Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing.
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ABSTRACT:
SUBMITTER: Smith AM
PROVIDER: S-EPMC6522004 | biostudies-literature | 2019
REPOSITORIES: biostudies-literature
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Project description:AIMS:To define epidemiology, clinical disease, and outcome of gemella bacteraemia by 16S rRNA gene sequencing. To examine the usefulness of the Vitek, API, and ATB systems in identifying two gemella species. METHODS:All alpha haemolytic streptococci other than Streptococcus pneumoniae isolated from blood cultures during a six year period were identified by conventional biochemical methods, the Vitek system, and the API system. 16S rRNA gene sequencing was performed on all isolates identified by both kits as gemella with >or= 95% confidence or by either kit as any bacterial species with < 95% confidence. The ATB expression system was used to identify the two isolates that were defined as gemella species by 16S rRNA gene sequencing. RESULTS:Of the 302 alpha haemolytic streptococci other than S pneumoniae isolated, one was identified as Gemella morbillorum, and another as Gemella haemolysans by 16S rRNA gene sequencing. The patient with monomicrobial G morbillorum bacteraemia was a 66 year old man with community acquired infective endocarditis with septic thromboemboli. The patient with G haemolysans bacteraemia was a 41 year old woman with hospital acquired polymicrobial bacteraemia during the neutropenic period of an autologous bone marrow transplant for non-Hodgkin's lymphoma, the first case of its kind in the English literature. The API and ATB expression systems only identified the second strain as G haemolysans at 94% and 99% confidence, respectively, whereas the Vitek system identified none of the two strains correctly at > 70% confidence. CONCLUSIONS:Gemella bacteraemia is uncommon. 16S rRNA gene sequencing is the method of choice for identification of gemella and gemella-like isolates.
| S-EPMC1770062 | biostudies-literature
Project description:BackgroundGlobicatella are streptococcus-like organisms that have been rarely isolated from clinical specimens. Their epidemiology and clinical significance remain largely unknown.AimsTo describe two cases of Globicatella bacteraemia identified by 16S ribosomal RNA (rRNA) gene sequencing.MethodsTwo unidentified streptococcus-like bacteria isolated from blood cultures of patients were subject to 16S rRNA gene sequencing.ResultsTwo cases of Globicatella bacteraemia were identified by 16S rRNA gene sequencing. In the first case, a gram positive coccus was isolated from the blood culture of an 80 year old woman with diabetes mellitus and nosocomial sepsis, who died the day after developing the bacteraemia. The bacterium was unidentified by conventional phenotypic tests, the Vitek (gram positive identification) and the ATB expression (ID32 Strep) systems. In the second case, a similar bacterium was isolated from the blood culture of a 92 year old woman with polymicrobial acute pyelonephritis complicated by septic shock, who subsequently recovered after antibiotic treatment. 16S rRNA gene sequencing of the two isolates showed 0.5% nucleotide difference from that of G. sulfidifaciens and 0.7% nucleotide difference from that of G. sanguinis, indicating that they were Globicatella species.ConclusionsBecause Globicatella is rarely encountered in clinical microbiology laboratories, it may have been overlooked or misidentified in these cases. 16S rRNA gene sequencing is a useful tool to better characterise the epidemiology and clinical significance of Globicatella.
