scholarly journals Improved taxonomic assignment of rumen bacterial 16S rRNA sequences using a revised SILVA taxonomic framework

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6496 ◽  
Author(s):  
Gemma Henderson ◽  
Pelin Yilmaz ◽  
Sandeep Kumar ◽  
Robert J. Forster ◽  
William J. Kelly ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Data ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Rumen Bacteria ◽  
Genus Level ◽  
Bacterial 16S Rrna Gene

The taxonomy and associated nomenclature of many taxa of rumen bacteria are poorly defined within databases of 16S rRNA genes. This lack of resolution results in inadequate definition of microbial community structures, with large parts of the community designated as incertae sedis, unclassified, or uncultured within families, orders, or even classes. We have begun resolving these poorly-defined groups of rumen bacteria, based on our desire to name these for use in microbial community profiling. We used the previously-reported global rumen census (GRC) dataset consisting of >4.5 million partial bacterial 16S rRNA gene sequences amplified from 684 rumen samples and representing a wide range of animal hosts and diets. Representative sequences from the 8,985 largest operational units (groups of sequence sharing >97% sequence similarity, and covering 97.8% of all sequences in the GRC dataset) were used to identify 241 pre-defined clusters (mainly at genus or family level) of abundant rumen bacteria in the ARB SILVA 119 framework. A total of 99 of these clusters (containing 63.8% of all GRC sequences) had no unique or had inadequate taxonomic identifiers, and each was given a unique nomenclature. We assessed this improved framework by comparing taxonomic assignments of bacterial 16S rRNA gene sequence data in the GRC dataset with those made using the original SILVA 119 framework, and three other frameworks. The two SILVA frameworks performed best at assigning sequences to genus-level taxa. The SILVA 119 framework allowed 55.4% of the sequence data to be assigned to 751 uniquely identifiable genus-level groups. The improved framework increased this to 87.1% of all sequences being assigned to one of 871 uniquely identifiable genus-level groups. The new designations were included in the SILVA 123 release (https://www.arb-silva.de/documentation/release-123/) and will be perpetuated in future releases.

scholarly journals Identification and Analysis of Informative Single Nucleotide Polymorphisms in 16S rRNA Gene Sequences of the Bacillus cereus Group

2016 ◽  
Vol 54 (11) ◽  
pp. 2749-2756 ◽  
Author(s):  
Janetta R. Hakovirta ◽  
Samantha Prezioso ◽  
David Hodge ◽  
Segaran P. Pillai ◽  
Linda M. Weigel
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacillus Cereus ◽  
Sequence Data ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Single Nucleotide ◽  
Content Type

Analysis of 16S rRNA genes is important for phylogenetic classification of known and novel bacterial genera and species and for detection of uncultivable bacteria. PCR amplification of 16S rRNA genes with universal primers produces a mixture of amplicons from all rRNA operons in the genome, and the sequence data generally yield a consensus sequence. Here we describe valuable data that are missing from consensus sequences, variable effects on sequence data generated from nonidentical 16S rRNA amplicons, and the appearance of data displayed by different software programs. These effects are illustrated by analysis of 16S rRNA genes from 50 strains of theBacillus cereusgroup, i.e.,Bacillus anthracis,Bacillus cereus,Bacillus mycoides, andBacillus thuringiensis. These species have 11 to 14 rRNA operons, and sequence variability occurs among the multiple 16S rRNA genes. A single nucleotide polymorphism (SNP) previously reported to be specific toB. anthraciswas detected in someB. cereusstrains. However, a different SNP, at position 1139, was identified as being specific toB. anthracis, which is a biothreat agent with high mortality rates. Compared with visual analysis of the electropherograms, basecaller software frequently missed gene sequence variations or could not identify variant bases due to overlapping basecalls. Accurate detection of 16S rRNA gene sequences that include intragenomic variations can improve discrimination among closely related species, improve the utility of 16S rRNA databases, and facilitate rapid bacterial identification by targeted DNA sequence analysis or by whole-genome sequencing performed by clinical or reference laboratories.

scholarly journals Three Cases of Anaerobiospirillum succiniciproducens Bacteremia Confirmed by 16S rRNA Gene Sequencing

1998 ◽  
Vol 36 (5) ◽  
pp. 1209-1213 ◽  
Author(s):  
Wee Tee ◽  
Tony M. Korman ◽  
Mary Jo Waters ◽  
Andrew Macphee ◽  
Adam Jenney ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Strain ◽  
Sequence Data ◽  
Infected Individual ◽  
The Other ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Anaerobiospirillum Succiniciproducens

