scholarly journals High-resolution ISR amplicon sequencing reveals personalized oral microbiome

2018 ◽  
Author(s):  
Chiranjit Mukherjee ◽  
Clifford J. Beall ◽  
Ann L. Griffen ◽  
Eugene J. Leys
Keyword(s):  
High Resolution ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Rrna Gene ◽  
Oral Microbiota ◽  
Community Fingerprinting

AbstractBackground:Sequencing of the 16S rRNA gene has been the standard for studying the composition of microbial communities. While it allows identification of bacteria at the level of species, it does not usually provide sufficient information to resolve at the sub-species level. Species-level resolution is not adequate for studies of transmission or stability, or for exploring subspecies variation in disease association. Current approaches using whole metagenome shotgun sequencing require very high coverage that can be cost-prohibitive and computationally challenging for diverse communities. Thus there is a need for high-resolution, yet cost-effective, high-throughput methods for characterizing microbial communities.Results:Significant improvement in resolution for amplicon-based bacterial community analysis was achieved by combining amplicon sequencing of a high-diversity marker gene, the ribosomal operon ISR, with a probabilistic error modeling algorithm, DADA2. The resolving power of this new approach was compared to that of both standard and high-resolution 16S-based approaches using a set of longitudinal subgingival plaque samples. The ISR strategy achieved a 5.2-fold increase in community richness compared to reference-based 16S rRNA gene analysis, and showed 100% accuracy in predicting the correct source of a clinical sample. Individuals’ microbial communities were highly personalized, and although they exhibited some drift in membership and levels over time, that difference was always smaller than the differences between any two subjects, even after one year. The construction of an ISR database from publicly available genomic sequences allowed us to explore genomic variationwithinspecies, resulting in the identification of multiple variants of the ISR for most species.Conclusions:The ISR approach resulted in significantly improved resolution of communities, and revealed a highly personalized, stable human oral microbiota. Multiple ISR types were observed for all species examined, demonstrating a high level of subspecies variation in the oral microbiota. The approach is high-throughput, high-resolution yet cost-effective, allowing subspecies-level community fingerprinting at a cost comparable to that of 16S rRNA gene amplicon sequencing. It will be useful for a range of applications that require high-resolution identification of organisms, including microbial tracking, community fingerprinting, and potentially for identification of virulence-associated strains.

scholarly journals Metagenome-validated Parallel Amplicon Sequencing and Text Mining-based Annotations for Simultaneous Profiling of Bacteria and Fungi: Vaginal Microbiome and Mycobiota in Healthy Women

2021 ◽  
Author(s):  
Seppo Virtanen ◽  
Schahzad Saqib ◽  
Tinja Kanerva ◽  
Pekka Nieminen ◽  
Ilkka Kalliala ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Extensive Literature ◽  
Metagenomic Sequencing ◽  
Text Extraction ◽  
Healthy Women ◽  
Bacteria And Fungi

Abstract Background: Amplicon sequencing of kingdom-specific tags such as 16S rRNA gene for bacteria and internal transcribed spacer (ITS) region for fungi are widely used for investigating microbial populations. So far most human studies have focused on bacteria while studies on host-associated fungi in health and disease have only recently started to accumulate. To enable cost-effective parallel analysis of bacterial and fungal communities in human and environmental samples, we developed a method where 16S rRNA gene and ITS-1 amplicons were pooled together for a single Illumina MiSeq or HiSeq run and analysed after primer-based segregation. Taxonomic assignments were performed with Blast in combination with an iterative text-extraction based filtration approach, which uses extensive literature records from public databases to select the most probable hits that were further validated by shotgun metagenomic sequencing. Results: Using 50 vaginal samples, we show that the combined run provides comparable results on bacterial composition and diversity to conventional 16S rRNA gene amplicon sequencing. The text-extraction-based taxonomic assignment guided tool provided ecosystem specific annotations that were confirmed by Metagenomic Phylogenetic Analysis (MetaPhlAn). The metagenome analysis revealed distinct functional differences between the bacterial community types while fungi were undetected, despite being identified in all samples based on ITS amplicons. Co-abundance analysis of bacteria and fungi did not show strong between-kingdom correlations within the vaginal ecosystem of healthy women.Conclusion: Combined amplicon sequencing for bacteria and fungi provides a simple and cost-effective method for simultaneous analysis of microbiota and mycobiota within the same samples. Text extraction-based annotation tool facilitates the characterization and interpretation of defined microbial communities from rapidly accumulating sequencing and metadata readily available through public databases.

scholarly journals 16S rRNA Gene Amplicon Sequencing of Microbial Communities Involved in Anaerobic Bulking in a Mesophilic Expanded Granular Sludge Bed Reactor Treating Wastewater Discharged from a Japanese-Style Thickened Worcestershire Sauce-Producing Factory

2019 ◽  
Vol 8 (36) ◽  
Author(s):  
Takeshi Yamada ◽  
Jun Harada ◽  
Yuki Okazaki ◽  
Tsuyoshi Yamaguchi ◽  
Atsushi Nakano
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Granular Sludge ◽  
Amplicon Sequencing ◽  
Organic Content ◽  
Rrna Gene ◽  
Sludge Bulking ◽  
High Organic Content

