Journal articles: '16S rRNA gene sequencing'

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Il Jun, Kang, Jangsup Moon, Taek Soo Kim, Chang Kyung Kang, Song Mi Moon, Kyoung-Ho Song, Pyoeng Gyun Choe, et al. "238. Direct identification of Bacterial Species with MinION Nanopore Sequencer In Clinical Specimens Suspected of Polybacterial Infection." Open Forum Infectious Diseases 6, Supplement_2 (October 2019): S136. http://dx.doi.org/10.1093/ofid/ofz360.313.

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Abstract Background Conventional culture tests usually identify only a few bacterial species, which can grow well in the culture system, in the cases of polybacterial infection. 16S rRNA gene nanopore sequencing enables semi-quantitative identification of bacterial genetic materials. We aimed to evaluate usefulness of 16s rRNA gene nanopore sequencing in the cases suspected of polybacterial infection. Methods The research was conducted in a single university hospital for one year. Conventional bacterial culture identification and nanopore sequencing of 16s rRNA gene were carried out simultaneously for cases where polybacterial infection is strongly suspected. Blood agar plate was used for conventional culture, and Microscan (Beckman Coulter, United States) and Vitek 2 (Biomerieux, FR) automated systems were used for identification. For nanopore sequencing, 16S rRNA gene PCR was performed from the clinical specimens, and sequencing libraries were generated from the PCR products using the rapid barcoding sequencing kit (Oxford nanopore technologies, UK). MinION sequencing was performed for 1–3 hours and the generated reads were analyzed using the EPI2ME 16S BLAST workflow. Results Specimens were obtained from 15 patients; 6 liver abscess, 2 psoas abscess, 2 thigh abcess, 1 paraspinal abscess, 1 mycotic aneurysm, 1 necrotizing fasciitis, 1 fingertip gangrene and 1 abscess in coccyx area. 16s rRNA gene nanopore sequencing showed monobacterial organism in 8 (53.3%) specimens and polybacterial organisms in 7 (46.6%) specimens. In three (37.5%) cases of 8 cases with monobacterial infections identified by 16s rRNA gene sequencing, no organism was grown in conventional culture, possibly due to previous antibiotic administration. Notably, among 8 cases with polybacterial infection by 16s rRNA gene nanopore sequencing test, traditional culture test showed polybacterial infection in only two (25%) cases and single bacterial organism was identified in the other 6 (75%) cases. Conclusion Nanopore sequencing of 16s rRNA gene using the MinION sequencer may be useful for identification of causing microorganism and differentiation between monobacterial and polybacterial infection when polybacterial infection is suspected. Disclosures All authors: No reported disclosures.

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Yoshimura, Kazuaki, Nobuo Morotomi, Kazumasa Fukuda, Masahiro Nakano, Masamichi Kashimura, Toru Hachisuga, and Hatsumi Taniguchi. "Intravaginal microbial flora by the 16S rRNA gene sequencing." American Journal of Obstetrics and Gynecology 205, no. 3 (September 2011): 235.e1–235.e9. http://dx.doi.org/10.1016/j.ajog.2011.04.018.

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Cloud, Joann L., Jay J. Meyer, June I. Pounder, Kenneth C. Jost, Amy Sweeney, Karen C. Carroll, and Gail L. Woods. "Mycobacterium arupense sp. nov., a non-chromogenic bacterium isolated from clinical specimens." International Journal of Systematic and Evolutionary Microbiology 56, no. 6 (June 1, 2006): 1413–18. http://dx.doi.org/10.1099/ijs.0.64194-0.

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Several Mycobacterium-like organisms related to the Mycobacterium terrae complex have been isolated from clinical samples. In the clinical microbiology laboratory, partial 16S rRNA gene sequencing (approximately the first 500 bp) rather than full 16S rRNA gene sequencing is often used to identify Mycobacterium species. Partial 16S rRNA gene sequence analysis revealed 100 % similarity between 65 clinical isolates and Mycobacterium sp. MCRO 6 (GenBank accession no. X93032). Even after sequencing the nearly full-length 16S rRNA gene, closest similarity was only 99.6 % to Mycobacterium nonchromogenicum ATCC 19530T. Sequencing of the nearly full-length 16S rRNA gene, the 16S–23S internal transcribed spacer region and the hsp65 gene did not reveal genotypic identity with the type strains of M. nonchromogenicum, M. terrae or Mycobacterium triviale. Although sequence analysis suggested that these clinical isolates represented a novel species, mycolic acid analysis by HPLC failed to distinguish them from M. nonchromogenicum. Therefore, phenotypic analysis including growth characterization, antibiotic susceptibility testing and biochemical testing was performed. These strains from clinical samples should be recognized as representing a novel species of the genus Mycobacterium, for which the name Mycobacterium arupense sp. nov. is proposed. The type strain is AR30097T (=ATCC BAA-1242T=DSM 44942T).

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Schloss, Patrick D., Matthew L. Jenior, Charles C. Koumpouras, Sarah L. Westcott, and Sarah K. Highlander. "Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system." PeerJ 4 (March 28, 2016): e1869. http://dx.doi.org/10.7717/peerj.1869.

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Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina’s MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3–V5, V1–V3, V1–V5, V1–V6, and V1–V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1–V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina’s MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

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Fujimoto, Naoshi, Keigo Mizuno, Tomoki Yokoyama, Akihiro Ohnishi, Masaharu Suzuki, Satoru Watanabe, Kenji Komatsu, et al. "Community analysis of picocyanobacteria in an oligotrophic lake by cloning 16S rRNA gene and 16S rRNA gene amplicon sequencing." Journal of General and Applied Microbiology 61, no. 5 (2015): 171–76. http://dx.doi.org/10.2323/jgam.61.171.

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Callahan, Benjamin J., Joan Wong, Cheryl Heiner, Steve Oh, Casey M. Theriot, Ajay S. Gulati, Sarah K. McGill, and Michael K. Dougherty. "High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution." Nucleic Acids Research 47, no. 18 (July 3, 2019): e103-e103. http://dx.doi.org/10.1093/nar/gkz569.

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AbstractTargeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

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Reischl, U., K. Feldmann, L. Naumann, B. J. M. Gaugler, B. Ninet, B. Hirschel, and S. Emler. "16S rRNA Sequence Diversity in Mycobacterium celatum Strains Caused by Presence of Two Different Copies of 16S rRNA Gene." Journal of Clinical Microbiology 36, no. 6 (1998): 1761–64. http://dx.doi.org/10.1128/jcm.36.6.1761-1764.1998.

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Direct sequencing of the 16S rRNA gene (16S rDNA) ofMycobacterium celatum isolates showed ambiguities, suggesting heterogeneity. Cloned 16S rDNA yielded two copies of the gene, which differed by insertion of a thymine at position 214 and by additional mismatches. Restriction fragment length polymorphism analysis confirmed the presence of two copies of 16S rDNA within the bacterial chromosome.

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Kennedy, Katherine, Michael W. Hall, Michael D. J. Lynch, Gabriel Moreno-Hagelsieb, and Josh D. Neufeld. "Evaluating Bias of Illumina-Based Bacterial 16S rRNA Gene Profiles." Applied and Environmental Microbiology 80, no. 18 (July 7, 2014): 5717–22. http://dx.doi.org/10.1128/aem.01451-14.

