scholarly journals Assessing and Maximizing Cultivated Diversity with Plate-Wash PCR and High Throughput Sequencing

2020 ◽  
Author(s):  
Emily N. Junkins ◽  
Bradley S. Stevenson
Keyword(s):  
High Throughput ◽  
Drug Targets ◽  
High Throughput Sequencing ◽  
Multidrug Resistant ◽  
Molecular Techniques ◽  
Bioactive Metabolites ◽  
Plate Count ◽  
Molecular Tools ◽  
Vast Number ◽  
Sequencing Studies

AbstractMolecular techniques continue to reveal a growing disparity between the immense diversity of microbial life and the small proportion that is in pure culture. The disparity, originally dubbed “the great plate count anomaly” by Staley and Konopka, has become even more vexing given our increased understanding of the importance of microbiomes to a host and the role of microorganisms in the vital biogeochemical functions of our biosphere. Searching for novel antimicrobial drug targets often focuses on screening a broad diversity of microorganisms. If diverse microorganisms are to be screened, they need to be cultivated. Recent innovative research has used molecular techniques to assess the efficacy of cultivation efforts, providing invaluable feedback to cultivation strategies for isolating targeted and/or novel microorganisms. Here, we aimed to determine the efficiency of cultivating representative microorganisms from a non-human, mammalian microbiome, identify those microorganisms, and determine the bioactivity of isolates. Molecular methods indicated that around 57% of the ASVs detected in the original inoculum were cultivated in our experiments, but nearly 53% of the total ASVs that were present in our cultivation experiments were not detected in the original inoculum. In light of our controls, our data suggests that when molecular tools were used to characterize our cultivation efforts, they provided a more complete, albeit more complex, understanding of which organisms were present compared to what was eventually cultivated. Lastly, about 3% of the isolates collected from our cultivation experiments showed inhibitory bioactivity against a multidrug-resistant pathogen panel, further highlighting the importance of informing and directing future cultivation efforts with molecular tools.ImportanceCultivation is the definitive tool to understand a microorganism’s physiology, metabolism, and ecological role(s). Despite continuous efforts to hone this skill, researchers are still observing yet-to-be cultivated organisms through high-throughput sequencing studies. Here, we use the very same tool that highlights biodiversity to assess cultivation efficiency. When applied to drug discovery, where screening a vast number of isolates for bioactive metabolites is common, cultivating redundant organisms is a hindrance. However, we observed that cultivating in combination with molecular tools can expand the observed diversity of an environment and its community, potentially increasing the number of microorganisms to be screened for natural products.

scholarly journals Using Plate-Wash PCR and High-Throughput Sequencing to Measure Cultivated Diversity for Natural Product Discovery Efforts

2021 ◽  
Vol 12 ◽  
Author(s):  
Emily N. Junkins ◽  
Bradley S. Stevenson
Keyword(s):  
Natural Product ◽  
High Throughput ◽  
Drug Targets ◽  
High Throughput Sequencing ◽  
Multidrug Resistant ◽  
Molecular Techniques ◽  
Plate Count ◽  
Molecular Tools ◽  
Microbial Life

Molecular techniques continue to reveal a growing disparity between the immense diversity of microbial life and the small proportion that is in pure culture. The disparity, originally dubbed “the great plate count anomaly” by Staley and Konopka, has become even more vexing given our increased understanding of the importance of microbiomes to a host and the role of microorganisms in the vital biogeochemical functions of our biosphere. Searching for novel antimicrobial drug targets often focuses on screening a broad diversity of microorganisms. If diverse microorganisms are to be screened, they need to be cultivated. Recent innovative research has used molecular techniques to assess the efficacy of cultivation efforts, providing invaluable feedback to cultivation strategies for isolating targeted and/or novel microorganisms. Here, we aimed to determine the efficiency of cultivating representative microorganisms from a non-human, mammalian microbiome, identify those microorganisms, and determine the bioactivity of isolates. Sequence-based data indicated that around 57% of the ASVs detected in the original inoculum were cultivated in our experiments, but nearly 53% of the total ASVs that were present in our cultivation experiments were not detected in the original inoculum. In light of our controls, our data suggests that when molecular tools were used to characterize our cultivation efforts, they provided a more complete and more complex, understanding of which organisms were present compared to what was eventually detected during cultivation. Lastly, about 3% of the isolates collected from our cultivation experiments showed inhibitory bioactivity against an already multidrug-resistant pathogen panel, further highlighting the importance of informing and directing future cultivation efforts with molecular tools.

scholarly journals Uncovering fungal community composition in natural habitat of Ophiocordyceps sinensis using high-throughput sequencing and culture-dependent approaches

2020 ◽  
Author(s):  
Chuanbo Zhang ◽  
Chao-Hui Ren ◽  
Yan-Li Wang ◽  
Qi-Qi Wang ◽  
Yun-Sheng Wang ◽  
...  
Keyword(s):  
Community Structure ◽  
High Throughput ◽  
Fungal Community ◽  
High Throughput Sequencing ◽  
Fungal Communities ◽  
Bioactive Metabolites ◽  
Fungal Community Structure ◽  
Ophiocordyceps Sinensis ◽  
Culturable Fungi ◽  
Culture Dependent