We describe three cases of Anaerobiospirillum succiniciproducens bacteremia from Australia. We believe one of these cases represents the first report of A. succiniciproducens bacteremia in a human immunodeficiency virus (HIV)-infected individual. The other two patients had an underlying disorder (one patient had bleeding esophageal varices complicating alcohol liver disease and one patient had non-Hodgkin’s lymphoma). A motile, gram-negative, spiral anaerobe was isolated by culturing blood from all patients. Electron microscopy showed a curved bacterium with bipolar tufts of flagella resembling Anaerobiospirillumspp. Sequencing of the 16S rRNA genes of the isolates revealed no close relatives (organisms likely to be in the same genus) in the sequence databases, nor were any sequence data available for A. succiniciproducens. This report presents for the first time the 16S rRNA gene sequence of the type strain of A. succiniciproducens, strain ATCC 29305. Two of the three clinical isolates have sequences identical to that of the type strain, while the sequence of the other strain differs from that of the type strain at 4 nucleotides.

scholarly journals An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Denise M. O’Sullivan ◽  
Ronan M. Doyle ◽  
Sasithon Temisak ◽  
Nicholas Redshaw ◽  
Alexandra S. Whale ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Relative Abundance ◽  
Amplicon Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Microbiome Analysis ◽  
The Impact

AbstractDespite the advent of whole genome metagenomics, targeted approaches (such as 16S rRNA gene amplicon sequencing) continue to be valuable for determining the microbial composition of samples. Amplicon microbiome sequencing can be performed on clinical samples from a normally sterile site to determine the aetiology of an infection (usually single pathogen identification) or samples from more complex niches such as human mucosa or environmental samples where multiple microorganisms need to be identified. The methodologies are frequently applied to determine both presence of micro-organisms and their quantity or relative abundance. There are a number of technical steps required to perform microbial community profiling, many of which may have appreciable precision and bias that impacts final results. In order for these methods to be applied with the greatest accuracy, comparative studies across different laboratories are warranted. In this study we explored the impact of the bioinformatic approaches taken in different laboratories on microbiome assessment using 16S rRNA gene amplicon sequencing results. Data were generated from two mock microbial community samples which were amplified using primer sets spanning five different variable regions of 16S rRNA genes. The PCR-sequencing analysis included three technical repeats of the process to determine the repeatability of their methods. Thirteen laboratories participated in the study, and each analysed the same FASTQ files using their choice of pipeline. This study captured the methods used and the resulting sequence annotation and relative abundance output from bioinformatic analyses. Results were compared to digital PCR assessment of the absolute abundance of each target representing each organism in the mock microbial community samples and also to analyses of shotgun metagenome sequence data. This ring trial demonstrates that the choice of bioinformatic analysis pipeline alone can result in different estimations of the composition of the microbiome when using 16S rRNA gene amplicon sequencing data. The study observed differences in terms of both presence and abundance of organisms and provides a resource for ensuring reproducible pipeline development and application. The observed differences were especially prevalent when using custom databases and applying high stringency operational taxonomic unit (OTU) cut-off limits. In order to apply sequencing approaches with greater accuracy, the impact of different analytical steps needs to be clearly delineated and solutions devised to harmonise microbiome analysis results.

Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene

2015 ◽  
Vol 41 (1) ◽  
pp. 51-58
Author(s):  
Mohammad Shamimul Alam ◽  
Hawa Jahan ◽  
Rowshan Ara Begum ◽  
Reza M Shahjahan
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Fish Larva ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Valuable Insight ◽  
Multiple Sequence ◽  
Pcr Rflp ◽  
Alternative Means

Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

scholarly journals Microbiological and Geochemical Heterogeneity in an In Situ Uranium Bioremediation Field Site

2005 ◽  
Vol 71 (10) ◽  
pp. 6308-6318 ◽  
Author(s):  
Helen A. Vrionis ◽  
Robert T. Anderson ◽  
Irene Ortiz-Bernad ◽  
Kathleen R. O'Neill ◽  
Charles T. Resch ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Acid Volatile Sulfide ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Geochemical Heterogeneity