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ABSTRACTMassively parallel sequencing of 16S rRNA genes enables the comparison of terrestrial, aquatic, and host-associated microbial communities with sufficient sequencing depth for robust assessments of both alpha and beta diversity. Establishing standardized protocols for the analysis of microbial communities is dependent on increasing the reproducibility of PCR-based molecular surveys by minimizing sources of methodological bias. In this study, we tested the effects of template concentration, pooling of PCR amplicons, and sample preparation/interlane sequencing on the reproducibility associated with paired-end Illumina sequencing of bacterial 16S rRNA genes. Using DNA extracts from soil and fecal samples as templates, we sequenced pooled amplicons and individual reactions for both high (5- to 10-ng) and low (0.1-ng) template concentrations. In addition, all experimental manipulations were repeated on two separate days and sequenced on two different Illumina MiSeq lanes. Although within-sample sequence profiles were highly consistent, template concentration had a significant impact on sample profile variability for most samples. Pooling of multiple PCR amplicons, sample preparation, and interlane variability did not influence sample sequence data significantly. This systematic analysis underlines the importance of optimizing template concentration in order to minimize variability in microbial-community surveys and indicates that the practice of pooling multiple PCR amplicons prior to sequencing contributes proportionally less to reducing bias in 16S rRNA gene surveys with next-generation sequencing.

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Szymczak, Aleksander, Stanisław Ferenc, Joanna Majewska, Paulina Miernikiewicz, Jan Gnus, Wojciech Witkiewicz, and Krystyna Dąbrowska. "Application of 16S rRNA gene sequencing in Helicobacter pylori detection." PeerJ 8 (May 13, 2020): e9099. http://dx.doi.org/10.7717/peerj.9099.

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Helicobacter pylori is one of the major stomach microbiome components, promoting development of inflammation and gastric cancer in humans. H. pylori has a unique ability to transform into a coccoidal form which is difficult to detect by many diagnostic methods, such as urease activity detection, and even histopathological examination. Here we present a comparison of three methods for H. pylori identification: histological assessment (with eosin, hematoxylin, and Giemsa staining), polymerase chain reaction (PCR) detection of urease (ureA specific primers), and detection by 16S rRNA gene sequencing. The study employed biopsies from the antral part of the stomach (N = 40). All samples were assessed histologically which revealed H. pylori in eight patients. Bacterial DNA isolated from the bioptates was used as a template for PCR reaction and 16S rRNA gene sequencing that revealed H. pylori in 13 and in 20 patients, respectively. Thus, 16S rRNA gene sequencing was the most sensitive method for detection of H. pylori in stomach biopsy samples.

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Angell, Inga Leena, Morten Nilsen, Karin C. Lødrup Carlsen, Kai-Håkon Carlsen, Gunilla Hedlin, Christine M. Jonassen, Benjamin Marsland, et al. "De novo species identification using 16S rRNA gene nanopore sequencing." PeerJ 8 (October 21, 2020): e10029. http://dx.doi.org/10.7717/peerj.10029.

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Nanopore sequencing is rapidly becoming more popular for use in various microbiota-based applications. Major limitations of current approaches are that they do not enable de novo species identification and that they cannot be used to verify species assignments. This severely limits applicability of the nanopore sequencing technology in taxonomic applications. Here, we demonstrate the possibility of de novo species identification and verification using hexamer frequencies in combination with k-means clustering for nanopore sequencing data. The approach was tested on the human infant gut microbiota of 3-month-old infants. Using the hexamer k-means approach we identified two new low abundant species associated with vaginal delivery. In addition, we confirmed both the vaginal delivery association for two previously identified species and the overall high levels of bifidobacteria. Taxonomic assignments were further verified by mock community analyses. Therefore, we believe our de novo species identification approach will have widespread application in analyzing microbial communities in the future.

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Valour, F., S. Boisset, L. Lebras, B. Martha, A. Boibieux, T. Perpoint, C. Chidiac, T. Ferry, and D. Peyramond. "Clostridium sordellii Brain Abscess Diagnosed by 16S rRNA Gene Sequencing." Journal of Clinical Microbiology 48, no. 9 (July 7, 2010): 3443–44. http://dx.doi.org/10.1128/jcm.00307-10.

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Abdul-Redha, Rawaa Jalil, Ulla Balslew, Jens Jørgen Christensen, and Michael Kemp. "Globicatella sanguinis bacteraemia identified by partial 16S rRNA gene sequencing." Scandinavian Journal of Infectious Diseases 39, no. 8 (January 2007): 745–48. http://dx.doi.org/10.1080/00365540701203527.

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McLaughlin, Heather P., Blake Cherney, Janetta R. Hakovirta, Rachael A. Priestley, Andrew Conley, Andrew Carter, David Hodge, et al. "Phylogenetic inference of Coxiella burnetii by 16S rRNA gene sequencing." PLOS ONE 12, no. 12 (December 29, 2017): e0189910. http://dx.doi.org/10.1371/journal.pone.0189910.

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Morey, R. E., R. L. Galloway, S. L. Bragg, A. G. Steigerwalt, L. W. Mayer, and P. N. Levett. "Species-Specific Identification of Leptospiraceae by 16S rRNA Gene Sequencing." Journal of Clinical Microbiology 44, no. 10 (October 1, 2006): 3510–16. http://dx.doi.org/10.1128/jcm.00670-06.

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Lau, S. K. P., P. C. Y. Woo, G. K. S. Woo, and K. Y. Yuen. "Catheter-Related Microbacterium Bacteremia Identified by 16S rRNA Gene Sequencing." Journal of Clinical Microbiology 40, no. 7 (July 1, 2002): 2681–85. http://dx.doi.org/10.1128/jcm.40.7.2681-2685.2002.

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Sulaiman, Imran, Benjamin G. Wu, Yonghua Li, Jun-Chieh Tsay, Maya Sauthoff, Adrienne S. Scott, Kun Ji, et al. "Functional lower airways genomic profiling of the microbiome to capture active microbial metabolism." European Respiratory Journal 58, no. 1 (January 14, 2021): 2003434. http://dx.doi.org/10.1183/13993003.03434-2020.

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BackgroundMicrobiome studies of the lower airways based on bacterial 16S rRNA gene sequencing assess microbial community structure but can only infer functional characteristics. Microbial products, such as short-chain fatty acids (SCFAs), in the lower airways have significant impact on the host's immune tone. Thus, functional approaches to the analyses of the microbiome are necessary.MethodsHere we used upper and lower airway samples from a research bronchoscopy smoker cohort. In addition, we validated our results in an experimental mouse model. We extended our microbiota characterisation beyond 16S rRNA gene sequencing with the use of whole-genome shotgun (WGS) and RNA metatranscriptome sequencing. SCFAs were also measured in lower airway samples and correlated with each of the sequencing datasets. In the mouse model, 16S rRNA gene and RNA metatranscriptome sequencing were performed.ResultsFunctional evaluations of the lower airway microbiota using inferred metagenome, WGS and metatranscriptome data were dissimilar. Comparison with measured levels of SCFAs shows that the inferred metagenome from the 16S rRNA gene sequencing data was poorly correlated, while better correlations were noted when SCFA levels were compared with WGS and metatranscriptome data. Modelling lower airway aspiration with oral commensals in a mouse model showed that the metatranscriptome most efficiently captures transient active microbial metabolism, which was overestimated by 16S rRNA gene sequencing.ConclusionsFunctional characterisation of the lower airway microbiota through metatranscriptome data identifies metabolically active organisms capable of producing metabolites with immunomodulatory capacity, such as SCFAs.

