scholarly journals High-resolution microbiome analysis enabled by linking of 16S rRNA gene sequences with adjacent genomic contexts

2021 ◽  
Vol 7 (9) ◽  
Author(s):  
Žana Kapustina ◽  
Justina Medžiūnė ◽  
Gediminas Alzbutas ◽  
Irmantas Rokaitis ◽  
Karolis Matjošaitis ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Sequencing ◽  
Gene Copy ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Copy Numbers ◽  
Rrna Sequencing ◽  
Metagenome Sequencing ◽  
Gene Copy Numbers

Sequence-based characterization of bacterial communities has long been a hostage of limitations of both 16S rRNA gene and whole metagenome sequencing. Neither approach is universally applicable, and the main efforts to resolve constraints have been devoted to improvement of computational prediction tools. Here, we present semi-targeted 16S rRNA sequencing (st16S-seq), a method designed for sequencing V1–V2 regions of the 16S rRNA gene along with the genomic locus upstream of the gene. By in silico analysis of 13 570 bacterial genome assemblies, we show that genome-linked 16S rRNA sequencing is superior to individual hypervariable regions or full-length gene sequences in terms of classification accuracy and identification of gene copy numbers. Using mock communities and soil samples we experimentally validate st16S-seq and benchmark it against the established microbial classification techniques. We show that st16S-seq delivers accurate estimation of 16S rRNA gene copy numbers, enables taxonomic resolution at the species level and closely approximates community structures obtainable by whole metagenome sequencing.

scholarly journals Correcting for 16S rRNA gene copy numbers in microbiome surveys remains an unsolved problem

Microbiome ◽  
2018 ◽  
Vol 6 (1) ◽  
Author(s):  
Stilianos Louca ◽  
Michael Doebeli ◽  
Laura Wegener Parfrey
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Unsolved Problem ◽  
Gene Copy ◽  
Rrna Gene ◽  
Copy Numbers ◽  
Gene Copy Numbers

scholarly journals Quantification of Burkholderia coxL Genes in Hawaiian Volcanic Deposits

2010 ◽  
Vol 76 (7) ◽  
pp. 2212-2217 ◽  
Author(s):  
C. F. Weber ◽  
G. M. King
Keyword(s):  
Carbon Monoxide ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Large Subunit ◽  
Dry Weight ◽  
Gene Copy ◽  
Rrna Gene ◽  
Copy Numbers ◽  
Volcanic Deposits ◽  
Gene Copy Numbers

ABSTRACT Isolation of multiple carbon monoxide (CO)-oxidizing Burkholderia strains and detection by culture-independent approaches suggest that Burkholderia may be an important component of CO-oxidizing communities in Hawaiian volcanic deposits. The absolute and relative abundance of the bacteria in these communities remains unknown, however. In this study, a quantitative PCR (Q-PCR) approach has been developed to enumerate Burkholderia coxL genes (large subunit of carbon monoxide dehydrogenase). This represents the first attempt to enumerate coxL genes from CO oxidizers in environmental samples. coxL copy numbers have been determined for samples from three sites representing a vegetation gradient on a 1959 volcanic deposit that included unvegetated cinders (bare), edges of vegetated sites (edge), and sites within tree stands (canopy). Q-PCR has also been used to estimate copy numbers of Betaproteobacteria 16S rRNA gene copy numbers and total Bacteria 16S rRNA. coxL genes could not be detected in the bare site (detection limit, ≥4.7 � 103 copies per reaction) but average 1.0 � 108 � 2.4 � 107 and 8.6 � 108 � 7.6 �107 copies g−1 (dry weight) in edge and canopy sites, respectively, which differ statistically (P = 0.0007). Average Burkholderia coxL gene copy numbers, expressed as a percentage of total Bacteria 16S rRNA gene copy numbers, are 6.2 and 0.7% for the edge and canopy sites, respectively. Although the percentage of Burkholderia coxL is lower in the canopy site, significantly greater gene copy numbers demonstrate that absolute abundance of coxL increases in vegetated sites and contributes to the expansion of CO oxidizer communities during biological succession on volcanic deposits.

scholarly journals Quantifying Genes and Transcripts To Assess the In Situ Physiology of “Dehalococcoides” spp. in a Trichloroethene-Contaminated Groundwater Site