ABSTRACT The geochemistry and microbiology of a uranium-contaminated subsurface environment that had undergone two seasons of acetate addition to stimulate microbial U(VI) reduction was examined. There were distinct horizontal and vertical geochemical gradients that could be attributed in large part to the manner in which acetate was distributed in the aquifer, with more reduction of Fe(III) and sulfate occurring at greater depths and closer to the point of acetate injection. Clone libraries of 16S rRNA genes derived from sediments and groundwater indicated an enrichment of sulfate-reducing bacteria in the order Desulfobacterales in sediment and groundwater samples. These samples were collected nearest the injection gallery where microbially reducible Fe(III) oxides were highly depleted, groundwater sulfate concentrations were low, and increases in acid volatile sulfide were observed in the sediment. Further down-gradient, metal-reducing conditions were present as indicated by intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and increased abundance of 16S rRNA gene sequences belonging to the dissimilatory Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI) reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene sequences in both groundwater and sediment; however, the sites at which these maxima occurred were spatially separated within the aquifer. The substantial microbial and geochemical heterogeneity at this site demonstrates that attempts should be made to deliver acetate in a more uniform manner and that closely spaced sampling intervals, horizontally and vertically, in both sediment and groundwater are necessary in order to obtain a more in-depth understanding of microbial processes and the relative contribution of attached and planktonic populations to in situ uranium bioremediation.

scholarly journals Extremely Acidophilic Protists from Acid Mine Drainage Host Rickettsiales-Lineage Endosymbionts That Have Intervening Sequences in Their 16S rRNA Genes

2003 ◽  
Vol 69 (9) ◽  
pp. 5512-5518 ◽  
Author(s):  
Brett J. Baker ◽  
Philip Hugenholtz ◽  
Scott C. Dawson ◽  
Jillian F. Banfield
Keyword(s):  
Acid Mine Drainage ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Mine Drainage ◽  
Close Association ◽  
Variable Region ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Acid Mine

ABSTRACT During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist Acanthamoeba. Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists. To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community. Approximately 4% of protists in the acid mine drainage samples contained endosymbionts. Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic. The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes. The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops. IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes. Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name “Candidatus Captivus acidiprotistae.” To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat.

scholarly journals Assessment of the Diversity, Abundance, and Ecological Distribution of Members of Candidate Division SR1 Reveals a High Level of Phylogenetic Diversity but Limited Morphotypic Diversity

2009 ◽  
Vol 75 (12) ◽  
pp. 4139-4148 ◽  
Author(s):  
James P. Davis ◽  
Noha H. Youssef ◽  
Mostafa S. Elshahed
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Clone Library ◽  
Phylogenetic Diversity ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Qpcr Analysis ◽  
Freshwater Pond ◽  
Candidate Division

ABSTRACT We used a combination of 16S rRNA gene clone library surveys, quantitative PCR (qPCR) analysis, and fluorescent in situ hybridization to investigate the diversity, abundance, and distribution of members of candidate division SR1 in multiple habitats. Using SR1-specific 16S rRNA gene primers, we identified multiple novel SR1 lineages in four different anaerobic environments: sediments from Zodletone Spring, a sulfide- and sulfur-rich spring in southwestern Oklahoma; inner layers of microbial mats obtained from Sperm Pool, a high-temperature, low-pH pool (55°C, pH 2.5) in Yellowstone National Park; fresh bovine ruminal contents; and anaerobic freshwater pond sediments (Duck Pond) in Norman, Oklahoma. qPCR analysis indicated that SR1 members constitute a small fraction (<0.01%) of the microbial communities in Duck Pond and ruminal samples but constitute a significant fraction (11.6 and 48.7%) of the total number of bacterial 16S rRNA genes in Zodletone Spring and the inner layers of Sperm Pool microbial mat samples, respectively. By using SR1-specific fluorescent probes, filamentous cells were identified as the sole SR1 morphotype in all environments examined, with the exception of Sperm Pool, where a second bacillus morphotype was also identified. Using a full-cycle 16S rRNA approach, we show that each of these two morphotypes corresponds to a specific phylogenetic lineage identified in the Sperm Pool clone library. This work greatly expands the intralineage phylogenetic diversity within candidate division SR1 and provides valuable quantification and visualization tools that could be used for investigating the ecological roles, dynamics, and genomics of this as-yet-uncultured bacterial phylum.