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Akihary, Claudia Valleria, and Beivy Jonathan Kolondam. "PEMANFAATAN GEN 16S rRNA SEBAGAI PERANGKAT IDENTIFIKASI BAKTERI UNTUK PENELITIAN-PENELITIAN DI INDONESIA." PHARMACON 9, no. 1 (February 28, 2020): 16. http://dx.doi.org/10.35799/pha.9.2020.27405.

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ABSTRACTThe 16S rRNA gene has hyper variable region and different for one bacterial species to another. The gene is being used as research tool to help for accurate identification of bacteria in many fields in Indonesia. As a useful tool, the 16S rRNA gene sequence is important as to explore the potencies of a bacterial species. Sequencing of this gene is very useful for research in clinical study, fisheries, marine science, agricultural science, and animal husbandry in Indonesia. Keywords: 16S rRNA gene, research tool, bacteria, Indonesia ABSTRAKGen 16S rRNA memiliki region yang sangat bervariasi dan berbeda setiap spesies bakteri. Penggunaannya sebagai perangkat penelitian, gen 16S rRNA telah banyak membantu dalam proses identifikasi berbagai jenis bakteri secara akurat untuk berbagai penelitian di Indonesia. Gen 16S rRNA tidak hanya dapat mengidentifikasi tetapi dapat dijadikan arahan dalam mengetahui potensi suatu bakteri. Sekuensing gen 16S rRNA telah digunakan secara luas untuk penelitian di bidang klinis, perikanan, kelautan, pertanian dan peternakan di Indonesia.Kata Kunci: Gen 16S rRNA, perangkat penelitian, Bakteri, Indonesia.

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Woo, Patrick C. Y., Jade L. L. Teng, Jeff K. L. Wu, Fion P. S. Leung, Herman Tse, Ami M. Y. Fung, Susanna K. P. Lau, and Kwok-yung Yuen. "Guidelines for interpretation of 16S rRNA gene sequence-based results for identification of medically important aerobic Gram-positive bacteria." Journal of Medical Microbiology 58, no. 8 (August 1, 2009): 1030–36. http://dx.doi.org/10.1099/jmm.0.008615-0.

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This study is believed to be the first to provide guidelines for facilitating interpretation of results based on full and 527 bp 16S rRNA gene sequencing and MicroSeq databases used for identifying medically important aerobic Gram-positive bacteria. Overall, full and 527 bp 16S rRNA gene sequencing can identify 24 and 40 % of medically important Gram-positive cocci (GPC), and 21 and 34 % of medically important Gram-positive rods (GPR) confidently to the species level, whereas the full-MicroSeq and 500-MicroSeq databases can identify 15 and 34 % of medically important GPC and 14 and 25 % of medically important GPR confidently to the species level. Among staphylococci, streptococci, enterococci, mycobacteria, corynebacteria, nocardia and members of Bacillus and related taxa (Paenibacillus, Brevibacillus, Geobacillus and Virgibacillus), the methods and databases are least useful for identification of staphylococci and nocardia. Only 0–2 and 2–13 % of staphylococci, and 0 and 0–10 % of nocardia, can be confidently and doubtfully identified, respectively. However, these methods and databases are most useful for identification of Bacillus and related taxa, with 36–56 and 11–14 % of Bacillus and related taxa confidently and doubtfully identified, respectively. A total of 15 medically important GPC and 18 medically important GPR that should be confidently identified by full 16S rRNA gene sequencing are not included in the full-MicroSeq database. A total of 9 medically important GPC and 21 medically important GPR that should be confidently identified by 527 bp 16S rRNA gene sequencing are not included in the 500-MicroSeq database. 16S rRNA gene sequence results of Gram-positive bacteria should be interpreted with basic phenotypic tests results. Additional biochemical tests or sequencing of additional gene loci are often required for definitive identification. To improve the usefulness of the MicroSeq databases, bacterial species that can be confidently identified by 16S rRNA gene sequencing but are not found in the MicroSeq databases should be included.

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Barney, Michael, Antonia Volgyi, Alfonso Navarro, and David Ryder. "Riboprinting and 16S rRNA Gene Sequencing for Identification of Brewery Pediococcus Isolates." Applied and Environmental Microbiology 67, no. 2 (February 1, 2001): 553–60. http://dx.doi.org/10.1128/aem.67.2.553-560.2001.

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ABSTRACT A total of 46 brewery and 15 ATCC Pediococcus isolates were ribotyped using a Qualicon RiboPrinter. Of these, 41 isolates were identified as Pediococcus damnosus using EcoRI digestion. Three ATCC reference strains had patterns similar to each other and matched 17 of the brewery isolates. Six other brewing isolates were similar to ATCC 25249. The other 18 P. damnosus brewery isolates had unique patterns. Of the remaining brewing isolates, one was identified as P. parvulus, two were identified as P. acidilactici, and two were identified as unique Pediococcus species. The use of alternate restriction endonucleases indicated that PstI andPvuII could further differentiate some strains having identical EcoRI profiles. An acid-resistant P. damnosus isolate could be distinguished from non-acid-resistant varieties of the same species using PstI instead ofEcoRI. 16S rRNA gene sequence analysis was compared to riboprinting for identifying pediococci. The complete 16S rRNA gene was PCR amplified and sequenced from seven brewery isolates and three ATCC references with distinctive riboprint patterns. The 16S rRNA gene sequences from six different brewery P. damnosus isolates were homologous with a high degree of similarity to the GenBank reference strain but were identical to each other and one ATCC strain with the exception of 1 bp in one strain. A slime-producing, beer spoilage isolate had 16S rRNA gene sequence homology to the P. acidilactici reference strain, in agreement with the riboprint data. Although 16S rRNA gene sequencing correctly identified the genus and species of the test Pediococcus isolates, riboprinting proved to be a better method for subspecies differentiation.

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Cuscó, Anna, Carlotta Catozzi, Joaquim Viñes, Armand Sanchez, and Olga Francino. "Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon." F1000Research 7 (November 6, 2018): 1755. http://dx.doi.org/10.12688/f1000research.16817.1.

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Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples often contain DNA from other sources, such as the host or the environment. The usual approach is sequencing specific hypervariable regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. Here, we aim to assess long-amplicon PCR-based approaches for assigning taxonomy at the genus and species level. We use Nanopore sequencing with two different markers: full-length 16S rRNA (~1,500 bp) and the whole rrn operon (16S rRNA–ITS–23S rRNA; 4,500 bp). Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities (HM-783D, Bei Resources; D6306, ZymoBIOMICS™) and two pools of low-biomass samples (dog skin from either the chin or dorsal back), using the MinION™ sequencer 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using the WIMP workflow on EPI2ME or Minimap2 software with rrn database. Results: The full-length 16S rRNA and the rrn operon were used to retrieve the microbiota composition at the genus and species level from the bacterial isolate, mock communities and complex skin samples. For the Staphylococcus pseudintermedius isolate, when using EPI2ME, the amplicons were assigned to the correct bacterial species in ~98% of the cases with the rrn operon marker, and in ~68% of the cases with the 16S rRNA gene. In both skin microbiota samples, we detected many species with an environmental origin. In chin, we found different Pseudomonas species in high abundance, whereas in dorsal skin there were more taxa with lower abundances. Conclusions: Both full-length 16S rRNA and 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, using the rrn operon would be the best choice.

