scholarly journals Use of universal primers for the 18S ribosomal RNA gene and whole soil DNAs to reveal the taxonomic structures of soil nematodes by high-throughput amplicon sequencing

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259842
Author(s):  
Harutaro Kenmotsu ◽  
Emi Takabayashi ◽  
Akinori Takase ◽  
Yuu Hirose ◽  
Toshihiko Eki
Keyword(s):  
High Throughput ◽  
Ribosomal Rna ◽  
Amplicon Sequencing ◽  
Pcr Primers ◽  
Universal Primers ◽  
Soil Nematode ◽  
Soil Nematodes ◽  
18S Ribosomal Rna ◽  
Nematode Communities ◽  
Taxonomic Profiling

Nematodes are abundant metazoans that play crucial roles in nutrient recycle in the pedosphere. Although high-throughput amplicon sequencing is a powerful tool for the taxonomic profiling of soil nematodes, polymerase chain reaction (PCR) primers for amplification of the 18S ribosomal RNA (SSU) gene and preparation of template DNAs have not been sufficiently evaluated. We investigated nematode community structure in copse soil using four nematode-specific (regions 1–4) and two universal (regions U1 and U2) primer sets for the SSU gene regions with two DNAs prepared from copse-derived mixed nematodes and whole soil. The major nematode-derived sequence variants (SVs) identified in each region was detected in both template DNAs. Order level taxonomy and feeding type of identified nematode-derived SVs were distantly related between the two DNA preparations, and the region U2 was closely related to region 4 in the non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarity. Thus, the universal primers for region U2 could be used to analyze soil nematode communities. We further applied this method to analyze the nematodes living in two sampling sites of a sweet potato-cultivated field, where the plants were differently growing. The structure of nematode-derived SVs from the two sites was distantly related in the principal coordinate analysis (PCoA) with weighted unifrac distances, suggesting their distinct soil environments. The resultant ecophysiological status of the nematode communities in the copse and field on the basis of feeding behavior and maturity indices was fairly consistent with those of the copse- and the cultivated house garden-derived nematodes in prior studies. These findings will be useful for the DNA metabarcoding of soil eukaryotes, including nematodes, using soil DNAs.

scholarly journals Distinct community structures of soil nematodes from three ecologically different sites revealed by high-throughput amplicon sequencing of four 18S ribosomal RNA gene regions

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0249571
Author(s):  
Harutaro Kenmotsu ◽  
Masahiro Ishikawa ◽  
Tomokazu Nitta ◽  
Yuu Hirose ◽  
Toshihiko Eki
Keyword(s):  
High Throughput ◽  
Ribosomal Rna ◽  
Amplicon Sequencing ◽  
Garden Soil ◽  
Soil Nematode ◽  
Field Soil ◽  
Ribosomal Rna Gene ◽  
Nematode Communities ◽  
18S Ribosomal Rna Gene ◽  
Dna Metabarcoding

Quantitative taxonomic compositions of nematode communities help to assess soil environments due to their rich abundance and various feeding habitats. DNA metabarcoding by the 18S ribosomal RNA gene (SSU) regions were preferentially used for analyses of soil nematode communities, but the optimal regions for high-throughput amplicon sequencing have not previously been well investigated. In this work, we performed Illumina-based amplicon sequencing of four SSU regions (regions 1–4) to identify suitable regions for nematode metabarcoding using the taxonomic structures of nematodes from uncultivated field, copse, and cultivated house garden soils. The fewest nematode-derived sequence variants (SVs) were detected in region 3, and the total nematode-derived SVs were comparable in regions 1 and 4. The relative abundances of reads in regions 1 and 4 were consistent in both orders and feeding groups with prior studies, thus suggesting that region 4 is a suitable target for the DNA barcoding of nematode communities. Distinct community structures of nematodes were detected in the taxon, feeding habitat, and life-history strategy of each sample; i.e., Dorylamida- and Rhabditida-derived plant feeders were most abundant in the copse soil, Rhabditida-derived bacteria feeders in the house garden soil, and Mononchida- and Dorylamida-derived omnivores and predators and Rhabditida-derived bacteria feeders in the field soil. Additionally, low- and high-colonizer–persister (cp) groups of nematodes dominated in the house garden and copse soils, respectively, whereas both groups were found in the field soil, suggesting bacteria-rich garden soil, undisturbed and plant-rich copse soil, and a transient status of nematode communities in the field soil. These results were also supported by the maturity indices of the three sampling sites. Finally, the influence of the primer tail sequences was demonstrated to be insignificant on amplification. These findings will be useful for DNA metabarcoding of soil nematode communities by amplicon sequencing.

