Deriving accurate microbiota profiles from human samples with low bacterial content through post-sequencing processing of Illumina MiSeq data

BackgroundThe rapid expansion of 16S rRNA gene sequencing in challenging clinical contexts has resulted in a growing body of literature of variable quality. To a large extent, this is due to a failure to address spurious signal that is characteristic of samples with low levels of bacteria and high levels of non-bacterial DNA. We have developed a workflow based on the paired-end read Illumina MiSeq-based approach, which enables significant improvement in data quality, post-sequencing. We demonstrate the efficacy of this methodology through its application to paediatric upper-respiratory samples from several anatomical sites.ResultsA workflow for processing sequence data was developed based on commonly available tools. Data generated from different sample types showed a marked variation in levels of non-bacterial signal and ‘contaminant’ bacterial reads. Significant differences in the ability of reference databases to accurately assign identity to operational taxonomic units (OTU) were observed. Three OTU-picking strategies were trialled as follows: de novo, open-reference and closed-reference, with open-reference performing substantially better. Relative abundance of OTUs identified as potential reagent contamination showed a strong inverse correlation with amplicon concentration allowing their objective removal. The removal of the spurious signal showed the greatest improvement in sample types typically containing low levels of bacteria and high levels of human DNA. A substantial impact of pre-filtering data and spurious signal removal was demonstrated by principal coordinate and co-occurrence analysis. For example, analysis of taxon co-occurrence in adenoid swab and middle ear fluid samples indicated that failure to remove the spurious signal resulted in the inclusion of six out of eleven bacterial genera that accounted for 80% of similarity between the sample types.ConclusionsThe application of the presented workflow to a set of challenging clinical samples demonstrates its utility in removing the spurious signal from the dataset, allowing clinical insight to be derived from what would otherwise be highly misleading output. While other approaches could potentially achieve similar improvements, the methodology employed here represents an accessible means to exclude the signal from contamination and other artefacts

Republished: Respiratory microbiota:Addressing clinical questions, informing clinical practice

Over the last decade, technological advances have revolutionised efforts to understand the role played by microbes in airways disease. With the application of ever more sophisticated techniques, the literature has become increasingly inaccessible to the non-specialist reader, potentially hampering the translation of these gains into improvements in patient care. In this article, we set out the key principles underpinning microbiota research in respiratory contexts and provide practical guidance on how best such studies can be designed, executed and interpreted. We examine how an understanding of the respiratory microbiota both challenges fundamental assumptions and provides novel clinical insights into lung disease, and we set out a number of important targets for ongoing research.</p

Astrophysical Limits on Very Light Axion-like Particles from Chandra Grating Spectroscopy of NGC 1275

Axions/axion-like particles (ALPs) are a well motivated extension of the Standard Model and are generic within String Theory. The X-ray transparency of the intracluster medium (ICM) in galaxy clusters is a powerful probe of light ALPs (with mass $<10^{-11}\,{\rm eV}$); as X-ray photons from an embedded or background source propagate through the magnetized ICM, they may undergo energy-dependent quantum mechanical conversion into ALPs (and vice versa), imprinting distortions on the X-ray spectrum. We present Chandra data for the active galactic nucleus NGC1275 at the center of the Perseus cluster. Employing a 490ks High-Energy Transmission Gratings (HETG) exposure, we obtain a high-quality 1-9keV spectrum free from photon pileup and ICM contamination. Apart from iron-band features, the spectrum is described by a power-law continuum, with any spectral distortions at the $<3\%$ level. We compute photon survival probabilities as a function of ALP mass $m_a$ and ALP-photon coupling constant $g_{a\gamma}$ for an ensemble of ICM magnetic field models, and then use the NGC1275 spectrum to constraint the $(m_a, g_{a\gamma})$-plane. Marginalizing over magnetic field realizations, the 99.7% credible region limits the ALP-photon coupling to $g_{a\gamma}<6-8\times 10^{-13}\, {\rm GeV}^{-1}$ (depending upon magnetic field model) for masses $m_a<1\times 10^{-12}\,{\rm eV}$. These are the most stringent limit to date on $g_{a\gamma}$ for these light ALPs, and have already reached the sensitivity limits of next-generation helioscopes and light-shining-through-wall experiments. We highlight the potential of these studies with the next-generation X-ray observatories Athena and Lynx, but note the critical importance of advances in relative calibration of these future X-ray spectrometers.Comment: Published in The Astrophysical Journal, 2020, vol 890, pp59. This posting corrects typos in equation (2) and the likelihood function just prior to equation (3). Typos were introduced into the draft at late stage and the analysis is correc

Method for culturing Candidatus Ornithobacterium hominis.

Candidatus Ornithobacterium hominis has been detected in nasopharyngeal microbiota sequence data from around the world. This report provides the first description of culture conditions for isolating this bacterium. The availability of an easily reproducible culture method is expected to facilitate deeper understanding of the clinical significance of this species

Quantitative PCR of ear discharge from Indigenous Australian children with acute otitis media with perforation supports a role for Alloiococcus otitidis as a secondary pathogen

Otitis media is endemic in remote Indigenous communities of Australia’s Northern Territory. Alloiococcus otitidis is an outer ear commensal and putative middle ear pathogen that has not previously been described in acute otitis media (AOM) in this population. The aims of this study were to determine the presence, antibiotic susceptibility and bacterial load of A. otitidis in nasopharyngeal and ear discharge swabs collected from Indigenous Australian children with AOM with perforation.Financial support for this study was provided by the Channel 7 Children’s Research Foundation; The Trust Foundation; and the National Health and Medical Research Council (Australia)

New constraints on light axion-like particles using Chandra transmission grating spectroscopy of the powerful cluster-hosted quasar H1821+643

Axion-like particles (ALPs) are predicted by several Beyond the Standard Model theories, in particular, string theory. In the presence of an external magnetic field perpendicular to the direction of propagation, ALPs can couple to photons. Therefore, if an X-ray source is viewed through a magnetized plasma, such as a luminous quasar in a galaxy cluster, we may expect spectral distortions that are well described by photon–ALP oscillations. We present a 571 ks combined high- and low-energy transmission grating Chandra observation of the powerful radio-quiet quasar H1821+643, hosted by a cool-core cluster at redshift 0.3. The spectrum is well described by a double power-law continuum and broad+narrow iron line emission typical of type-1 active galactic nuclei (AGNs), with remaining spectral features 6.3 × 10−13 GeV−1 for most ALP masses <10−12 eV. Our results are moderately more sensitive to constraining ALPs than the best previous result from Chandra observations of the Perseus cluster, albeit with a less constrained field model. We reflect on the promising future of ALP studies with bright AGNs embedded in rich clusters, especially with the upcoming Athena mission