2008 ◽  
Vol 74 (9) ◽  
pp. 2728-2739 ◽  
Author(s):  
Patrick K. H. Lee ◽  
Tamzen W. Macbeth ◽  
Kent S. Sorenson ◽  
Rula A. Deeb ◽  
Lisa Alvarez-Cohen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy ◽  
Rrna Gene ◽  
Copy Numbers ◽  
Field Samples ◽  
Rdase Genes ◽  
Gene Copy Numbers ◽  
The 16S Rrna Gene

ABSTRACT Quantitative PCR (qPCR) was coupled with reverse transcription (RT) to analyze both gene copy numbers and transcripts of the 16S rRNA gene and three reductive dehalogenase (RDase) genes (tceA, vcrA, and bvcA) as biomarkers of “Dehalococcoides” spp. in the groundwater of a trichloroethene-dense nonaqueous-phase liquid site at Fort Lewis, WA, that was sequentially subjected to biostimulation and bioaugmentation. Dehalococcoides cells carrying the tceA, vcrA, and bvcA genes were indigenous to the site. The sum of the three identified RDase gene copy numbers closely correlated to 16S rRNA gene copy numbers throughout the biostimulation and bioaugmentation activity, suggesting that these RDase genes represented the major Dehalococcoides metabolic functions at this site. Biomarker quantification revealed an overall increase of more than 3 orders of magnitude in the total Dehalococcoides population through the 1-year monitoring period (spanning biostimulation and bioaugmentation), and measurement of the respective RDase gene concentrations indicated different growth dynamics among Dehalococcoides cells. The Dehalococcoides cells containing the tceA gene consistently lagged behind other Dehalococcoides cells in population numbers and made up less than 5% of the total Dehalococcoides population, whereas the vcrA- and bvcA-containing cells represented the dominant fractions. Quantification of transcripts in groundwater samples verified that the 16S rRNA gene and the bvcA and vcrA genes were consistently highly expressed in all samples examined, while the tceA transcripts were detected inconsistently, suggesting a less active physiological state of the cells with this gene. The production of vinyl chloride and ethene toward the end of treatment supported the physiological activity of the bvcA- and vcrA-carrying cells. A clone library of the expressed RDase genes in field samples produced with degenerate primers revealed the expression of two putative RDase genes that were not previously monitored with RT-qPCR. The level of abundance of one of the putative RDase genes (FtL-RDase-1638) identified in the cDNA clone library tracked closely in field samples with abundance of the bvcA gene, suggesting that the FtL-RDase-1638 gene was likely colocated in genomes containing the bvcA gene. Overall, results from this study demonstrate that quantification of biomarker dynamics at field sites can provide useful information about the in situ physiology of Dehalococcoides strains and their associated activity.

scholarly journals Archaeal Abundance across a pH Gradient in an Arable Soil and Its Relationship to Bacterial and Fungal Growth Rates

2012 ◽  
Vol 78 (16) ◽  
pp. 5906-5911 ◽  
Author(s):  
Per Bengtson ◽  
Anna E. Sterngren ◽  
Johannes Rousk
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Soil Ph ◽  
Fungal Growth ◽  
Ph Gradient ◽  
Gene Copy ◽  
Rrna Gene ◽  
Copy Numbers ◽  
Ph Range ◽  
Gene Copy Numbers

ABSTRACTSoil pH is one of the most influential factors for the composition of bacterial and fungal communities, but the influence of soil pH on the distribution and composition of soil archaeal communities has yet to be systematically addressed. The primary aim of this study was to determine how total archaeal abundance (quantitative PCR [qPCR]-based estimates of 16S rRNA gene copy numbers) is related to soil pH across a pH gradient (pH 4.0 to 8.3). Secondarily, we wanted to assess how archaeal abundance related to bacterial and fungal growth rates across the same pH gradient. We identified two distinct and opposite effects of pH on the archaeal abundance. In the lowest pH range (pH 4.0 to 4.7), the abundance of archaea did not seem to correspond to pH. Above this pH range, there was a sharp, almost 4-fold decrease in archaeal abundance, reaching a minimum at pH 5.1 to 5.2. The low abundance of archaeal 16S rRNA gene copy numbers at this pH range then sharply increased almost 150-fold with pH, resulting in an increase in the ratio between archaeal and bacterial copy numbers from a minimum of 0.002 to more than 0.07 at pH 8. The nonuniform archaeal response to pH could reflect variation in the archaeal community composition along the gradient, with some archaea adapted to acidic conditions and others to neutral to slightly alkaline conditions. This suggestion is reinforced by observations of contrasting outcomes of the (competitive) interactions between archaea, bacteria, and fungi toward the lower and higher ends of the examined pH gradient.