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Cuscó, Anna, Carlotta Catozzi, Joaquim Viñes, Armand Sanchez, and Olga Francino. "Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon." F1000Research 7 (August 1, 2019): 1755. http://dx.doi.org/10.12688/f1000research.16817.2.

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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.

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Wu, Xueling, Hong Duan, Hongwei Fan, Zhenzhen Zhang, and Lili Liu. "Comparative Study of PCR-Based Approaches for the Genetic Characterization of Three Strains of Acidithiobacillus caldus Isolated from Different Sites in China." Polish Journal of Microbiology 62, no. 4 (2013): 351–58. http://dx.doi.org/10.33073/pjm-2013-048.

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Comparative study of the genetic characteristics among three Acidithiobacillus caldus strains isolated from different typical environments in China was performed using a combination of molecular methods, namely sequencing analysis of PCR-amplified 16S rRNA genes and 16S-23S rRNA gene intergenic spacers (ITS), repetitive element PCR (rep-PCR), arbitrarily primed PCR (AP-PCR) fingerprinting and random amplified polymorphic DNA (RAPD). Both of the 16S rRNA gene and 16S-23S rRNA gene intergenic spacers sequences of the three strains exhibited small variations, with 99.9-100%, 99.7-100% identity respectively. In contrast, according to the analysis of bacterial diversity based on rep-PCR and AP-PCR fingerprinting, they produced highly discriminatory banding patterns, and the similarity values between them varied from 61.97% to 71.64%. RAPD analysis showed that banding profiles of their genomic DNA exhibited obvious differences from each other with 53.44-75% similarity. These results suggested that in contrast to 16S rRNA genes and 16S-23S rRNA gene intergenic spacers sequencing analysis, rep-PCR, AP-PCR fingerprinting and RAPD analysis possessed higher discriminatory power in identifying these closely related strains. And they could be used as rapid and highly discriminatory typing techniques in studying bacterial diversity, especially in differentiating bacteria within Acidithiobacillus caldus.

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Blackwood, Kym S., Cheng He, James Gunton, Christine Y. Turenne, Joyce Wolfe, and Amin M. Kabani. "Evaluation of recA Sequences for Identification of Mycobacterium Species." Journal of Clinical Microbiology 38, no. 8 (2000): 2846–52. http://dx.doi.org/10.1128/jcm.38.8.2846-2852.2000.

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16S rRNA sequence data have been used to provide a molecular basis for an accurate system for identification of members of the genusMycobacterium. Previous studies have shown thatMycobacterium species demonstrate high levels (>94%) of 16S rRNA sequence similarity and that this method cannot differentiate between all species, i.e., M. gastri and M. kansasii. In the present study, we have used the recAgene as an alternative sequencing target in order to complement 16S rRNA sequence-based genetic identification. The recA genes of 30 Mycobacterium species were amplified by PCR, sequenced, and compared with the published recA sequences of M. tuberculosis, M. smegmatis, and M. leprae available from GenBank. By recA sequencing the species showed a lower degree of interspecies similarity than they did by 16S rRNA gene sequence analysis, ranging from 96.2% betweenM. gastri and M. kansasii to 75.7% betweenM. aurum and M. leprae. Exceptions to this were members of the M. tuberculosis complex, which were identical. Two strains of each of 27 species were tested, and the intraspecies similarity ranged from 98.7 to 100%. In addition, we identified new Mycobacterium species that contain a protein intron in their recA genes, similar to M. tuberculosis and M. leprae. We propose thatrecA gene sequencing offers a complementary method to 16S rRNA gene sequencing for the accurate identification of theMycobacterium species.

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Kozich, James J., Sarah L. Westcott, Nielson T. Baxter, Sarah K. Highlander, and Patrick D. Schloss. "Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform." Applied and Environmental Microbiology 79, no. 17 (June 21, 2013): 5112–20. http://dx.doi.org/10.1128/aem.01043-13.

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ABSTRACTRapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.

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Aksentijević, Ksenija, Jelena Ašanin, Dušan Milivojević, Svetlana Čolović, Ana Butorac, Mario Cindrić, and Dušan Mišić. "Differentiation between Pseudomonas and Stenotrophomonas Species Isolated from Fish Using Molecular and MALDI-TOF Method." Acta Veterinaria 66, no. 3 (September 1, 2016): 304–16. http://dx.doi.org/10.1515/acve-2016-0027.

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Abstract For the purpose of precise antibiotic susceptibility testing it is necessary to clearly distinguish Pseudomonas and Stenotrophomonas genera, considering acquired resistance of Pseudomonas species, as well as the intrinsic resistance of Stenotrophomonas species. This is why in the identification of the 51 isolates originated from fish, the following methods were used: standard PCR, 16S rRNA gene sequencing, and MALDI-TOF. The results of the standard PCR test, 16S rRNA gene sequencing and MALDI-TOF analysis confirmed 35 strains to belong to the Pseudomonas genus. Standard PCR test and VITEK MS device confirmed that 10 strains belong to Stenotrophomonas maltophilia species. Three strains were positive in both standard PCR tests for Pseudomonas and Stenotrpohomonas. 16S rRNA gene sequencing identified these 3 strains to be 99% Pseudomonas sp. and 99% Stenotrophomonas sp. VITEK MS first identified these three strains as 99% Stenotrophomonas, and in the repeated identification it identified them as 99% Pseudomonas. MALDI TOF/TOF 4800 Plus device identified these strains as Stenotrophomonas. Three strains were negative in both standard PCR tests for Pseudomonas and Stenotrpohomonas. 16S rRNA gene sequencing identified these 3 strains to be 99% Pseudomonas sp. and 99% Stenotrophomonas sp. VITEK MS first identified these three strains as 99% Stenotrophomonas, and in the repeated identification it identified them as 99% Pseudomonas. MALDI TOF/TOF 4800 Plus device identified these strains as Stenotrophomonas. Although modern test methods that have very high specificity (PCR, 16S rRNA gene sequencing, MALDI TOF) were used in this study, precise differentiation between Pseudomonas and Stenotrophomonas species for 6 isolates could not be reached using the above mentioned methods.

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Dixit, Kunal. "Benchmarking of 16S rRNA gene databases using known strain sequences." Bioinformation 17, no. 3 (March 31, 2021): 377–91. http://dx.doi.org/10.6026//97320630017377.

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16S rRNA gene analysis is the most convenient and robust method for microbiome studies. Inaccurate taxonomic assignment of bacterial strains could have deleterious effects as all downstream analyses rely heavily on the accurate assessment of microbial taxonomy. The use of mock communities to check the reliability of the results has been suggested. However, often the mock communities used in most of the studies represent only a small fraction of taxa and are used mostly as validation of sequencing run to estimate sequencing artifacts. Moreover, a large number of databases and tools available for classification and taxonomic assignment of the 16S rRNA gene make it challenging to select the best-suited method for a particular dataset. In the present study, we used authentic and validly published 16S rRNA gene type strain sequences (full length, V3-V4 region) and analyzed them using a widely used QIIME pipeline along with different parameters of OTU clustering and QIIME compatible databases. Data Analysis Measures (DAM) revealed a high discrepancy in ratifying the taxonomy at different taxonomic hierarchies. Beta diversity analysis showed clear segregation of different DAMs. Limited differences were observed in reference data set analysis using partial (V3-V4) and full-length 16S rRNA gene sequences, which signify the reliability of partial 16S rRNA gene sequences in microbiome studies. Our analysis also highlights common discrepancies observed at varioustaxonomic levels using various methods and databases.