scholarly journals Distribution of nematodes in wetland soils with difference distance from the Bohai sea 359–366

2008 ◽  
Vol 54 (No. 8) ◽  
pp. 359-366 ◽  
Author(s):  
H.Y. Wu ◽  
X.X. Li ◽  
L.B. Shi ◽  
Z.H. Wang ◽  
F.Y. Ma
Keyword(s):  
Community Structure ◽  
Bohai Sea ◽  
Coastal Wetland ◽  
Yellow River ◽  
Nematode Community ◽  
Soil Nematode ◽  
Soil Nematodes ◽  
Nematode Communities ◽  
Plant Parasitism ◽  
Soil Nematode Communities

In order to elucidate the distribution of soil nematodes in coastal wetlands and the effect of different distance from the sea line on soil nematode communities, we investigated the community structure of soil nematodes in one wetland perpendicularly oriented from Bohai sea coastline. In June 2006, soil samples were collected from the Yellow River Delta wetlands, in Dongying city of Shandong Province, China. Soil nematode communities were analyzed at the depths of 0–10 and 10–20 cm. The results showed that plant parasite nematodes were the most abundant trophic groups in both depths and at four sites. The average relative abundance was 91.33% of the nematode community. Several ecological indices which reflected soil nematode community structure, diversity, maturity and plant parasitism were compared in these four sites. The results indicated that the maturity index (MI) and plant parasitism index (PPI) were more sensitive than the other indices for assessing the response of soil nematode communities to soil of coastal wetland.

scholarly journals Effect of transgenic insect-resistant maize to the community structure of soil nematodes in two field trials

2015 ◽  
Vol 52 (1) ◽  
pp. 41-49
Author(s):  
A. Čerevková ◽  
L Cagáň
Keyword(s):  
Community Structure ◽  
Food Web ◽  
Field Trials ◽  
Soil Nematode ◽  
Soil Nematodes ◽  
Soil Food Web ◽  
Bt Maize ◽  
Maize Hybrid ◽  
Nematode Communities ◽  
Soil Nematode Communities

Summary The aim of this study was to determine the effects of Bt maize hybrid cultivation on soil nematode communities in two field trials, as well as to analyse other factors (fertilisation and moisture) responsible for the community structure of soil nematodes. Nematode communities were studied in maize plots at the locality of Borovce in western Slovakia. During 2012 and 2013, hybrids DK440 and DKC3871 (conventional) or DKC4442YG and DKC3872YG (Bt maize, event MON810) were sown in 10 repetitions each. Nematodes were extracted from soil samples collected at the maize flowering (July 11, 2012 and July 30, 2013). Altogether, 39 nematode species belonged to 35 genera were identified in two maize variants. The dominant taxa in both variants were Acrobeloides nanus, Ce-phalobus persegnis, Aphelenchoides composticola, Aphelenchus avenae, Eudorylaimus carteri and Filenchus vulgaris. Calculation of the maturity index, plant parasitic index, enrichment index and structure index did not confirm any clear influence of year or hybrid type on soil nematode communities. The proportional representation of cp-1, cp-2 and cp-3-5 groups of nematode fauna indicated conditions of low stability and high stress. Faunal profiles representing the structure and enrichment conditions of the soil food web showed an environment with a high C:N ratio and high levels of fungal feeders. Based on the calculation of the metabolic footprint of nematodes in the soil food web, a difference between the isoline maize variant and Bt maize variant in 2012 was found, but this difference was not readily apparent in 2013. The occurrence of nematodes, their abundance, proportion of feeding types and selected ecological indices did not depend on the type of maize hybrid (Bt or non-Bt). Thus, the cultivation of genetically modified maize did not directly influence nematode populations. The application of fertiliser at certain periods does not influence the nematode community. The observed significant higher abundance of nematodes was correlated with soil moisture.