| S-EPMC1860353 | biostudies-literature
Project description:BackgroundOwing to problems in accurate species identification of the diverse genus clostridium, the epidemiology and pathogenicity of many species are not fully understood. Moreover, previous studies on clostridium bacteraemia have been limited and relied only on phenotypic species identification.AimsTo characterise the epidemiology, disease spectrum, and outcome of clostridium bacteraemia using 16S ribosomal RNA (rRNA) gene sequencing.MethodDuring a four year period (1998-2001), all cases of clostridium bacteraemia were prospectively studied and all "non-perfringens" clostridium isolates identified to the species level by 16S rRNA gene sequencing.ResultsFifty one blood culture isolates were identified as Clostridium perfringens and 17 belonged to 11 other clostridium species. The first case of C disporicum infection and two cases of clostridium bacteraemia in children with intussusception were also described. Of the 68 clostridium isolates from 68 different patients, 38 were associated with clinically relevant bacteraemia. The gastrointestinal and hepatobiliary tracts were common sites of both underlying disease and portal of entry in these patients. Clostridium perfringens accounted for 79% of all clinically relevant bacteraemia, with the remainder caused by a diversity of species. The attributable mortality rate of clinically relevant clostridium bacteraemia was 29%. Younger age and underlying gastrointestinal/hepatobiliary tract disease were associated with mortality (p < 0.05).ConclusionsPatients with clinically relevant clostridium bacteraemia should be investigated for the presence of underlying disease processes in the gastrointestinal or hepatobiliary tracts. 16S rRNA gene analysis will continue to be useful in further understanding the pathogenicity of various clostridium species.
| S-EPMC1770585 | biostudies-literature
Project description:Corynebacterium species are non-fermentous Gram-positive bacilli that are normal flora of human skin and mucous membranes and are commonly isolated in clinical specimens. Non-diphtheriae Corynebacterium are regarded as contaminants when found in blood culture. Currently, Corynebacterium striatum is considered one of the emerging nosocomial agents implicated in endocarditis and serious infections. We report a case of native-valve infective endocarditis caused by C. striatum, which was misidentified by automated identification system but identified accurately by 16S ribosomal RNA sequencing, in a 55-year-old male patient. The patient had two mobile vegetations on his mitral valve, both of which had high embolic risk. Through surgical valve replacement and an antibiotic regimen, the patient recovered completely. In unusual clinical scenarios, C. striatum should not be simply dismissed as a contaminant when isolated from clinical specimens. The possibility of C. striatum infection should be considered even in an immunocompetent patient, and we suggest a genotypic assay, such as 16S rRNA sequencing, to confirm species identity.
| S-EPMC5048009 | biostudies-literature
Project description:High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes.
| S-EPMC7768885 | biostudies-literature
Project description:Ribosomal RNAs are decorated by numerous post-transcriptional modifications whose exact roles in ribosome biogenesis, function, and human pathophysiology remain largely unknown. Here, we report a targeted direct rRNA sequencing approach involving a substrate selection step and demonstrate its suitability to identify differential modification sites in combination with the JACUSA2 software. We compared JACUSA2 to other tools designed for RNA modification detection and show that JACUSA2 outperforms other software with regard to detection of base modifications such as methylation, acetylation and aminocarboxypropylation. To illustrate its widespread usability, we applied our method to a collection of CRISPR-Cas9 engineered colon carcinoma cells lacking specific enzymatic activities responsible for particular rRNA modifications and systematically compared them to isogenic wild-type RNAs. Besides the numerous 2'-O methylated riboses and pseudouridylated residues, our approach was suitable to reliably identify differential base methylation and acetylation events. Importantly, our method does not require any prior knowledge of modification sites or the need to train complex models. We further report for the first time detection of human rRNA modifications by direct RNA-sequencing on Flongle flow cells, the smallest-scale nanopore flow cell available to date. The use of these smaller flow cells reduces RNA input requirements, making our workflow suitable for the analysis of samples with limited availability and clinical work.
| S-EPMC10464549 | biostudies-literature
Project description:In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization.