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Dixit, Kunal. "Benchmarking of 16S rRNA gene databases using known strain sequences." Bioinformation 17, no. 3 (March 31, 2021): 377–91. http://dx.doi.org/10.6026/97320630017377.

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16S rRNA gene analysis is the most convenient and robust method for microbiome studies. Inaccurate taxonomic assignment of bacterial strains could have deleterious effects as all downstream analyses rely heavily on the accurate assessment of microbial taxonomy. The use of mock communities to check the reliability of the results has been suggested. However, often the mock communities used in most of the studies represent only a small fraction of taxa and are used mostly as validation of sequencing run to estimate sequencing artifacts. Moreover, a large number of databases and tools available for classification and taxonomic assignment of the 16S rRNA gene make it challenging to select the best-suited method for a particular dataset. In the present study, we used authentic and validly published 16S rRNA gene type strain sequences (full length, V3-V4 region) and analyzed them using a widely used QIIME pipeline along with different parameters of OTU clustering and QIIME compatible databases. Data Analysis Measures (DAM) revealed a high discrepancy in ratifying the taxonomy at different taxonomic hierarchies. Beta diversity analysis showed clear segregation of different DAMs. Limited differences were observed in reference data set analysis using partial (V3-V4) and full-length 16S rRNA gene sequences, which signify the reliability of partial 16S rRNA gene sequences in microbiome studies. Our analysis also highlights common discrepancies observed at varioustaxonomic levels using various methods and databases.

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Marotz, Clarisse, Anukriti Sharma, Greg Humphrey, Neil Gottel, Christopher Daum, Jack A. Gilbert, Emiley Eloe-Fadrosh, and Rob Knight. "Triplicate PCR reactions for 16S rRNA gene amplicon sequencing are unnecessary." BioTechniques 67, no. 1 (July 2019): 29–32. http://dx.doi.org/10.2144/btn-2018-0192.

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Wang, Wen-Jia, You-Lian Zhou, Jie He, Zhi-Qiang Feng, Long Zhang, Xiao-Bo Lai, Jun-Xiao Zhou, and Hong Wang. "Characterizing the composition of intestinal microflora by 16S rRNA gene sequencing." World Journal of Gastroenterology 26, no. 6 (February 14, 2020): 614–26. http://dx.doi.org/10.3748/wjg.v26.i6.614.

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Woo, P. C. Y., A. M. Y. Fung, S. K. P. Lau, and K. Y. Yuen. "Identification by 16S rRNA Gene Sequencing of Lactobacillus salivarius Bacteremic Cholecystitis." Journal of Clinical Microbiology 40, no. 1 (January 1, 2002): 265–67. http://dx.doi.org/10.1128/jcm.40.1.265-267.2002.

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Gorkiewicz, G., G. Feierl, C. Schober, F. Dieber, J. Kofer, R. Zechner, and E. L. Zechner. "Species-Specific Identification of Campylobacters by Partial 16S rRNA Gene Sequencing." Journal of Clinical Microbiology 41, no. 6 (June 1, 2003): 2537–46. http://dx.doi.org/10.1128/jcm.41.6.2537-2546.2003.

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Eda, Haruka, Ho Namkoong, Yoshifumi Uwamino, Takanori Ohata, Shinji Sakaguchi, Fumitake Saito, Yoshihito Otsuka, Naoki Hasegawa, and Hideki Yuki. "A Case of Pulmonary Actinomycosis Identified by 16S rRNA Gene Sequencing." Nihon Naika Gakkai Zasshi 107, no. 10 (October 10, 2018): 2162–69. http://dx.doi.org/10.2169/naika.107.2162.

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Fida, Madiha, Sarwat Khalil, Omar Abu Saleh, Douglas W. Challener, Muhammad Rizwan Sohail, Joshua N. Yang, Bobbi S. Pritt, Audrey N. Schuetz, and Robin Patel. "Diagnostic Value of 16S Ribosomal RNA Gene Polymerase Chain Reaction/Sanger Sequencing in Clinical Practice." Clinical Infectious Diseases 73, no. 6 (August 18, 2021): 961–68. http://dx.doi.org/10.1093/cid/ciab167.

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Abstract Background Accurate microbiologic diagnosis is important for appropriate management of infectious diseases. Sequencing-based molecular diagnostics are increasingly used for precision diagnosis of infections. However, their clinical utility is unclear. Methods We conducted a retrospective analysis of specimens that underwent 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR) followed by Sanger sequencing at our institution from April 2017 through March 2019. Results A total of 566 specimens obtained from 460 patients were studied. Patients were considered clinically infected or noninfected based on final diagnosis and management. In 17% of patients, 16S rRNA PCR/sequencing was positive and in 5% of patients, this test led to an impact on clinical care. In comparison, bacterial cultures were positive in 21% of patients. Specimens with a positive Gram stain had 12 times greater odds of having a positive molecular result than those with a negative Gram stain (95% confidence interval for odds ratio, 5.2–31.4). Overall, PCR positivity was higher in cardiovascular specimens (37%) obtained from clinically infected patients, with bacterial cultures being more likely to be positive for musculoskeletal specimens (P < .001). 16S rRNA PCR/sequencing identified a probable pathogen in 10% culture-negative specimens. Conclusion 16S rRNA PCR/sequencing can play a role in the diagnostic evaluation of patients with culture-negative infections, especially those with cardiovascular infections.

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Korczak, Bożena M., Regina Stieber, Stefan Emler, André P. Burnens, Joachim Frey, and Peter Kuhnert. "Genetic relatedness within the genus Campylobacter inferred from rpoB sequences." International Journal of Systematic and Evolutionary Microbiology 56, no. 5 (May 1, 2006): 937–45. http://dx.doi.org/10.1099/ijs.0.64109-0.

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The genus Campylobacter comprises 17 species, some of which are important animal and human pathogens. To gain more insight into the genetic relatedness of this genus and to improve the molecular tools available for diagnosis, a universal sequencing approach was established for the gene encoding the beta-subunit of RNA polymerase (rpoB) for the genus Campylobacter. A total of 59 strains, including the type strains of currently recognized species as well as field isolates, were investigated in the study. A primer set specific for Campylobacter species enabled straightforward amplification and sequencing of a 530 bp fragment of the rpoB gene. The 16S rRNA gene sequences of all of the strains were determined in parallel. A good congruence was obtained between 16S rRNA and rpoB gene sequence-based trees within the genus Campylobacter. The branching of the rpoB tree was similar to that of the 16S rRNA gene tree, even though a few discrepancies were observed for certain species. The resolution of the rpoB gene within the genus Campylobacter was generally much higher than that of the 16S rRNA gene sequence, resulting in a clear separation of most species and even some subspecies. The universally applicable amplification and sequencing approach for partial rpoB gene sequence determination provides a powerful tool for DNA sequence-based discrimination of Campylobacter species.

