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.

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 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 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 rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

2019 ◽  
Author(s):  
Jean-Claude OGIER ◽  
Sylvie Pagès ◽  
Maxime Galan ◽  
Matthieu Barret ◽  
Sophie Gaudriault
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Taxonomic Resolution ◽  
Taxonomic Structure ◽  
Rrna Gene ◽  
Specificity And Sensitivity ◽  
The 16S Rrna Gene ◽  
Mock Communities

Abstract Background Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3V4 hypervariable region of the 16S rRNA gene. Results We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3V4. We applied both primers to infective juveniles of the nematode Steinernema glaseri. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium Xenorhabdus poinarii. Conclusions Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene.

scholarly journals rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

2019 ◽  
Author(s):  
Jean-Claude OGIER ◽  
Sylvie Pagès ◽  
Maxime Galan ◽  
Matthieu Barret ◽  
Sophie Gaudriault
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Taxonomic Resolution ◽  
Taxonomic Structure ◽  
Rrna Gene ◽  
Specificity And Sensitivity ◽  
The 16S Rrna Gene ◽  
Mock Communities

Abstract Background Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3V4 hypervariable region of the 16S rRNA gene. Results We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3V4. We applied both primers to infective juveniles of the nematode Steinernema glaseri. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium Xenorhabdus poinarii. Conclusions Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene.

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 Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon

F1000Research ◽  
2019 ◽  
Vol 7 ◽  
pp. 1755 ◽  
Author(s):  
Anna Cuscó ◽  
Carlotta Catozzi ◽  
Joaquim Viñes ◽  
Armand Sanchez ◽  
Olga Francino
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Genetic Marker ◽  
Species Level ◽  
Full Length ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Microbiota Composition ◽  
The 16S Rrna Gene ◽  
Mock Communities

Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples are prone to contain DNA from other sources (e.g. host or environment). The usual approach is sequencing short regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. To achieve an increased taxonomic resolution, we aim to develop long-amplicon PCR-based approaches using Nanopore sequencing. We assessed two different genetic markers: the full-length 16S rRNA (~1,500 bp) and the 16S-ITS-23S region from the rrn operon (4,300 bp). Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities and two pools of low-biomass samples (dog skin). Nanopore sequencing was performed on MinION™ using the 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using EPI2ME or Minimap2 with rrn database. Consensus sequences of the 16S-ITS-23S genetic marker were obtained using canu. Results: The full-length 16S rRNA and the 16S-ITS-23S region of the rrn operon were used to retrieve the microbiota composition of the samples at the genus and species level. For the Staphylococcus pseudintermedius isolate, the amplicons were assigned to the correct bacterial species in ~98% of the cases with the16S-ITS-23S genetic marker, and in ~68%, with the 16S rRNA gene when using EPI2ME. Using mock communities, we found that the full-length 16S rRNA gene represented better the abundances of a microbial community; whereas, 16S-ITS-23S obtained better resolution at the species level. Finally, we characterized low-biomass skin microbiota samples and detected species with an environmental origin. Conclusions: Both full-length 16S rRNA and the 16S-ITS-23S of the rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, targeting the 16S-ITS-23S of the rrn operon would be the best choice.

scholarly journals rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

2019 ◽  
Author(s):  
Jean-Claude Ogier ◽  
Sylvie Pagès ◽  
Maxime Galan ◽  
Mathieu Barret ◽  
Sophie Gaudriault
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Taxonomic Resolution ◽  
Taxonomic Structure ◽  
Rrna Gene ◽  
Specificity And Sensitivity ◽  
The 16S Rrna Gene ◽  
Mock Communities

AbstractBackgroundMicrobiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3V4 hypervariable region of the 16S rRNA gene.ResultsWe first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3V4. We applied both primers to infective juveniles of the nematode Steinernema glaseri. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium Xenorhabdus poinarii.ConclusionsOur results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene.

scholarly journals Quantification of the Detrimental Effect of a Single Primer-Template Mismatch by Real-Time PCR Using the 16S rRNA Gene as an Example

2008 ◽  
Vol 74 (5) ◽  
pp. 1660-1663 ◽  
Author(s):  
D. Bru ◽  
F. Martin-Laurent ◽  
L. Philippot
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy Number ◽  
Pcr Amplification ◽  
Gene Copy ◽  
Rrna Gene ◽  
Template Dna ◽  
Single Primer ◽  
The 16S Rrna Gene ◽  
Extension Sequence

ABSTRACT We investigated the effects of internal primer-template mismatches on the efficiency of PCR amplification using the 16S rRNA gene as the model template DNA. We observed that the presence of a single mismatch in the second half of the primer extension sequence can result in an underestimation of up to 1,000-fold of the gene copy number, depending on the primer and position of the mismatch.

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