| S-EPMC4204986 | biostudies-literature
Project description:Little is known about the relative importance of the four species of Lancefield group G beta-hemolytic streptococci in causing bacteremia and the factors that determine the outcome for patients with group G beta-hemolytic streptococcal bacteremia. From 1997 to 2000, 75 group G beta-hemolytic streptococcal strains were isolated from the blood cultures of 66 patients. Sequencing of the 16S rRNA genes of the group G beta-hemolytic streptococci showed that all 75 isolates were Streptococcus dysgalactiae subspecies equisimilis. The API system (20 STREP) and Vitek system (GPI) successfully identified 65 (98.5%) and 62 (93.9%) isolates, respectively, as S. dysgalactiae subspecies equisimilis with >95% confidence, whereas the ATB Expression system (ID32 STREP) only successfully identified 49 isolates (74.2%) as S. dysgalactiae subspecies equisimilis with >95% confidence. The median age of the patients was 76 years (range, 33 to 99 years). Fifty-six patients (85%) were over 60 years old. All patients had underlying diseases. No source of the bacteremia was identified (primary bacteremia) in 34 patients (52%), whereas 17 (26%) had cellulitis and 8 (12%) had bed sore or wound infections. Fifty-eight patients (88%) had community-acquired group G streptococcal bacteremia. Sixty-two patients (94%) had group G Streptococcus recovered in one blood culture, whereas 4 patients (6%) had it recovered in multiple blood cultures. Fifty-nine patients (89%) had group G Streptococcus as the only bacterium recovered in their blood cultures, whereas in 7 patients other bacteria were recovered concomitantly with the group G Streptococcus in the blood cultures (Staphylococcus aureus in 3, Clostridium perfringens in 2, Citrobacter freundii in 1, and Bacteroides fragilis in 1). Overall, 10 patients (15%) died. Male sex, diagnosis other than cellulitis, hospital-acquired bacteremia, and multiple positive blood cultures were associated with mortality [P < 0.005 (relative risk [RR] = 7.6), P < 0.05 (RR = 3.7), P < 0.005 (RR = 5.6), and P < 0.05 (RR = 5.6), respectively]. Unlike group C beta-hemolytic streptococcal bacteremia, group G beta-hemolytic streptococcal bacteremia is not a zoonotic infection in Hong Kong.
| S-EPMC88311 | biostudies-literature
Project description:A platform for highly parallel direct sequencing of native RNA strands was recently described by Oxford Nanopore Technologies, but despite initial efforts it remains crucial to further investigate the technology for quantification of complex transcriptomes. Here we undertake native RNA sequencing of polyA + RNA from two human cell lines, analysing ~5.2 million aligned native RNA reads. To enable informative comparisons, we also perform relevant ONT direct cDNA- and Illumina-sequencing. We find that while native RNA sequencing does enable some of the anticipated advantages, key unexpected aspects currently hamper its performance, most notably the quite frequent inability to obtain full-length transcripts from single reads, as well as difficulties to unambiguously infer their true transcript of origin. While characterising issues that need to be addressed when investigating more complex transcriptomes, our study highlights that with some defined improvements, native RNA sequencing could be an important addition to the mammalian transcriptomics toolbox.
| S-EPMC6668388 | biostudies-literature
Project description:BackgroundSingle-cell RNA-sequencing (scRNA-seq) relies on PCR amplification to retrieve information from vanishingly small amounts of starting material. To selectively enrich mRNA from abundant non-polyadenylated transcripts, poly(A) selection is a key step during library preparation. However, some transcripts, such as mitochondrial genes, can escape this elimination and overwhelm libraries. Often, these transcripts are removed in silico, but whether physical depletion improves detection of rare transcripts in single cells is unclear.ResultsWe find that a single 16S ribosomal RNA is widely enriched in planarian scRNA-seq datasets, independent of the library preparation method. To deplete this transcript from scRNA-seq libraries, we design 30 single-guide RNAs spanning its length. To evaluate the effects of depletion, we perform a side-by-side comparison of the effects of eliminating the 16S transcript and find a substantial increase in the number of genes detected per cell, coupled with virtually complete loss of the 16S RNA. Moreover, we systematically determine that library complexity increases with a limited number of PCR cycles following CRISPR treatment. When compared to in silico depletion of 16S, physically removing it reduces dropout rates, retrieves more clusters, and reveals more differentially-expressed genes.ConclusionsOur results show that abundant transcripts reduce the retrieval of informative transcripts in scRNA-seq and distort the analysis. Physical removal of these contaminants enables the detection of rare transcripts at lower sequencing depth, and also outperforms in silico depletion. Importantly, this method can be easily customized to deplete any abundant transcript from scRNA-seq libraries.
| S-EPMC10246003 | biostudies-literature