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Böttger, Sebastian, Silke Zechel-Gran, Philipp Streckbein, Michael Knitschke, Torsten Hain, Markus Weigel, Jan-Falco Wilbrand, Eugen Domann, Hans-Peter Howaldt, and Sameh Attia. "A New Type of Chronic Wound Infection after Wisdom Tooth Extraction: A Diagnostic Approach with 16S-rRNA Gene Analysis, Next-Generation Sequencing, and Bioinformatics." Pathogens 9, no. 10 (September 28, 2020): 798. http://dx.doi.org/10.3390/pathogens9100798.

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Delayed-onset infections are rare postoperative complications of lower third molar extractions. This article presents a case of a chronic combined hard and soft tissue infection after the extraction of a third molar, where the causative organisms could only be elucidated by molecular methods. Experimental 16S-rRNA gene analysis with next-generation sequencing and bioinformatics was used to identify the bacterial spectrum of the infection. 16S-rRNA gene analysis delivered the microbiome of the abscessing inflammation while standard culture and laboratory examinations were all sterile. The microbiome showed a mixed bacterial infection with a dominance of Delftia and Alcanivorax (spp.) besides other bacteria of the normal oral flora. Using 16S-rRNA-gene analysis, next-generation sequencing, and bioinformatics, a new type of chronic wound infection after wisdom tooth extraction was found. The property of Delftia and Alcanivorax (spp.) as water-affine environmental bacteria raises suspicion of infection from contaminated water from a dental unit. Thus, osteotomies of teeth should only be done with sterile cooling water. The 16S-rRNA gene analysis should become a part of the routine diagnostics in medical microbiology.

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Hamdan, Mahmoud, and Wafa Masoud. "Characterization of Bacterial Communities in Palestinian Lamb Meat by Phenotyping and 16S rRNA Gene Sequence Analysis." مجلة جامعة فلسطين التقنية خضوري للأبحاث 8, no. 2 (September 1, 2020): 12–22. http://dx.doi.org/10.53671/ptukrj.v8i2.89.

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The main purpose of the present study was to isolate, identify and quantify bacteria in Palestinian fresh lamb meat. Phenotyping and 16S rRNA gene sequence analysis was used to identify bacteria present in lamb meat samples. Thirty-four bacterial isolates were obtained from 20 samples of fresh lamb meat collected from 4 meat shops in Tulkarem city in Palestine. Bacterial counts were in a range of 3 x 103 - 1.5 x 105 cfu / g with Staphylococcus aureus being the highest in numbers among other bacteria. Enterobacteriaceae and Staphylococcaceae were the predominant bacterial families detected in fresh lamb meat samples. Two bacterial isolates, which were not identified by phenotyping, were identified by 16S rRNA gene sequence analysis. There was an agreement between phenotyping and 16S rRNA gene sequencing in identification of 19 bacterial isolates. On the other hand, a disagreement was observed between phenotyping and 16S rRNA gene sequencing in identification of the remaining bacterial isolates. Fresh lamb meat seems to be a good medium for growth of various bacterial species

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Hamdan, Mahmoud, and Wafa Masoud. "Characterization of Bacterial Communities in Palestinian Lamb Meat by Phenotyping and 16S rRNA Gene Sequence Analysis." مجلة جامعة فلسطين التقنية للأبحاث 8, no. 2 (September 1, 2020): 12–22. http://dx.doi.org/10.53671/pturj.v8i2.89.

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The main purpose of the present study was to isolate, identify and quantify bacteria in Palestinian fresh lamb meat. Phenotyping and 16S rRNA gene sequence analysis was used to identify bacteria present in lamb meat samples. Thirty-four bacterial isolates were obtained from 20 samples of fresh lamb meat collected from 4 meat shops in Tulkarem city in Palestine. Bacterial counts were in a range of 3 x 103 - 1.5 x 105 cfu / g with Staphylococcus aureus being the highest in numbers among other bacteria. Enterobacteriaceae and Staphylococcaceae were the predominant bacterial families detected in fresh lamb meat samples. Two bacterial isolates, which were not identified by phenotyping, were identified by 16S rRNA gene sequence analysis. There was an agreement between phenotyping and 16S rRNA gene sequencing in identification of 19 bacterial isolates. On the other hand, a disagreement was observed between phenotyping and 16S rRNA gene sequencing in identification of the remaining bacterial isolates. Fresh lamb meat seems to be a good medium for growth of various bacterial species

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Carr, Charles, Hannah Wilcox, Jeremy P. Burton, Sharanya Menon, Kait F. Al, David O’Gorman, Brent A. Lanting, Edward M. Vasarhelyi, Michael Neufeld, and Matthew G. Teeter. "Deciphering the low abundance microbiota of presumed aseptic hip and knee implants." PLOS ONE 16, no. 9 (September 14, 2021): e0257471. http://dx.doi.org/10.1371/journal.pone.0257471.

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16S rRNA gene sequencing of DNA extracted from clinically uninfected hip and knee implant samples has revealed polymicrobial populations. However, previous studies assessed 16S rRNA gene sequencing as a technique for the diagnosis of periprosthetic joint infections, leaving the microbiota of presumed aseptic hip and knee implants largely unstudied. These communities of microorganisms might play important roles in aspects of host health, such as aseptic loosening. Therefore, this study sought to characterize the bacterial composition of presumed aseptic joint implant microbiota using next generation 16S rRNA gene sequencing, and it evaluated this method for future investigations. 248 samples were collected from implants of 41 patients undergoing total hip or knee arthroplasty revision for presumed aseptic failure. DNA was extracted using two methodologies—one optimized for high throughput and the other for human samples—and amplicons of the V4 region of the 16S rRNA gene were sequenced. Sequencing data were analyzed and compared with ancillary specific PCR and microbiological culture. Computational tools (SourceTracker and decontam) were used to detect and compensate for environmental and processing contaminants. Microbial diversity of patient samples was higher than that of open-air controls and differentially abundant taxa were detected between these conditions, possibly reflecting a true microbiota that is present in clinically uninfected joint implants. However, positive control-associated artifacts and DNA extraction methodology significantly affected sequencing results. As well, sequencing failed to identify Cutibacterium acnes in most culture- and PCR-positive samples. These challenges limited characterization of bacteria in presumed aseptic implants, but genera were identified for further investigation. In all, we provide further support for the hypothesis that there is likely a microbiota present in clinically uninfected joint implants, and we show that methods other than 16S rRNA gene sequencing may be ideal for its characterization. This work has illuminated the importance of further study of microbiota of clinically uninfected joint implants with novel molecular and computational tools to further eliminate contaminants and artifacts that arise in low bacterial abundance samples.

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Chatellier, Sonia, Nathalie Mugnier, Françoise Allard, Bertrand Bonnaud, Valérie Collin, Alex van Belkum, Jean-Baptiste Veyrieras, and Stefan Emler. "Comparison of two approaches for the classification of 16S rRNA gene sequences." Journal of Medical Microbiology 63, no. 10 (October 1, 2014): 1311–15. http://dx.doi.org/10.1099/jmm.0.074377-0.

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The use of 16S rRNA gene sequences for microbial identification in clinical microbiology is accepted widely, and requires databases and algorithms. We compared a new research database containing curated 16S rRNA gene sequences in combination with the lca (lowest common ancestor) algorithm (RDB-LCA) to a commercially available 16S rDNA Centroid approach. We used 1025 bacterial isolates characterized by biochemistry, matrix-assisted laser desorption/ionization time-of-flight MS and 16S rDNA sequencing. Nearly 80 % of isolates were identified unambiguously at the species level by both classification platforms used. The remaining isolates were mostly identified correctly at the genus level due to the limited resolution of 16S rDNA sequencing. Discrepancies between both 16S rDNA platforms were due to differences in database content and the algorithm used, and could amount to up to 10.5 %. Up to 1.4 % of the analyses were found to be inconclusive. It is important to realize that despite the overall good performance of the pipelines for analysis, some inconclusive results remain that require additional in-depth analysis performed using supplementary methods.