scholarly journals Discrimination of Multiple Dehalococcoides Strains in a Trichloroethene Enrichment by Quantification of Their Reductive Dehalogenase Genes

2006 ◽  
Vol 72 (9) ◽  
pp. 5877-5883 ◽  
Author(s):  
Victor F. Holmes ◽  
Jianzhong He ◽  
Patrick K. H. Lee ◽  
Lisa Alvarez-Cohen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy ◽  
Rrna Gene ◽  
Sequence Matching ◽  
Relative Copy Number ◽  
Reductive Dehalogenase ◽  
Copy Numbers ◽  
Reductase Gene ◽  
Reductive Dehalogenase Genes

ABSTRACT While many anaerobic microbial communities are capable of reductively dechlorinating tetrachloroethene (PCE) and trichloroethene (TCE) to dichloroethene (DCE), vinyl chloride (VC), and finally ethene, the accumulation of the highly toxic intermediates, cis-DCE (cDCE) and VC, presents a challenge for bioremediation processes. Members of the genus Dehalococcoides are apparently solely responsible for dechlorination beyond DCE, but isolates of Dehalococcoides each metabolize only a subset of PCE dechlorination intermediates and the interactions among distinct Dehalococcoides strains that result in complete dechlorination are not well understood. Here we apply quantitative PCR to 16S rRNA and reductase gene sequences to discriminate and track Dehalococcoides strains in a TCE enrichment derived from soil taken from the Alameda Naval Air Station (ANAS) using a four-gene plasmid standard. This standard increased experimental accuracy such that 16S rRNA and summed reductase gene copy numbers matched to within 10%. The ANAS culture was found to contain only a single Dehalococcoides 16S rRNA gene sequence, matching that of D. ethenogenes 195, but both the vcrA and tceA reductive dehalogenase genes. Quantities of these two genes in the enrichment summed to the quantity of the Dehalococcoides 16S rRNA gene. Further, between ANAS subcultures enriched on TCE, cDCE, or VC, the relative copy number of the two dehalogenases shifted 14-fold, indicating that the genes are present in two different Dehalococcoides strains. Comparison of cell yields in VC-, cDCE-, and TCE-enriched subcultures suggests that the tceA-containing strain is responsible for nearly all of the TCE and cDCE metabolism in ANAS, whereas the vcrA-containing strain is responsible for all of the VC metabolism.

scholarly journals Correlation of Dehalococcoides 16S rRNA and Chloroethene-Reductive Dehalogenase Genes with Geochemical Conditions in Chloroethene-Contaminated Groundwater

2009 ◽  
Vol 76 (3) ◽  
pp. 843-850 ◽  
Author(s):  
Bas van der Zaan ◽  
Fredericke Hannes ◽  
Nanne Hoekstra ◽  
Huub Rijnaarts ◽  
Willem M. de Vos ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gene Copy ◽  
Rrna Gene ◽  
Chlorinated Ethene ◽  
Monitoring Wells ◽  
Geochemical Conditions ◽  
Copy Numbers ◽  
Gene Copies ◽  
Reductase Gene ◽  
Gene Copy Numbers

ABSTRACT Quantitative analysis of genes that code for Dehalococcoides 16S rRNA and chloroethene-reductive dehalogenases TceA, VcrA, and BvcA was done on groundwater sampled from 150 monitoring wells spread over 11 chlorinated ethene polluted European locations. Redundancy analysis was used to relate molecular data to geochemical conditions. Dehalococcoides 16S rRNA- and vinyl chloride (VC)-reductase genes were present at all tested locations in concentrations up to 106 gene copies per ml of groundwater. However, differences between and also within locations were observed. Variation in Dehalococcoides 16S rRNA gene copy numbers were most strongly correlated to dissolved organic carbon concentration in groundwater and to conditions appropriate for biodegradation of chlorinated ethenes (U.S. Environmental Protection Agency score). In contrast, vcrA gene copy numbers correlated most significantly to VC and chlorinated ethene concentrations. Interestingly, bvcA and especially tceA were more correlated with oxidizing conditions. In groundwater microcosms, dechlorination of 1 mM VC was correlated to an increase of vcrA and/or bvcA gene copies by 2 to 4 orders of magnitude. Interestingly, in 34% of the monitoring wells and in 40% of the active microcosms, the amount of individual VC-reductase gene copies exceeded that of Dehalococcoides 16S rRNA gene copies. It is concluded that the geographical distribution of the genes was not homogeneous, depending on the geochemical conditions, whereby tceA and bvcA correlated to more oxidized conditions than Dehalococcoides 16S rRNA and vcrA. Because the variation in VC-reductase gene numbers was not directly correlated to variation in Dehalococcoides spp., VC-reductase genes are better monitoring parameters for VC dechlorination capacity than Dehalococcoides spp.

