Evaluation of nanopore-based sequencing technology for gene marker based analysis of complex microbial communities. Method development for accurate 16S rRNA gene amplicon sequencing

Nucleic acid sequencing can provide a detailed overview of microbial communities in comparison with standard plate-culture methods. Expansion of high-throughput sequencing (HTS) technologies and reduction in analysis costs has allowed for detailed exploration of various habitats with use of amplicon, metagenomics, and metatranscriptomics approaches. However, due to a capital cost of HTS platforms and requirements for batch analysis, genomics-based studies are still not being used as a standard method for the comprehensive examination of environmental or clinical samples for microbial characterization. This research project investigated the potential of a novel nanopore-based sequencing platform from Oxford Nanopore Technologies (ONT) for rapid and accurate analysis of various environmentally complex samples. ONT is an emerging company that developed the first-ever portable nanopore-based sequencing platform called MinIONTM. Portability and miniaturised size of the device gives an immense opportunity for de-centralised, in-field, and real-time analysis of environmental and clinical samples. Nonetheless, benchmarking of this new technology against the current gold-standard platform (i.e., Illumina sequencers) is necessary to evaluate nanopore data and understand its benefits and limitations. The focus of this study is on the evaluation of nanopore sequencing data: read quality, sequencing errors, alignment quality but also bacterial community structure. For this reason, mock bacterial community samples were generated, sequenced and analysed with use of multiple bioinformatics approaches. Furthermore, this study developed sophisticated library preparation and data analyses methods to enable high-accuracy analysis of amplicon libraries from complex microbial communities for sequencing on the nanopore platform. Besides, the best performing library preparation and data analyses methods were used for analysis of environmental samples and compared to high-quality Illumina metagenomics data. This work opens a new possibility for accurate, in-field amplicon analysis of complex samples with the use of MinIONTM and for the development of autonomous biosensing technology for culture-free detection of pathogenic and non-pathogenic microorganisms in water, soil, food, drinks or blood

NanoAmpli-Seq: a workflow for amplicon sequencing for mixed microbial communities on the nanopore sequencing platform

Background: Amplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity but is limited in genetic resolution due to short read lengths. While Oxford Nanopore or Pacific Biosciences sequencing platforms overcome this limitation, their application has been limited due to higher error rates or lower data output. Results: In this study, we introduce an amplicon sequencing workflow, i.e., NanoAmpli-Seq, that builds on the intramolecular-ligated nanopore consensus sequencing (INC-Seq) approach and demonstrate its application for full-length 16S rRNA gene sequencing. NanoAmpli-Seq includes vital improvements to the INC-Seq protocol that reduces sample processing time while significantly improving sequence accuracy. The developed protocol includes chopSeq software for fragmentation and read orientation correction of INC-Seq consensus reads while nanoClust algorithm was designed for read partitioning-based de novo clustering and within cluster consensus calling to obtain accurate full-length 16S rRNA gene sequences. Conclusions: NanoAmpli-Seq accurately estimates the diversity of tested mock communities with average consensus sequence accuracy of 99.5% for 2D and 1D2 sequencing on the nanopore sequencing platform. Nearly all residual errors in NanoAmpli-Seq sequences originate from deletions in homopolymer regions, indicating that homopolymer aware base calling or error correction may allow for sequencing accuracy comparable to short-read sequencing platforms

The distinct features of microbial 'dysbiosis' of Crohn's disease do not occur to the same extent in their unaffected, genetically linked kindred

Background/Aims: Studying the gut microbiota in unaffected relatives of people with Crohn’s disease (CD) may advance our understanding of the role of bacteria in disease aetiology. Methods: Faecal microbiota composition (16S rRNA gene sequencing), genetic functional capacity (shotgun metagenomics) and faecal short chain fatty acids (SCFA) were compared in unaffected adult relatives of CD children (CDR, n = 17) and adult healthy controls, unrelated to CD patients (HUC, n = 14). The microbiota characteristics of 19 CD children were used as a benchmark of CD ‘dysbiosis’. Results: The CDR microbiota was less diverse (p = 0.044) than that of the HUC group. Local contribution of β-diversity analysis showed no difference in community structure between the CDR and HUC groups. Twenty one of 1,243 (1.8%) operational taxonomic units discriminated CDR from HUC. The metagenomic functional capacity (p = 0.207) and SCFA concentration or pattern were similar between CDR and HUC (p>0.05 for all SCFA). None of the KEGG metabolic pathways were different between these two groups. Both of these groups (HUC and CDR) had a higher microbiota α-diversity (CDR, p = 0.026 and HUC, p<0.001) with a community structure (β-diversity) distinct from that of children with CD. Conclusions: While some alterations were observed, a distinct microbial ‘dysbiosis’, characteristic of CD patients, was not observed in their unaffected, genetically linked kindred

Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.