scholarly journals Nasopharyngeal Microbiota in SARS-CoV-2 Positive and Negative Patients

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Phillip A. Engen ◽  
Ankur Naqib ◽  
Cheryl Jennings ◽  
Stefan J. Green ◽  
Alan Landay ◽  
...  
Keyword(s):  
Community Structure ◽  
Microbial Community ◽  
Disease Progression ◽  
High Throughput ◽  
Ribosomal Rna ◽  
Microbial Community Structure ◽  
Amplicon Sequencing ◽  
Proof Of Concept ◽  
Viral Transport ◽  
16S Ribosomal Rna Gene

AbstractWe investigated nasopharyngeal microbial community structure in COVID-19-positive and -negative patients. High-throughput 16S ribosomal RNA gene amplicon sequencing revealed significant microbial community structure differences between COVID-19-positive and -negative patients. This proof-of-concept study demonstrates that: (1) nasopharyngeal microbiome communities can be assessed using collection samples already collected for SARS-CoV-2 testing (viral transport media) and (2) SARS-CoV-2 infection is associated with altered dysbiotic microbial profiles which could be a biomarker for disease progression and prognosis in SARS-CoV-2.

scholarly journals Large-Scale Patterns of Soil Nematodes across Grasslands on the Tibetan Plateau: Relationships with Climate, Soil and Plants

Diversity ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 369
Author(s):  
Han Chen ◽  
Shuaiwei Luo ◽  
Guixin Li ◽  
Wanyanhan Jiang ◽  
Wei Qi ◽  
...  
Keyword(s):  
Tibetan Plateau ◽  
Large Scale ◽  
The Tibetan Plateau ◽  
Soil Nematode ◽  
Soil Nematodes ◽  
Soil Factors ◽  
Alpine Meadows ◽  
Nematode Communities ◽  
Soil Nematode Community ◽  
Genus Richness

Soil nematodes are important contributors to soil biodiversity. Nonetheless, the distribution patterns and environmental drivers of soil nematode communities are poorly understood, especially at the large scale, where multiple environmental variables covary. We collected 520 soil samples from 104 sites representing alpine meadow and steppe ecosystems. First, we explored the soil nematode community characteristics and compared community patterns between the ecosystems. Then, we examined the contributions of aboveground and belowground factors on these patterns. The genus richness and abundance of nematodes on the Tibetan Plateau are lower than other alpine ecosystems, but are comparable to desert or polar ecosystems. Alpine meadows supported a higher nematode abundance and genus richness than alpine steppes; bacterial-based energy channels were pre-dominant in both the ecosystems. Soil factors explained the most variation in the soil nematode community composition in the alpine meadows, while plant factors were as essential as soil factors in the alpine steppes. Unexpectedly, the climate variables barely impacted the nematode communities. This is the first study to explore the spatial patterns of soil nematode compositions on the Tibetan Plateau, and we found that the contributions of climate, plants, and soil properties on soil nematodes community were essentially different from the previous knowledge for well-studied plant and animal communities.

scholarly journals The effect of reverse transcription enzymes and conditions on high throughput amplicon sequencing of the 16S rRNA

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7608
Author(s):  
Adam Šťovíček ◽  
Smadar Cohen-Chalamish ◽  
Osnat Gillor
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
Ribosomal Rna ◽  
Reverse Transcription ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Amplicon Sequencing ◽  
Soil Microbiology ◽  
Active Population ◽  
Dna And Rna