Clover proliferation phytoplasma: ‘Candidatus Phytoplasma trifolii’

2004 ◽  
Vol 54 (4) ◽  
pp. 1349-1353 ◽  
Author(s):  
Chuji Hiruki ◽  
Keri Wang
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Reference Strain ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Beet Leafhopper ◽  
Signature Sequences ◽  
Elm Yellows ◽  
The 16S Rrna Gene

Clover proliferation phytoplasma (CPR) is designated as the reference strain for the CP phylogenetic group or subclade, on the basis of molecular analyses of genomic DNA, the 16S rRNA gene and the 16S–23S spacer region. Other strains related to CPR include alfalfa witches'-broom (AWB), brinjal little leaf (BLL), beet leafhopper-transmitted virescence (BLTV), Illinois elm yellows (ILEY), potato witches'-broom (PWB), potato yellows (PY), tomato big bud in California (TBBc) and phytoplasmas from Fragaria multicipita (FM). Phylogenetic analysis of the 16S rRNA gene sequences of BLL, CPR, FM and ILEY, together with sequences from 16 other phytoplasmas that belong to the ash yellows (AshY), jujube witches'-broom (JWB) and elm yellows (EY) groups that were available in GenBank, produced a tree on which these phytoplasmas clearly clustered as a discrete group. Three subgroups have been classified on the basis of sequence homology and the collective RFLP patterns of amplified 16S rRNA genes. AWB, BLTV, PWB and TBBc are assigned to taxonomic subgroup CP-A, FM belongs to subgroup CP-B and BLL and ILEY are assigned to subgroup CP-C. Genetic heterogeneity between different isolates of AWB, CPR and PWB has been observed from heteroduplex mobility assay analysis of amplified 16S rRNA genes and the 16S–23S spacer region. Two unique signature sequences that can be utilized to distinguish the CP group from others were present. On the basis of unique properties of the DNA from clover proliferation phytoplasma, the name ‘Candidatus Phytoplasma trifolii’ is proposed for the CP group.

scholarly journals A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2492 ◽  
Author(s):  
Catherine M. Burke ◽  
Aaron E. Darling
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Single Molecule ◽  
Illumina Miseq ◽  
Full Length ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Bacterial Taxonomy

BackgroundThe bacterial 16S rRNA gene has historically been used in defining bacterial taxonomy and phylogeny. However, there are currently no high-throughput methods to sequence full-length 16S rRNA genes present in a sample with precision.ResultsWe describe a method for sequencing near full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform and test it using DNA from human skin swab samples. Proof of principle of the approach is demonstrated, with the generation of 1,604 sequences greater than 1,300 nt from a single Nano MiSeq run, with accuracy estimated to be 100-fold higher than standard Illumina reads. The reads were chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection.ConclusionsThis method could be scaled up to generate many thousands of sequences per MiSeq run and could be applied to other sequencing platforms. This has great potential for populating databases with high quality, near full-length 16S rRNA gene sequences from under-represented taxa and environments and facilitates analyses of microbial communities at higher resolution.

scholarly journals REP-PCR Analysis to Study Prokaryotic Biodiversity from Lake Meyghan

2017 ◽  
Vol 61 ◽  
pp. 69-84 ◽  
Author(s):  
Ali Naghoni ◽  
Giti Emtiazi ◽  
Mohammad Ali Amoozegar ◽  
Zahra Etemadifar ◽  
Seyed Abolhassan Shahzadeh Fazeli
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
White Region ◽  
Rrna Gene Sequencing

Repetitive extragenic palindromic elements-polymerase chain reaction (rep-PCR) with 16S ribosomal ribonucleic acid (16S rRNA) genes sequences successfully used for the analysis of microbial community. In this study, the prokaryotic community in Lake Meyghan described by using rep-PCR analysis along with 16S rRNA gene sequencing. The water samples were collected from Lake Meyghan in November 2013. All samples were diluted and cultured on three different media. To estimate the number of prokaryotes per milliliter of the lake we used quantitative real‑time PCR (qPCR). Rep-PCR combination with 16S rRNA gene sequencing was performed to investigate prokaryotes biodiversity in the lake. 305 strains were isolated in this work; 113 isolates for green region, 102 isolates for red region, and 90 isolates for white region. The dendrograms generated 10, 7, and 9 clusters for a 70 % similarity cut-off for green, red, and white regions, respectively. Based on rep-PCR and 16S rRNA gene sequencing, the recovered isolates were dominated by (77.5 %)Halobacteriacaeand many isolates were related to the generaHalorubrum,Haloarcula,Haloterrigena,Natrinema, andHalovivaxin the white region. In the red region more isolated strains (57.5 %) belonged toBacillaceaeand the remaining 42.5 % of isolates belonged to archaea domain,Halorubrum, andHaloarcula. In the green region members ofGammaproteobacteriawere recoverd, this region was dominant withPseudoalteromonas,Salinivibrio, andAliidiomarina.

Sign in / Sign up

Export Citation Format

Share Document