Abstract Background The fungal communities inhabiting natural Ophiocordyceps sinensis play critical ecological roles in alpine meadow ecosystem, contribute to infect host insect, influence the occurrence of O. sinensis, and are repertoire of potential novel metabolites discovery. However, a comprehensive understanding of fungal communities of O. sinensis remain elusive. Therefore, the present study aimed to unravel fungal communities of natural O. sinensis using combination of high-throughput sequencing and culture-dependent approach. Results A total of 280,519 high-quality sequences, belonging to 5 fungal phyla, 15 classes, 41 orders, 79 families, 112 genera, and 352 putative operational taxonomic units (OTUs) were obtained from natural O. sinensis using high-throughput sequencing. Among of which, 43 genera were identified in external mycelial cortices (EMC), Ophiocordyceps, Sebacinia, Archaeorhizomyces were predominant genera with the abundance of 95.86%, 1.14%, 0.85%, respectively. Total 66 genera were identified from soil microhabitat, Inocybe, Archaeorhizomyces, Unclassified Thelephoraceae, Tomentella, Thelephora, Sebacina, Unclassified Ascomycota, Unclassified Fungi were predominant genera with an average abundance of 53.32%, 8.69%, 8.12%, 8.12%, 7.21%, 4.6%, 3.08% and 3.05%, respectively. The fungal communities in external mycelial cortices (EMC) were significantly distinct from the soil microhabitat (Soil). Meanwhile, seven culture media that benefit for the growth of O. sinensis were used to isolate culturable fungi at 16 °C, resulted in 77 fungal strains isolated for rDNA ITS sequence analysis, belonging to 33 genera, including Ophiocordyceps, Trichoderma, Cytospora, Truncatella, Dactylonectria, Isaria, Cephalosporium, Fusarium, Cosmospora, Paecilomyces, etc.. Among all culturable fungi, Mortierella and Trichoderma were predominant genera of total isolates. Conclusions The significantly distinction and overlap in fungal community structure between two approaches highlight that integration of approaches would generate more information than either of them. Our finding is the first investigation of fungal community structure of natural O. sinensis by two approachs, provide new insight into O. sinensis associated fungi, and support that microbiota of O. sinensis is an untapped source for novel bioactive metabolites discovery.

scholarly journals Beyond the Ribosome: Extra-translational Functions of tRNA Fragments

2016 ◽  
Vol 11s1 ◽  
pp. BMI.S35904 ◽  
Author(s):  
Kevin W. Diebel ◽  
Kun Zhou ◽  
Aaron B. Clarke ◽  
Lynne T. Bemis
Keyword(s):  
Data Analysis ◽  
High Throughput ◽  
Biological Samples ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Degradation Products ◽  
Noncoding Rnas ◽  
Transfer Rnas ◽  
Sequencing Studies ◽  
Domains Of Life

High-throughput sequencing studies of small RNAs reveal a complex milieu of noncoding RNAs in biological samples. Early data analysis was often limited to microRNAs due to their regulatory nature and potential as biomarkers; however, many more classes of noncoding RNAs are now being recognized. A class of fragments initially excluded from analysis were those derived from transfer RNAs (tRNAs) because they were thought to be degradation products. More recently, critical cellular function has been attributed to tRNA fragments (tRFs), and their conservation across all domains of life has propelled them into an emerging area of scientific study. The biogenesis of tRFs is currently being elucidated, and initial studies show that a diverse array of tRFs are genera ted from all parts of a tRNA molecule. The goal of this review was to present what is currently known about tRFs and their potential as biomarkers for the earlier detection of disease.

scholarly journals Fulcrum: condensing redundant reads from high-throughput sequencing studies

2012 ◽  
Vol 28 (10) ◽  
pp. 1324-1327 ◽  
Author(s):  
Matthew S. Burriesci ◽  
Erik M. Lehnert ◽  
John R. Pringle
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequencing Studies

scholarly journals Using familial information for variant filtering in high-throughput sequencing studies

2014 ◽  
Vol 133 (11) ◽  
pp. 1331-1341 ◽  
Author(s):  
Melanie Bahlo ◽  
Rick Tankard ◽  
Vesna Lukic ◽  
Karen L. Oliver ◽  
Katherine R. Smith
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Variant Filtering ◽  
Sequencing Studies

scholarly journals Hiding in plain sight: structure and sequence analysis reveals the importance of the antibody DE loop for antibody-antigen binding

2020 ◽  
Author(s):  
Simon P. Kelow ◽  
Jared Adolf-Bryfogle ◽  
Roland L. Dunbrack
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Data Bank ◽  
Antigen Binding ◽  
Sequence Variability ◽  
Antigen Binding Site ◽  
Sequencing Studies ◽  
Variable Domains ◽  
Complementarity Determining Regions ◽  
Hiv 1