It is assumed that the sequencing of ribosomes better reflects the active microbial community than the sequencing of the ribosomal RNA encoding genes. Yet, many studies exploring microbial communities in various environments, ranging from the human gut to deep oceans, questioned the validity of this paradigm due to the discrepancies between the DNA and RNA based communities. Here, we focus on an often neglected key step in the analysis, the reverse transcription (RT) reaction. Previous studies showed that RT may introduce biases when expressed genes and ribosmal rRNA are quantified, yet its effect on microbial diversity and community composition was never tested. High throughput sequencing of ribosomal RNA is a valuable tool to understand microbial communities as it better describes the active population than DNA analysis. However, the necessary step of RT may introduce biases that have so far been poorly described. In this manuscript, we compare three RT enzymes, commonly used in soil microbiology, in two temperature modes to determine a potential source of bias due to non-standardized RT conditions. In our comparisons, we have observed up to six fold differences in bacterial class abundance. A temperature induced bias can be partially explained by G-C content of the affected bacterial groups, thus pointing toward a need for higher reaction temperatures. However, another source of bias was due to enzyme processivity differences. This bias is potentially hard to overcome and thus mitigating it might require the use of one enzyme for the sake of cross-study comparison.

scholarly journals The effect of reverse transcription enzymes and conditions on high throughput amplicon sequencing of the 16S rRNA

2019 ◽  
Author(s):  
Adam Šťovíček ◽  
Smadar Cohen-Chalamish ◽  
Osnat Gillor
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
Ribosomal Rna ◽  
Reverse Transcription ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Amplicon Sequencing ◽  
Soil Microbiology ◽  
Active Population ◽  
Dna And Rna

It is assumed that the sequencing of ribosomes better reflects the active microbial community than the sequencing of the ribosomal RNA encoding genes. Yet, many studies exploring microbial communities in various environments, ranging from the human gut to deep oceans, questioned the validity of this paradigm due to the discrepancies between the DNA and RNA based communities. Here we focus on an often neglected key step in the analysis, the reverse transcription (RT) reaction. Previous studies showed that RT may introduce biases when expressed genes and ribosmal rRNA are quantified, yet its effect on microbial diversity and community composition was never tested. High throughput sequencing of ribosomal RNA is a valuable tool to understand microbial communities as it better describes the active population than DNA analysis. However, the necessary step of RT may introduce biases that have so far been poorly described. In this manuscript, we compare three RT enzymes, commonly used in soil microbiology, in two temperature modes to determine a potential source of bias due to non-standardized RT conditions. In our comparisons, we have observed up to 6 fold differences in bacterial class abundance. A temperature induced bias can be partially explained by G-C content of the affected bacterial groups, thus pointing towards a need for higher reaction temperatures. However, another source of bias was due to enzyme processivity differences. This bias is potentially hard to overcome and thus mitigating it might require the use of one enzyme for the sake of cross-study comparison.

scholarly journals The effect of reverse transcription enzymes and conditions on high throughput amplicon sequencing of the 16S rRNA

2019 ◽  
Author(s):  
Adam Šťovíček ◽  
Smadar Cohen-Chalamish ◽  
Osnat Gillor
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
Ribosomal Rna ◽  
Reverse Transcription ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Amplicon Sequencing ◽  
Soil Microbiology ◽  
Active Population ◽  
Dna And Rna

It is assumed that the sequencing of ribosomes better reflects the active microbial community than the sequencing of the ribosomal RNA encoding genes. Yet, many studies exploring microbial communities in various environments, ranging from the human gut to deep oceans, questioned the validity of this paradigm due to the discrepancies between the DNA and RNA based communities. Here we focus on an often neglected key step in the analysis, the reverse transcription (RT) reaction. Previous studies showed that RT may introduce biases when expressed genes and ribosmal rRNA are quantified, yet its effect on microbial diversity and community composition was never tested. High throughput sequencing of ribosomal RNA is a valuable tool to understand microbial communities as it better describes the active population than DNA analysis. However, the necessary step of RT may introduce biases that have so far been poorly described. In this manuscript, we compare three RT enzymes, commonly used in soil microbiology, in two temperature modes to determine a potential source of bias due to non-standardized RT conditions. In our comparisons, we have observed up to 6 fold differences in bacterial class abundance. A temperature induced bias can be partially explained by G-C content of the affected bacterial groups, thus pointing towards a need for higher reaction temperatures. However, another source of bias was due to enzyme processivity differences. This bias is potentially hard to overcome and thus mitigating it might require the use of one enzyme for the sake of cross-study comparison.

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