Evaluating the Efficiency of RRN (16S-ITS-23S) Operon Sequencing
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB72454 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR12650353.fastq.gz | Fastqsanger.gz | |||
| ERR12650354.fastq.gz | Fastqsanger.gz | |||
| ERR12650355.fastq.gz | Fastqsanger.gz | |||
| ERR12650356.fastq.gz | Fastqsanger.gz | |||
| ERR12650357.fastq.gz | Fastqsanger.gz |
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Project description:Rapid advancements in long-read sequencing have facilitated species-level microbial profiling through full-length 16S rRNA sequencing (~ 1500 bp), and more notably, by the newer 16S-ITS-23S ribosomal RNA operon (RRN) sequencing (~ 4500 bp). RRN sequencing is emerging as a superior method for species resolution, exceeding the capabilities of short-read and full-length 16S rRNA sequencing. However, being in its early stages of development, RRN sequencing has several underexplored or understudied elements, highlighting the need for a critical and thorough examination of its methodologies. Key areas that require detailed analysis include understanding how primer pairs, sequencing platforms, and classifiers and databases affect the accuracy of species resolution achieved through RRN sequencing. Our study addresses these gaps by evaluating the effect of primer pairs using four RRN primer combinations, and that of sequencing platforms by employing PacBio and Oxford Nanopore Technologies (ONT) systems. Furthermore, two classification methods (Minimap2 and OTU clustering), in combination with four RRN reference databases (MIrROR, rrnDB, and two versions of GROND) were compared to identify consistent and accurate classification methods with RRN sequencing. Here we demonstrate that RRN primer pair choice and sequencing platform do not substantially bias taxonomic profiles for most of the tested mock communities, while classification methods significantly impact the accuracy of species-level assignments. Of the classification methods tested, Minimap2 classifier in combination with the GROND database most consistently provided accurate species-level classification across the communities tested, irrespective of sequencing platform.
| S-EPMC11754871 | biostudies-literature
Project description:Full-length 16S-23S rRNA gene sequences from pre and post PrEP specimens
| PRJEB29019 | ENA
Project description:The 16S-23S spacer regions of two ribosomal operons (rrnA and rrnE) have been sequenced in seven representatives of the Salmonella enterica subspecies. Isolated nucleotide substitutions were found at the same sites as in Escherichia coli but the number of polymorphic sites was much larger, as could be expected for a more heterogeneous species. Still, as in E. coli, most of the variation found was due to insertions and/or deletions affecting blocks of nucleotides generally located at equivalent regions of the putative secondary structure for both species. Isolated polymorphic sites generated phylogenetic trees generally consistent with the subspecies structure and the accepted relationships among the subspecies. However, the sequences of rrnE put subspecies I closer to E. coli K-12 than to the other S. enterica subspecies. The distribution of polymorphisms affecting blocks of nucleotides was much more random, and the presence of equivalent sequences in distantly related subspecies, and even in E. coli, could reflect relatively frequent horizontal transfer. The smallest 16S-23S spacers in other genera of the family Enterobacteriaceae were also sequenced. As expected, the level of variation was much larger. Still, the phylogenetic tree inferred is consistent with those of 16S rRNA or housekeeping genes.
| S-EPMC107142 | biostudies-literature
Project description:Pseudomonas entomophila strain:23S Genome sequencing
| PRJNA669330 | ENA
Project description: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.
| S-EPMC6372929 | biostudies-literature
Project description:Sequence comparison of 16S rRNA PCR amplicons is an established approach to taxonomically identify bacterial isolates and profile complex microbial communities. One potential application of recent advances in long-read sequencing technologies is to sequence entire rRNA operons and capture significantly more phylogenetic information compared to sequencing of the 16S rRNA (or regions thereof) alone, with the potential to increase the proportion of amplicons that can be reliably classified to lower taxonomic ranks. Here we describe GROND (Genome-derived Ribosomal Operon Database), a publicly available database of quality-checked 16S-ITS-23S rRNA operons, accompanied by multiple taxonomic classifications. GROND will aid researchers in analysis of their data and act as a standardised database to allow comparison of results between studies.
| S-EPMC11261877 | biostudies-literature
Project description:This study explores the effects of prolonged stationary phase, on whole genome expression of Escherichia coli. In summary these results imply that prolonged stationary phase cells have shown less growth rate compared to fresh cells due to the low abundance of transcripts for translation initiation factor infC, and for all the seven rrn operons of 5S, 16S, 23S ribosomal RNA which further delayed in protein synthesis leading to low biomass in prolonged stationary phase cells.
2013-09-12 | GSE50828 | GEO
Project description:Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286-37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput.
| S-EPMC6849856 | biostudies-literature
Project description:Clostridium difficile is a major spore-forming environmental pathogen that causes serious health problems in patients undergoing antibiotic therapy. Consequently, reliable and sensitive methods for typing individual strains are required for epidemiological and environmental studies. Ribotyping is generally considered the best method, but it fails to account for sequence diversity which might exist in intergenic 16S-23S rRNA spacer regions (ISRs) within and among strains of this organism. Therefore, this study was undertaken to compare the sequence of each individual ISR in five strains of C. difficile to explore the extent of this diversity and see whether such information might provide the basis for more sensitive and discriminatory strain typing methods. After targeted PCR amplification, cloning, and sequencing, the diversity of the ISRs was used as a measure of rRNA operon copy number. In C. difficile strains 630, ATCC 43593, A, and B, 11, 11, 7, and 8 ISR length variants, respectively, were found (containing different combinations of sequence groups [i to xiii]), suggesting 11, 11, 7, and 8 rrn copies in the respective strains. Many ISRs of the same length differed markedly in their sequences, and some of these were restricted in occurrence to a single strain. Most of these ISRs did not contain any tRNA genes, and only single copies of the tRNA(Ala) gene were found in those that did. The presence of ISR sequence groups (i to xiii) varied between strains, with some found in one, two, three, four, or all five strains. We conclude that the intergenic 16S-23S rRNA spacer regions showed a high degree of diversity, not only among the rrn operons in different strains and different rrn copies in a single strain but also among ISRs of the same length. It appears that C. difficile ISRs vary more at the inter- and intragenic levels than those of other species as determined by empirical comparison of sequences. The precise characterization of these sequences has demonstrated a high level of mosaic sequence block rearrangements that are present or absent in multiple strain-variable rrn copies within and between five different strains of C. difficile.
| S-EPMC1636144 | biostudies-literature