AbstractAntibody variable domains contain “complementarity determining regions” (CDRs), the loops that form the antigen binding site. CDRs1-3 are recognized as the canonical CDRs. However, a fourth loop sits adjacent to CDR1 and CDR2 and joins the D and E strands on the antibody v-type fold. This “DE loop” is usually treated as a framework region, even though mutations in the loop affect the conformation of the CDRs and residues in the DE loop occasionally contact antigen. We analyzed the length, structure, and sequence features of all DE loops in the Protein Data Bank, as well as millions of sequences from HIV-1 infected and naïve patients. We refer to the DE loop as H4 and L4 in the heavy and light chain respectively. Clustering the backbone conformations of the most common length of L4 (6 residues) reveals four conformations: two κ-only clusters, one λ-only cluster, and one mixed κ/λ cluster. The vast majority of H4 loops are length-8 and exist primarily in one conformation; a secondary conformation represents a small fraction of H4-8 structures. H4 sequence variability exceeds that of the antibody framework in naïve human high-throughput sequences, and both L4 and H4 sequence variability from λ and heavy germline sequences exceed that of germline framework regions. Finally, we identified dozens of structures in the PDB with insertions in the DE loop, all related to broadly neutralizing HIV-1 antibodies, as well as antibody sequences from high-throughput sequencing studies of HIV-infected individuals, illuminating a possible role in humoral immunity to HIV-1.

scholarly journals Female dietary bias towards large migratory moths in the European free-tailed bat ( Tadarida teniotis )

2016 ◽  
Vol 12 (3) ◽  
pp. 20150988 ◽  
Author(s):  
Vanessa A. Mata ◽  
Francisco Amorim ◽  
Martin F. V. Corley ◽  
Gary F. McCracken ◽  
Hugo Rebelo ◽  
...  
Keyword(s):  
High Throughput ◽  
Resource Use ◽  
Diet Composition ◽  
High Throughput Sequencing ◽  
Food Resource ◽  
Migratory Behaviour ◽  
Molecular Tools ◽  
Autographa Gamma ◽  
Males And Females ◽  
Dietary Variation

In bats, sexual segregation has been described in relation to differential use of roosting and foraging habitats. It is possible that variation may also exist between genders in the use of different prey types. However, until recently this idea was difficult to test owing to poorly resolved taxonomy of dietary studies. Here, we use high-throughput sequencing to describe gender-related variation in diet composition of the European free-tailed bat ( Tadarida teniotis ), while controlling for effects of age and season. We analysed guano pellets collected from 143 individuals mist-netted from April to October 2012 and 2013, in northeast Portugal. Moths (Lepidoptera; mainly Noctuidae and Geometridae) were by far the most frequently recorded prey, occurring in nearly all samples and accounting for 96 out of 115 prey taxa. There were significant dietary differences between males and females, irrespective of age and season. Compared to males, females tended to consume larger moths and more moths of migratory behaviour (e.g. Autographa gamma ). Our study provides the first example of gender-related dietary variation in bats, illustrating the value of novel molecular tools for revealing intraspecific variation in food resource use in bats and other insectivores.

scholarly journals Functional genomics to explore cancer cell vulnerabilities

2010 ◽  
Vol 28 (1) ◽  
pp. E5 ◽  
Author(s):  
Kristopher T. Kahle ◽  
David Kozono ◽  
Kimberly Ng ◽  
Grace Hsieh ◽  
Pascal O. Zinn ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
High Throughput Sequencing ◽  
Chromosomal Abnormalities ◽  
Cost Effective ◽  
Gene Copy ◽  
Tumor Initiation ◽  
Cancer Proliferation ◽  
Synthetically Lethal

Our understanding of glioblastoma multiforme (GBM), the most common form of primary brain cancer, has been significantly advanced by recent efforts to characterize the cancer genome using unbiased high-throughput sequencing analyses. While these studies have documented hundreds of mutations, gene copy alterations, and chromosomal abnormalities, only a subset of these alterations are likely to impact tumor initiation or maintenance. Furthermore, genes that are not altered at the genomic level may play essential roles in tumor initiation and maintenance. Identification of these genes is critical for therapeutic development and investigative methodologies that afford insight into biological function. This requirement has largely been fulfilled with the emergence of RNA interference (RNAi) and high-throughput screening technology. In this article, the authors discuss the application of genome-wide, high-throughput RNAi-based genetic screening as a powerful tool for the rapid and cost-effective identification of genes essential for cancer proliferation and survival. They describe how these technologies have been used to identify genes that are themselves selectively lethal to cancer cells, or synthetically lethal with other oncogenic mutations. The article is intended to provide a platform for how RNAi libraries might contribute to uncovering glioma cell vulnerabilities and provide information that is highly complementary to the structural characterization of the glioblastoma genome. The authors emphasize that unbiased, systems-level structural and functional genetic approaches are complementary efforts that should facilitate the identification of genes involved in the pathogenesis of GBM and permit the identification of novel drug targets.

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