scholarly journals Gene copy normalization of the 16S rRNA gene cannot outweigh the methodological biases of sequencing

2019 ◽  
Author(s):  
Robert Starke ◽  
Daniel Morais
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy ◽  
Rrna Gene ◽  
16S Sequencing ◽  
Copy Numbers ◽  
Α Diversity ◽  
Golden Standard ◽  
The 16S Rrna Gene ◽  
Mock Communities

The 16S rRNA gene is the golden standard target of sequencing to uncover the composition of bacterial communities but the presence of multiple copies of the gene makes gene copy normalization (GCN) inevitable. Even though GCN resulted in abundances closer to the metagenome, it should be validated by communities with known composition as both amplicon and shotgun sequencing are prone to methodological biases. Here we compared the composition of three mock communities to the composition derived from 16S sequencing without and with GCN. In all of them, the 16S composition was different from the mock community and GCN improved the picture only in the community with the lowest Shannon diversity. Albeit with low abundance, half of the identified genera were not present in the mock communities. Our approach provides empirical evidence to the methodological biases introduced by sequencing that was only counteracted by GCN in the case of low α-diversity, potentially due to the small number of bacterial taxa with known gene copy numbers. We thus cannot recommend the use of GCN moving forward and it is questionable whether a complete catalogue of 16S rRNA copy numbers can outweigh the methodological biases of sequencing.

Monoglobus pectinilyticus gen. nov., sp. nov., a pectinolytic bacterium isolated from human faeces.

2020 ◽  
Author(s):  
CC Kim ◽  
WJ Kelly ◽  
ML Patchett ◽  
GW Tannock ◽  
Z Jordens ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Middle Lamella ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Citrus Peel ◽  
Human Faeces ◽  
The Family

© 2017 IUMS. A novel anaerobic pectinolytic bacterium (strain 14T) was isolated from human faeces. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 14T belonged to the family Ruminococcaceae, but was located separately from known clostridial clusters within the taxon. The closest cultured relative of strain 14T was Acetivibrio cellulolyticus (89.7% sequence similarity). Strain 14T shared ~99% sequence similarity with cloned 16S rRNA gene sequences from uncultured bacteria derived from the human gut. Cells were Gram-stain-positive, non-motile cocci approximately 0.6μm in diameter. Strain 14T fermented pectins from citrus peel, apple, and kiwifruit as well as carbohydrates that are constituents of pectins and hemicellulose, such as galacturonic acid, xylose, and arabinose. TEM images of strain 14T, cultured in association with plant tissues, suggested extracellular fibrolytic activity associated with the bacterial cells, forming zones of degradation in the pectin-rich regions of middle lamella. Phylogenetic and phenotypic analysis supported the differentiation of strain 14T as a novel genus in the family Ruminococcaceae. The name Monoglobus pectinilyticus gen. nov., sp. nov. is proposed; the type strain is 14T (JCM 31914T=DSM 104782T).

scholarly journals The Termite Group I Phylum Is Highly Diverse and Widespread in the Environment

2007 ◽  
Vol 73 (20) ◽  
pp. 6682-6685 ◽  
Author(s):  
Daniel P. R. Herlemann ◽  
Oliver Geissinger ◽  
Andreas Brune
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Specific Primers ◽  
Environmental Distribution ◽  
Data Set ◽  
Group I

ABSTRACT The bacterial candidate phylum Termite Group I (TG-1) presently consists mostly of “Endomicrobia,” which are endosymbionts of flagellate protists occurring exclusively in the hindguts of termites and wood-feeding cockroaches. Here, we show that public databases contain many, mostly undocumented 16S rRNA gene sequences from other habitats that are affiliated with the TG-1 phylum but are only distantly related to “Endomicrobia.” Phylogenetic analysis of the expanded data set revealed several diverse and deeply branching lineages comprising clones from many different habitats. In addition, we designed specific primers to explore the diversity and environmental distribution of bacteria in the TG-1 phylum.

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