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Naghoni, Ali, Giti Emtiazi, Mohammad Ali Amoozegar, Zahra Etemadifar, and Seyed Abolhassan Shahzadeh Fazeli. "REP-PCR Analysis to Study Prokaryotic Biodiversity from Lake Meyghan." International Letters of Natural Sciences 61 (January 2017): 69–84. http://dx.doi.org/10.18052/www.scipress.com/ilns.61.69.

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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.

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Bharadwaj, R., M. Fairfax, H. Salimnia, T. Painter, and P. Chandrasekhar. "P182 Clinical significance of 16S ribosomal gene sequencing (16S rRNA) method for rapid bacterial identification." International Journal of Antimicrobial Agents 34 (July 2009): S85. http://dx.doi.org/10.1016/s0924-8579(09)70401-7.

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Bartram, Andrea K., Michael D. J. Lynch, Jennifer C. Stearns, Gabriel Moreno-Hagelsieb, and Josh D. Neufeld. "Generation of Multimillion-Sequence 16S rRNA Gene Libraries from Complex Microbial Communities by Assembling Paired-End Illumina Reads." Applied and Environmental Microbiology 77, no. 11 (April 1, 2011): 3846–52. http://dx.doi.org/10.1128/aem.02772-10.

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ABSTRACTMicrobial communities host unparalleled taxonomic diversity. Adequate characterization of environmental and host-associated samples remains a challenge for microbiologists, despite the advent of 16S rRNA gene sequencing. In order to increase the depth of sampling for diverse bacterial communities, we developed a method for sequencing and assembling millions of paired-end reads from the 16S rRNA gene (spanning the V3 region; ∼200 nucleotides) by using an Illumina genome analyzer. To confirm reproducibility and to identify a suitable computational pipeline for data analysis, sequence libraries were prepared in duplicate for both a defined mixture of DNAs from known cultured bacterial isolates (>1 million postassembly sequences) and an Arctic tundra soil sample (>6 million postassembly sequences). The Illumina 16S rRNA gene libraries represent a substantial increase in number of sequences over all extant next-generation sequencing approaches (e.g., 454 pyrosequencing), while the assembly of paired-end 125-base reads offers a methodological advantage by incorporating an initial quality control step for each 16S rRNA gene sequence. This method incorporates indexed primers to enable the characterization of multiple microbial communities in a single flow cell lane, may be modified readily to target other variable regions or genes, and demonstrates unprecedented and economical access to DNAs from organisms that exist at low relative abundances.

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Beau, Frédéric, Claude Bollet, Thierry Coton, Eric Garnotel, and Michel Drancourt. "Molecular Identification of a Nocardiopsis dassonvillei Blood Isolate." Journal of Clinical Microbiology 37, no. 10 (1999): 3366–68. http://dx.doi.org/10.1128/jcm.37.10.3366-3368.1999.

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Nocardiopsis dassonvillei is an environmental aerobic actinomycete seldom isolated in cutaneous and pulmonary infections. We herein report the first N. dassonvillei blood isolate in a patient hospitalized for cholangitis. Although morphological characteristics and biochemical tests allowed a presumptive identification of this isolate, cell wall fatty acid chromatographic analysis confirmed identification at the genus level, and 16S rRNA gene sequencing achieved definite identification. This study illustrates the usefulness of 16S rRNA gene sequencing as a routine method for the identification of actinomycetes.

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Nayak, Bina S., Brian Badgley, and Valerie J. Harwood. "Comparison of Genotypic and Phylogenetic Relationships of Environmental Enterococcus Isolates by BOX-PCR Typing and 16S rRNA Gene Sequencing." Applied and Environmental Microbiology 77, no. 14 (May 27, 2011): 5050–55. http://dx.doi.org/10.1128/aem.00130-11.

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ABSTRACTEnvironmentalEnterococcusspp. were compared by BOX-PCR genotyping and 16S rRNA gene sequencing to clarify the predictive relationship of BOX-PCR fingerprints to species designation. BOX-PCR and 16S rRNA gene relationships agreed for 77% of strains. BOX-PCR provided superior intraspecies discrimination but incorrectly identified some strains to the species level and divided some species into multiple groups.

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Shin, Jeong Hwan, Si Hyun Kim, Haeng Soon Jeong, Seung Hwan Oh, Hye Ran Kim, Jeong Nyeo Lee, Young Chul Yoon, Yang Wook Kim, and Yeong Hoon Kim. "Identification of Coagulase-Negative Staphylococci Isolated from Continuous Ambulatory Peritoneal Dialysis Fluid using 16s Ribosomal RNA,tuf, andSodAGene Sequencing." Peritoneal Dialysis International: Journal of the International Society for Peritoneal Dialysis 31, no. 3 (May 2011): 340–46. http://dx.doi.org/10.3747/pdi.2010.00073.

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IntroductionCoagulase-negative staphylococcus (CoNS) is the most common pathogen in continuous ambulatory peritoneal dialysis (CAPD)–associated peritonitis. There is no well-organized, standardized database for CoNS, and few studies have used gene sequencing in reporting species distribution in CAPD peritonitis. In the present study, we used 3 housekeeping genes to evaluate the prevalence of CoNS isolated from CAPD peritonitis episodes and to estimate the accuracy of, and the characteristic differences between, these genes for species identification.MethodsAll 51 non-duplicated CoNS isolates obtained from CAPD peritonitis between April 2006 and May 2008 were used. The strains were identified by polymerase chain reaction and by direct sequencing using the 16S ribosomal RNA (rRNA), tuf, and sodA genes. We determined species distribution, and using selected databases, we analyzed the characteristics and diagnostic utility of the individual genes for species identification.ResultsIn GenBank (National Institutes of Health, Bethesda, MD, USA), we found 49 type or reference strains for CoNS 16S rRNA, 17 for tuf, and 46 for sodA, and we used those data for sequence-similarity comparisons with CAPD isolates. Among our 51 strains, S. epidermidis (66.7%) was the most common, followed by S. haemolyticus (11.8%), S. warneri (7.8%), S. caprae (5.9%), S. capitis (3.9%), and S. pasteuri (2.0%). For 1 strain, different species results were obtained with each gene. The identification rates with 16S rRNA, sodA, and tuf gene sequencing were 84.0%, 96.0%, and 92.2% respectively. The discrimination capability of 16S rRNA gene was lower in a few individual species, and for the sodA gene, the percentage similarity to sequences from reference strains was also lower. The tuf gene had excellent identification capacity, but relatively few type strains are available in public databases. The 16S rRNA gene did not discriminate between S. caprae and S. capitis. The sodA gene showed a similarity rate that was lower than that for sequences of the 16S rRNA gene. The tuf type strain sequences for S. caprae and S. pasteuri are not available in public databases.ConclusionsThe sodA, tuf, and 16S rRNA genes were very useful for CoNS identification. Each has its own characteristics of similarity, discriminative power, and inclusion in databases.

