplexed high throughput DNA sequencing (HTS-DNA-SIP), it is now possible to identify patterns of isotope incorporation for thousands of microbial taxa. HTS-DNA-SIP has enormous potential to reveal patterns of carbon and nitrogen exchange within microbial food webs. A current limitation is that, due to the expense of these experiments, it has been impossible to evaluate the accuracy of DNA-SIP methods. We have developed a model that simulates DNA-SIP data, and we use the model to systematically evaluate and validate the accuracy of DNA-SIP analyses. This model can determine the analytical accuracy of DNA-SIP experiments in a range of contexts. Furthermore, the ability to predict experimental outcomes, as a function of experimental design and community characteristics, should be of great use in the design and interpretation DNA-SIP experiments.SummaryDNA Stable isotope probing (DNA-SIP) is a powerful method that identifiesin situisotope assimilation by microbial taxa. Combining DNA-SIP with multiplexed high throughput DNA sequencing (HTS-DNA-SIP) creates the potential to mapin situassimilation dynamics for thousands of microbial taxonomic units. However, the accuracy of methods for analyzing DNA-SIP data has never been evaluated. We have developed a toolset (SIPSim) for simulating HTS-DNA-SIP datasets and evaluating the accuracy of methods for analyzing HTS-DNA-SIP data. We evaluated two different approaches to analyzing HTS-DNA-SIP data: “high resolution stable isotope probing” (HR-SIP) and “quantitative stable isotope probing” (q-SIP). HR-SIP was highly specific and moderately sensitive, with very few false positives but potential for false negatives. In contrast, q-SIP had fewer false negatives but many false positives. We also found HR-SIP more robust than q-SIP with respect to experimental variance. Furthermore, we found that the detection sensitivity of HTS-DNA-SIP can be increased without compromising specificity by evaluating evidence of isotope incorporation over multiple windows of buoyant density (MW-HR-SIP). SIPSim provides a platform for determining the accuracy of HTS-DNA-SIP methods across a range of experimental parameters, which will be useful in the design, analysis, and validation of DNA-SIP experiments.

scholarly journals HTSSIP: an R package for analysis of high throughput sequencing data from nucleic acid stable isotope probing (SIP) experiments

2017 ◽  
Author(s):  
Nicholas D. Youngblut ◽  
Samuel E. Barnett ◽  
Daniel H. Buckley
Keyword(s):  
High Resolution ◽  
Stable Isotope ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
R Package ◽  
Stable Isotope Probing ◽  
Sequencing Data ◽  
Metabolic Processes ◽  
Link Type ◽  
High Throughput Sequencing Data

AbstractCombining high throughput sequencing with stable isotope probing (HTS-SIP) is a powerful method for mapping in situ metabolic processes to thousands of microbial taxa. However, accurately mapping metabolic processes to taxa is complex and challenging. Multiple HTS-SIP data analysis methods have been developed, including high-resolution stable isotope probing (HR-SIP), multi-window high-resolution stable isotope probing (MW-HR-SIP), quantitative stable isotope probing (q-SIP), and ΔBD. Currently, the computational tools to perform these analyses are either not publicly available or lack documentation, testing, and developer support. To address this shortfall, we have developed the HTSSIP R package, a toolset for conducting HTS-SIP analyses in a straightforward and easily reproducible manner. The HTSSIP package, along with full documentation and examples, is available from CRAN at https://cran.r-project.org/web/packages/HTSSIP/index.html and Github at https://github.com/nick-youngblut/HTSSIP.

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