BACKGROUND: The study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents. RESULTS: In this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination. CONCLUSIONS: These results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised

Rapid draft sequencing and real-time nanopore sequencing in a hospital outbreak of Salmonella

Background: Foodborne outbreaks of Salmonella remain a pressing public health concern. We recently detected a large outbreak of Salmonella enterica serovar Enteritidis phage type 14b affecting more than 30 patients in our hospital. This outbreak was linked to community, national and European-wide cases. Hospital patients with Salmonella are at high risk, and require a rapid response. We initially investigated this outbreak by whole-genome sequencing using a novel rapid protocol on the Illumina MiSeq; we then integrated these data with whole-genome data from surveillance sequencing, thereby placing the outbreak in a national context. Additionally, we investigated the potential of a newly released sequencing technology, the MinION from Oxford Nanopore Technologies, in the management of a hospital outbreak of Salmonella. Results: We demonstrate that rapid MiSeq sequencing can reduce the time to answer compared to the standard sequencing protocol with no impact on the results. We show, for the first time, that the MinION can acquire clinically relevant information in real time and within minutes of a DNA library being loaded. MinION sequencing permits confident assignment to species level within 20 min. Using a novel streaming phylogenetic placement method samples can be assigned to a serotype in 40 min and determined to be part of the outbreak in less than 2 h. Conclusions: Both approaches yielded reliable and actionable clinical information on the Salmonella outbreak in less than half a day. The rapid availability of such information may facilitate more informed epidemiological investigations and influence infection control practices

Mycobacterial dihydrofolate reductase inhibitors identified using chemogenomic methods and in vitro validation.

The lack of success in target-based screening approaches to the discovery of antibacterial agents has led to reemergence of phenotypic screening as a successful approach of identifying bioactive, antibacterial compounds. A challenge though with this route is then to identify the molecular target(s) and mechanism of action of the hits. This target identification, or deorphanization step, is often essential in further optimization and validation studies. Direct experimental identification of the molecular target of a screening hit is often complex, precisely because the properties and specificity of the hit are not yet optimized against that target, and so many false positives are often obtained. An alternative is to use computational, predictive, approaches to hypothesize a mechanism of action, which can then be validated in a more directed and efficient manner. Specifically here we present experimental validation of an in silico prediction from a large-scale screen performed against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. The two potent anti-tubercular compounds studied in this case, belonging to the tetrahydro-1,3,5-triazin-2-amine (THT) family, were predicted and confirmed to be an inhibitor of dihydrofolate reductase (DHFR), a known essential Mtb gene, and already clinically validated as a drug target. Given the large number of similar screening data sets shared amongst the community, this in vitro validation of these target predictions gives weight to computational approaches to establish the mechanism of action (MoA) of novel screening hit

A P. aeruginosa serotype-defining single read from our first Oxford Nanopore run

<p>Here is, I think, the first publically-available Oxford Nanopore read to be published. This came off our MinION instrument this morning (Wednesday 11th June). The DNA was derived from Pseudomonas aeruginosa strain 910 which originally came from hospital water. DNA was fragmented with Covaris G-Tube as per the Oxford Nanopore standard genomic library preparation protocol. The read maps to part of the P. aeruginosa O6-antigen determining region.</p> <p> </p

The effect of DNA extraction methodology on gut microbiota research applications

Background: The effect that traditional and modern DNA extraction methods have on applications to study the role of gut microbiota in health and disease is a topic of current interest. Genomic DNA was extracted from three faecal samples and one probiotic capsule using three popular methods; chaotropic (CHAO) method, phenol/chloroform (PHEC) extraction, proprietary kit (QIAG). The performance of each of these methods on DNA yield and quality, microbiota composition using quantitative PCR, deep sequencing of the 16S rRNA gene, and sequencing analysis pipeline was evaluated. Results: The CHAO yielded the highest and the QIAG kit the lowest amount of double-stranded DNA, but the purity of isolated nucleic acids was better for the latter method. The CHAO method yielded a higher concentration of bacterial taxa per mass (g) of faeces. Sequencing coverage was higher in CHAO method but a higher proportion of the initial sequencing reads were retained for assignments to operational taxonomic unit (OTU) in the QIAG kit compared to the other methods. The QIAG kit appeared to have longer trimmed reads and shorter regions of worse quality than the other two methods. A distinct separation of α-diversity indices between different DNA extraction methods was not observed. When compositional dissimilarities between samples were explored, a strong separation was observed according to sample type. The effect of the extraction method was either marginal (Bray–Curtis distance) or none (unweighted Unifrac distance). Taxon membership and abundance in each sample was independent of the DNA extraction method used. Conclusions: We have benchmarked several DNA extraction methods commonly used in gut microbiota research and their differences depended on the downstream applications intended for use. Caution should be paid when the intention is to pool and analyse samples or data from studies which have used different DNA extraction methods

Consulta favorable a la concesión de viático al doctor don Reimundo Geraldino, rector del Colegio de San Jorge de Irlandeses en Alcalá, para ir a la Misión de Irlanda

Fecha del documento: 1673-07-18. 2 páginasConsulta del Consejo de Estado a la regente doña Mariana de Austria sobre el memorial del doctor don Reimundo Geraldino, rector del Colegio de San Jorge de Irlandeses en la Universidad de Alcalá, en la que refiere desea pasar a Irlanda a predicar el Santo Evangelio. El Consejo propone dar 100 ducados para el viaje en la forma en que se estila. Respuesta de la regente: "Está bien y así lo he mandado".Proyecto Proyección Política y Social de la Comunidad Irlandesa en la Monarquía hispánica y en la América Colonial de la Edad Moderna(siglos XVI-XVIII) (HAR2009-11339 - subprograma HIST) del Ministerio de Economía y Competitividad en colaboración con el Consejo Superior de Investigaciones Científicas (CSIC), Embajada de Irlanda en Madrid, National University of Ireland (NUI) Maynooth, University College Dublin y Trinity College DublinConsejo de EstadoMariana de AustriaColegio de San Jorge de Irlandeses de AlcaláNo100 ducadosNoN

Additional file 1: Table S1. of Staphylococcal species heterogeneity in the nasal microbiome following antibiotic prophylaxis revealed by tuf gene deep sequencing

Primer sequences used in the initial PCR step of tuf gene fragment amplicon sequencing. (DOC 35 kb