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Ramiro-Garcia, Javier, Gerben D. A. Hermes, Christos Giatsis, Detmer Sipkema, Erwin G. Zoetendal, Peter J. Schaap, and Hauke Smidt. "NG-Tax, a highly accurate and validated pipeline for analysis of 16S rRNA amplicons from complex biomes." F1000Research 5 (November 23, 2018): 1791. http://dx.doi.org/10.12688/f1000research.9227.2.

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Background: Massive high-throughput sequencing of short, hypervariable segments of the 16S ribosomal RNA (rRNA) gene has transformed the methodological landscape describing microbial diversity within and across complex biomes. However, several studies have shown that the methodology rather than the biological variation is responsible for the observed sample composition and distribution. This compromises meta-analyses, although this fact is often disregarded. Results: To facilitate true meta-analysis of microbiome studies, we developed NG-Tax, a pipeline for 16S rRNA gene amplicon sequence analysis that was validated with different mock communities and benchmarked against QIIME as a frequently used pipeline. The microbial composition of 49 independently amplified mock samples was characterized by sequencing two variable 16S rRNA gene regions, V4 and V5-V6, in three separate sequencing runs on Illumina’s HiSeq2000 platform. This allowed for the evaluation of important causes of technical bias in taxonomic classification: 1) run-to-run sequencing variation, 2) PCR–error, and 3) region/primer specific amplification bias. Despite the short read length (~140 nt) and all technical biases, the average specificity of the taxonomic assignment for the phylotypes included in the mock communities was 97.78%. On average 99.95% and 88.43% of the reads could be assigned to at least family or genus level, respectively, while assignment to ‘spurious genera’ represented on average only 0.21% of the reads per sample. Analysis of α- and β-diversity confirmed conclusions guided by biology rather than the aforementioned methodological aspects, which was not achieved with QIIME. Conclusions: Different biological outcomes are commonly observed due to 16S rRNA region-specific performance. NG-Tax demonstrated high robustness against choice of region and other technical biases associated with 16S rRNA gene amplicon sequencing studies, diminishing their impact and providing accurate qualitative and quantitative representation of the true sample composition. This will improve comparability between studies and facilitate efforts towards standardization.

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Clarridge, Jill E. "Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases." Clinical Microbiology Reviews 17, no. 4 (October 2004): 840–62. http://dx.doi.org/10.1128/cmr.17.4.840-862.2004.

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SUMMARY The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. Comparison of the bacterial 16S rRNA gene sequence has emerged as a preferred genetic technique. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, can be routinely used for identification of mycobacteria, and can lead to the recognition of novel pathogens and noncultured bacteria. Problems remain in that the sequences in some databases are not accurate, there is no consensus quantitative definition of genus or species based on 16S rRNA gene sequence data, the proliferation of species names based on minimal genetic and phenotypic differences raises communication difficulties, and microheterogeneity in 16S rRNA gene sequence within a species is common. Despite its accuracy, 16S rRNA gene sequence analysis lacks widespread use beyond the large and reference laboratories because of technical and cost considerations. Thus, a future challenge is to translate information from 16S rRNA gene sequencing into convenient biochemical testing schemes, making the accuracy of the genotypic identification available to the smaller and routine clinical microbiology laboratories.

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Plouzeau, Chloé, Pascale Bémer, Anne Sophie Valentin, Geneviève Héry-Arnaud, Didier Tandé, Anne Jolivet-Gougeon, Pascal Vincent, et al. "First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections." Journal of Clinical Microbiology 53, no. 2 (November 19, 2014): 419–24. http://dx.doi.org/10.1128/jcm.02413-14.

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The objective of this study was to assess the performance of seven French laboratories for 16S rRNA gene detection by real-time PCR in the diagnosis of bone and joint infection (BJI) to validate a large multicenter study. External quality control (QC) was required owing to the differences in extraction procedures and the molecular equipment used in the different laboratories. Three proficiency sets were organized, including four bacterial DNA extracts and four bead mill-pretreated osteoarticular specimens. Extraction volumes, 16S rRNA gene primers, and sequencing interpretation rules were standardized. In order to assess each laboratory's ability to achieve the best results, scores were assigned, and each QC series was classified as optimal, acceptable, or to be improved. A total of 168 QCs were sent, and 160 responses were analyzed. The expected results were obtained for 93.8%, with the same proportion for extracts (75/80) and clinical specimens (75/80). For the specimens, there was no significant difference between manual and automated extraction. This QC demonstrated the ability to achieve good and homogeneous results using the same 16S rRNA gene PCR with different equipment and validates the possibility of high-quality multicenter studies using molecular diagnosis for BJI.

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Khalil, Sarwat, Madiha Fida, Douglas W. Challener, Omar Abu Saleh, Muhammad R. Sohail, Joshua Yang, Bobbi Pritt, Audrey Schuetz, Robin Patel, and Robin Patel. "1834. Incremental Diagnostic Value of 16S Ribosomal RNA Gene Polymerase Chain Reaction/Sanger Sequencing in Clinical Practice." Open Forum Infectious Diseases 6, Supplement_2 (October 2019): S44. http://dx.doi.org/10.1093/ofid/ofz359.096.

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Abstract Background Polymerase chain reaction (PCR)/sequencing targeting the 16S ribosomal RNA (rRNA) gene to detect bacteria in normally sterile tissues and fluids has become increasingly popular in clinical medicine. This culture-independent technique can detect bacteria that are nonviable or difficult to cultivate using conventional methods. The clinical value of this type of testing is not well defined. We aimed to assess the diagnostic value of 16S rRNA PCR/Sanger sequencing as a clinical diagnostic assay at Mayo Clinic. Methods This is an interim analysis of the first 173 of 478 patients who had 16S rRNA PCR/Sanger sequencing done on sterile tissues or fluids at our institution from April, 2017 to November, 2018 as part of routine clinical practice. Medical records are being retrospectively reviewed, with results compared with those of culture. Results We reviewed 207 specimens from 173 patients (musculoskeletal 79%, cardiovascular 7%, central nervous system 4%, other 9%) that underwent 16S rRNA PCR/Sanger sequencing by clinical request (Table 1). In 90% of these specimens, the test was pre-planned rather than added-on. Nine specimens were excluded from analysis, as cultures were not performed. Overall concordance of culture with PCR/sequencing was 81% (160/197; P < 0.0001). Of 44 culture-positive specimens, PCR detected the same bacterium in 21 (48%) (Table 2). 45% (20/44) of those with positive cultures and 46% of those with positive PCR/sequencing results had received prior antimicrobial therapy (Table 3). PCR was negative in 139/144 specimens that were culture-negative (97%). PCR/sequencing was helpful in detecting a putative bacterial pathogen in 4 patients with negative cultures (Table 4). Conclusion Overall, 16S rRNA PCR/Sanger sequencing improved diagnostic yield compared with culture in a minority of cases. The described assay is limited by its inability to detect polymicrobial infections, a technical limitation that could possibly be addressed using massive parallel sequencing. Careful selection of cases and a save and add-on approach may be more cost-effective than upfront testing, although this was requested in a minority of cases. Disclosures Robin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.

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Burke, Catherine M., and Aaron E. Darling. "A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq." PeerJ 4 (September 20, 2016): e2492. http://dx.doi.org/10.7717/peerj.2492.

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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.

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Journal articles on the topic '16S rRNA gene sequencing'

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