Employment of Near Full-Length Ribosome Gene TA-Cloning and Primer-Blast to Detect Multiple Species in a Natural Complex Microbial Community Using Species-Specific Primers Designed with Their Genome Sequences

2016 ◽  
Vol 58 (11) ◽  
pp. 729-737 ◽  
Author(s):  
Huimin Zhang ◽  
Hongkui He ◽  
Xiujuan Yu ◽  
Zhaohui Xu ◽  
Zhizhou Zhang
Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Benjamin J. Callahan ◽  
Dmitry Grinevich ◽  
Siddhartha Thakur ◽  
Michael A. Balamotis ◽  
Tuval Ben Yehezkel

Abstract Background Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Methods Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. Results LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. Conclusions The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.


2021 ◽  
Author(s):  
Cecilia Di Bernardi ◽  
Camilla Wikenros ◽  
Eva Hedmark ◽  
Luigi Boitani ◽  
Paolo Ciucci ◽  
...  

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Michelle Nordkvist ◽  
Maartje J. Klapwijk ◽  
La rs Edenius ◽  
Christer Björkman

AbstractMost plants are subjected to damage from multiple species of herbivores, and the combined impact on plant growth can be non-additive. Since plant response to herbivores tends to be species specific, and change with repeated damage, the outcome likely depend on the sequence and number of attacks. There is a high likelihood of non-additive effects on plant growth by damage from mammals and insects, as mammalian herbivory can alter insect herbivore damage levels, yet few studies have explored this. We report the growth response of young Scots pine trees to sequential mammal and insect herbivory, varying the sequence and number of damage events, using an ungulate-pine-sawfly system. Combined sawfly and ungulate herbivory had both additive and non-additive effects on pine growth—the growth response depended on the combination of ungulate browsing and sawfly defoliation (significant interaction effect). Repeated sawfly herbivory reduced growth (compared to single defoliation) on un-browsed trees. However, on browsed trees, depending on when sawfly defoliation was combined with browsing, trees exposed to repeated sawfly herbivory had both higher, lower and the same growth as trees exposed to a single defoliation event. We conclude that the sequence of attacks by multiple herbivores determine plant growth response.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
María-Eugenia DeCastro ◽  
Michael P. Doane ◽  
Elizabeth Ann Dinsdale ◽  
Esther Rodríguez-Belmonte ◽  
María-Isabel González-Siso

AbstractIn the present study we investigate the microbial community inhabiting As Burgas geothermal spring, located in Ourense (Galicia, Spain). The approximately 23 Gbp of Illumina sequences generated for each replicate revealed a complex microbial community dominated by Bacteria in which Proteobacteria and Aquificae were the two prevalent phyla. An association between the two most prevalent genera, Thermus and Hydrogenobacter, was suggested by the relationship of their metabolism. The high relative abundance of sequences involved in the Calvin–Benson cycle and the reductive TCA cycle unveils the dominance of an autotrophic population. Important pathways from the nitrogen and sulfur cycle are potentially taking place in As Burgas hot spring. In the assembled reads, two complete ORFs matching GH2 beta-galactosidases were found. To assess their functional characterization, the two ORFs were cloned and overexpressed in E. coli. The pTsbg enzyme had activity towards o-Nitrophenyl-β-d-galactopyranoside (ONPG) and p-Nitrophenyl-β-d-fucopyranoside, with high thermal stability and showing maximal activity at 85 °C and pH 6, nevertheless the enzyme failed to hydrolyze lactose. The other enzyme, Tsbg, was unable to hydrolyze even ONPG or lactose. This finding highlights the challenge of finding novel active enzymes based only on their sequence.


2017 ◽  
Vol 65 (4) ◽  
pp. 327 ◽  
Author(s):  
Saskia Grootemaat ◽  
Ian J. Wright ◽  
Peter M. van Bodegom ◽  
Johannes H. C. Cornelissen ◽  
Veronica Shaw

Bark shedding is a remarkable feature of Australian trees, yet relatively little is known about interspecific differences in bark decomposability and flammability, or what chemical or physical traits drive variation in these properties. We measured the decomposition rate and flammability (ignitibility, sustainability and combustibility) of bark from 10 common forest tree species, and quantified correlations with potentially important traits. We compared our findings to those for leaf litter, asking whether the same traits drive flammability and decomposition in different tissues, and whether process rates are correlated across tissue types. Considerable variation in bark decomposability and flammability was found both within and across species. Bark decomposed more slowly than leaves, but in both tissues lignin concentration was a key driver. Bark took longer to ignite than leaves, and had longer mass-specific flame durations. Variation in flammability parameters was driven by different traits in the different tissues. Decomposability and flammability were each unrelated, when comparing between the different tissue types. For example, species with fast-decomposing leaves did not necessarily have fast-decomposing bark. For the first time, we show how patterns of variation in decomposability and flammability of bark diverge across multiple species. By taking species-specific bark traits into consideration there is potential to make better estimates of wildfire risks and carbon loss dynamics. This can lead to better informed management decisions for Australian forests, and eucalypt plantations, worldwide.


Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.


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