We analyzed the prokaryotes in bulking and healthy sludge from a mesophilic expanded granular sludge bed reactor treating wastewater with high organic content by 16S rRNA gene amplicon sequencing. We tabulated the microbiota at the phylum level, providing a framework for avoiding sludge bulking.

scholarly journals Species-level resolution of 16S rRNA gene amplicons sequenced through the MinIONTM portable nanopore sequencer

2015 ◽  
Author(s):  
Alfonso Benítez-Páez ◽  
Kevin J. Portune ◽  
Yolanda Sanz
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Diversity ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Taxonomic Resolution ◽  
Universal Primers ◽  
Rrna Gene ◽  
Rdna Sequences ◽  
Dna Sequencer

AbstractBackgroundThe miniaturised and portable DNA sequencer MinIONTM has been released to the scientific community within the framework of an early access programme to evaluate its application for a wide variety of genetic approaches. This technology has demonstrated great potential, especially in genome-wide analyses. In this study, we tested the ability of the MinIONTM system to perform amplicon sequencing in order to design new approaches to study microbial diversity using nearly full-length 16S rDNA sequences.ResultsUsing R7.3 chemistry, we generated more than 3.8 million events (nt) during a single sequencing run. These data were sufficient to reconstruct more than 90% of the 16S rRNA gene sequences for 20 different species present in a mock reference community. After read mapping and 16S rRNA gene assembly, consensus sequences and 2d reads were recovered to assign taxonomic classification down to the species level. Additionally, we were able to measure the relative abundance of all the species present in a mock community and detected a biased species distribution originating from the PCR reaction using ‘universal’ primers.ConclusionsAlthough nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, the MinIONTM DNA sequencer is valuable for both high taxonomic resolution and microbial diversity analysis. Improvements in nanopore chemistry, such as minimising base-calling errors and the nucleotide bias reported here for 16S amplicon sequencing, will further deliver more reliable information that is useful for the specific detection of microbial species and strains in complex ecosystems.

scholarly journals Rabbit microbiota across the whole body revealed by 16S rRNA gene amplicon sequencing

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xiaofen Hu ◽  
Fei Wang ◽  
Shanshan Yang ◽  
Xu Yuan ◽  
Tingyu Yang ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Amplicon Sequencing ◽  
Whole Body ◽  
Rrna Gene ◽  
Microbial Function ◽  
Relationship Of ◽  
The Relationship ◽  
Microbial Samples

Abstract Background Rabbit can produce meat, fur and leather, and serves as an important biomedical animal model. Understanding the microbial community of rabbits helps to raise rabbits healthily and better support their application as animal models. Results In this study, we selected 4 healthy Belgium gray rabbits to collect the microbial samples from 12 body sites, including skin, lung, uterus, mouth, stomach, duodenum, ileum, jejunum, colon, cecum, cecal appendix and rectum. The microbiota across rabbit whole body was investigated via 16S rRNA gene amplicon sequencing. After quality control, 46 samples were retained, and 3,148 qualified ASVs were obtained, representing 23 phyla and 264 genera. Based on the weighted UniFrac distances, these samples were divided into the large intestine (Lin), stomach and small intestine (SSin), uterus (Uter), and skin, mouth and lung (SML) groups. The diversity of Lin microbiota was the highest, followed by those of the SSin, Uter and SML groups. In the whole body, Firmicutes (62.37%), Proteobacteria (13.44%) and Bacteroidota (11.84%) were the most predominant phyla. The relative abundance of Firmicutes in the intestinal tract was significantly higher than that in the non-intestinal site, while Proteobacteria was significantly higher in the non-intestinal site. Among the 264 genera, 35 were the core microbiota distributed in all body sites. Sixty-one genera were specific in the SML group, while 13, 8 and 1 were specifically found in the Lin, SSin and Uter groups, respectively. The Lin group had the most difference with other groups, there were average 72 differential genera between the Lin and other groups. The functional prediction analysis showed that microbial function within each group was similar, but there was a big difference between the intestinal tracts and the non-intestinal group. Notably, the function of microorganism in uterus and mouth were the most different from those in the gastrointestinal sites; rabbit’s coprophagy of consuming soft feces possibly resulted in little differences of microbial function between stomach and large intestinal sites. Conclusion Our findings improve the knowledge about rabbit microbial communities throughout whole body and give insights into the relationship of microbial communities among different body sites in health rabbits.

scholarly journals The Human Oral Microbiome

2010 ◽  
Vol 192 (19) ◽  
pp. 5002-5017 ◽  
Author(s):  
Floyd E. Dewhirst ◽  
Tuste Chen ◽  
Jacques Izard ◽  
Bruce J. Paster ◽  
Anne C. R. Tanner ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Species Level ◽  
Oral Microbiome ◽  
Rrna Gene ◽  
Oral Microbiota ◽  
16S Rrna Gene Sequences ◽  
Culture Independent ◽  
Health And Disease

ABSTRACT The human oral cavity contains a number of different habitats, including the teeth, gingival sulcus, tongue, cheeks, hard and soft palates, and tonsils, which are colonized by bacteria. The oral microbiome is comprised of over 600 prevalent taxa at the species level, with distinct subsets predominating at different habitats. The oral microbiome has been extensively characterized by cultivation and culture-independent molecular methods such as 16S rRNA cloning. Unfortunately, the vast majority of unnamed oral taxa are referenced by clone numbers or 16S rRNA GenBank accession numbers, often without taxonomic anchors. The first aim of this research was to collect 16S rRNA gene sequences into a curated phylogeny-based database, the Human Oral Microbiome Database (HOMD), and make it web accessible (www.homd.org ). The HOMD includes 619 taxa in 13 phyla, as follows: Actinobacteria, Bacteroidetes, Chlamydiae, Chloroflexi, Euryarchaeota, Firmicutes, Fusobacteria, Proteobacteria, Spirochaetes, SR1, Synergistetes, Tenericutes, and TM7. The second aim was to analyze 36,043 16S rRNA gene clones isolated from studies of the oral microbiota to determine the relative abundance of taxa and identify novel candidate taxa. The analysis identified 1,179 taxa, of which 24% were named, 8% were cultivated but unnamed, and 68% were uncultivated phylotypes. Upon validation, 434 novel, nonsingleton taxa will be added to the HOMD. The number of taxa needed to account for 90%, 95%, or 99% of the clones examined is 259, 413, and 875, respectively. The HOMD is the first curated description of a human-associated microbiome and provides tools for use in understanding the role of the microbiome in health and disease.

scholarly journals High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

2014 ◽  
Vol 80 (12) ◽  
pp. 3568-3575 ◽  
Author(s):  
Mathis Hjort Hjelmsø ◽  
Lars Hestbjerg Hansen ◽  
Jacob Bælum ◽  
Louise Feld ◽  
William E. Holben ◽  
...  
Keyword(s):  
High Resolution ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Community Composition ◽  
Bacterial Community Composition ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
High Resolution Melt ◽  
Hrm Analysis

ABSTRACTIn the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.

scholarly journals Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yoshiyuki Matsuo ◽  
Shinnosuke Komiya ◽  
Yoshiaki Yasumizu ◽  
Yuki Yasuoka ◽  
Katsura Mizushima ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Full Length ◽  
Rrna Gene ◽  
Short Read ◽  
Short Read Sequencing ◽  
Long Read

Abstract Background Species-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. Results We modified our existing protocol for full-length 16S rRNA gene amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S rRNA gene amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition. Conclusions Our present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene.

scholarly journals Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

2020 ◽  
Author(s):  
Yoshiyuki Matsuo ◽  
Shinnosuke Komiya ◽  
Yoshiaki Yasumizu ◽  
Yuki Yasuoka ◽  
Katsura Mizushima ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Full Length ◽  
Rrna Gene ◽  
Short Read ◽  
Short Read Sequencing ◽  
16S Amplicon Sequencing ◽  
Long Read

AbstractBackgroundSpecies-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples.ResultsWe modified our existing protocol for full-length 16S amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition.ConclusionsOur present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene, which provided the requisite species-level resolution and accuracy in clinical settings.

scholarly journals PSXIV-23 Characterization of microbial communities associated with the rumen lining, digesta and rumen fluid from beef cattle consuming a high energy diet using 16S rRNA gene amplicon sequencing

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 446-446
Author(s):  
Arquimides Reyes ◽  
Margaret Weinroth ◽  
Cory Wolfe ◽  
Robert Delmore ◽  
Terry Engle ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Beef Cattle ◽  
Microbial Communities ◽  
Relative Abundance ◽  
Rumen Fluid ◽  
Amplicon Sequencing ◽  
High Energy ◽  
Feedlot Cattle ◽  
Rrna Gene

Abstract The true etiology of liver abscesses is not well known. Therefore, the objective of this study was to characterize the microbial communities in the rumen lining, digesta, and rumen fluid from beef cattle consuming a high energy diet, using 16S rRNA gene amplicon sequencing. Twelve crossbred feedlot steers (450 ±10 kg; ~ 3.0 years of age) fitted with ruminal fistulas, consuming a high energy finishing diet (1.43 NEg, Mcal/kg DM) for 21 d were utilized in this experiment. Microbial DNA from three regions within the rumen [rumen lining (ventral/lateral), digesta (geometric center of the rumen), and rumen fluid] was extracted and the V4 region of the 16S rRNA gene was amplified and sequenced. Across all sample regions, bacterial sequences were classified into 34 phyla, 76 classes, 143 orders, and 254 families. Bacteroidetes and Firmicutes were the predominant phyla present across all samples. The relative abundance of Bacteroidetes detected in rumen fluid was lesser (P < 0.05) when compared to bacteria sampled from the rumen lining and digesta. In contrast, the relative abundance of Firmicutes were greater (P < 0.05) in rumen fluid and the rumen lining when compared to digesta samples. There are very few publications describing the complex community of the rumen microbiome. To our knowledge this is the first publication categorizing microbial populations in three distinct locations within the rumen using next generation sequencing in feedlot cattle.

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