Cloud Computing for Metagenomics: Building a Personalized Computational Platform for Pipeline Analyses.

Cloud Computing services such as Microsoft Azure, Amazon Web Services, and Google Cloud provide a range of tools and services that enable scientists to rapidly prototype, build, and deploy platforms for their computational experiments.This chapter describes a protocol to deploy and configure an Ubuntu Linux Virtual Machine in the Microsoft Azure cloud, which includes Minconda Python, a Jupyter Lab server, and the QIIME toolkit configured for access through a web browser to facilitate a typical metagenomics analysis pipeline.

Manipulating and Basic Analysis of Tabular Metagenomics Datasets Using R.

Handling and manipulating tabular datasets is a critical step in every metagenomics analysis pipeline. The R statistical programming language offers a variety of versatile tools for working with tabular data that allow for the development of computationally efficient and reproducible workflows. Here we outline the basics of the R programming language and showcase a number of tools for data manipulation and basic analysis of metagenomics datasets.

SpheroidAnalyseR-an online platform for analyzing data from 3D spheroids or organoids grown in 96-well plates.

Spheroids and organoids are increasingly popular three-dimensional (3D) cell culture models. Spheroid models are more physiologically relevant to a tumor compared to two-dimensional (2D) cultures and organoids are a simplified version of an organ with similar composition. Spheroids are often only formed from a single cell type which does not represent the situation in vivo. However, despite this, both spheroids and organoids can be used in cell migration studies, disease modelling and drug discovery. A drawback of these models is, however, the lack of appropriate analytical tools for high throughput imaging and analysis over a time course. To address this, we have developed an R Shiny app called SpheroidAnalyseR: a simple, fast, effective open-source app that allows the analysis of spheroid or organoid size data generated in a 96-well format. SpheroidAnalyseR processes and analyzes datasets of image measurements that can be obtained via a bespoke software, described herein, that automates spheroid imaging and quantification using the Nikon A1R Confocal Laser Scanning Microscope. However, templates are provided to enable users to input spheroid image measurements obtained by user-preferred methods. SpheroidAnalyseR facilitates outlier identification and removal followed by graphical visualization of spheroid measurements across multiple predefined parameters such as time, cell-type and treatment(s). Spheroid imaging and analysis can, thus, be reduced from hours to minutes, removing the requirement for substantial manual data manipulation in a spreadsheet application. The combination of spheroid generation in 96-well ultra-low attachment microplates, imaging using our bespoke software, and analysis using SpheroidAnalyseR toolkit allows high throughput, longitudinal quantification of 3D spheroid growth whilst minimizing user input and significantly improving the efficiency and reproducibility of data analysis. Our bespoke imaging software is available from https://github.com/GliomaGenomics. SpheroidAnalyseR is available at https://spheroidanalyser.leeds.ac.uk, and the source code found at https://github.com/GliomaGenomics.

Prevalence and genetic analysis of phenotypically Vi- negative Salmonella typhi isolates in children from Kathmandu, Nepal.

The Vi capsular polysaccharide (ViPS) protects Salmonella enterica subspecies enterica serotype Typhi (S.Typhi) in vivo by multiple mechanisms. Recent microbiological reports from typhoid endemic countries suggest that acapsulate S.Typhi may occur in nature and contribute to clinical typhoid fever that is indistinguishable from disease caused by capsulate strains. The prevalence and genetic basis of ViPS-negative S.Typhi isolates in children from Kathmandu, Nepal, were tested in 68 isolates. Although 5.9% of isolates tested negative for capsular expression by slide agglutination tests, a novel multiplex PCR assay and individual PCR analyses demonstrated the presence of all 14 genes responsible for the synthesis, transportation and regulation of the ViPS. These data suggest that phenotypically acapsulate S.Typhi may not have a genetic basis for the same.

Construction of Opa-positive and Opa-negative strains of Neisseria meningitidis to evaluate a novel meningococcal vaccine.

Neisseria meningitidis is a major global pathogen causing invasive disease with a mortality of 5-10%. Most disease in developed countries is caused by serogroup B infection, against which there is no universal vaccine. Opacity-associated adhesin (Opa) proteins are major meningococcal outer membrane proteins, which have shown recent promise as a potential novel vaccine. Immunisation of mice with different Opa variants elicited high levels of meningococcal-specific bactericidal antibodies, demonstrating proof in principle for this approach. Opa proteins are critical in meningococcal pathogenesis, mediating bacterial adherence to host cells, and modulating human cellular immunity via interactions with T cells and neutrophils, although there are conflicting data regarding their effects on CD4(+) T cells. We constructed Opa-positive and Opa-negative meningococcal strains to allow further evaluation of Opa as a vaccine component. All four opa genes from N. meningitidis strain H44/76 were sequentially disrupted to construct all possible combinations of N. meningitidis strains deficient in one, two, three, or all four opa genes. The transformations demonstrated that homologous recombination of exogenous DNA into the meningococcal chromosome can occur with as little as 80 bp, and that minor sequence differences are permissible. Anti-Opa bactericidal antibody responses following immunisation of mice with recombinant Opa were specific to the Opa variant used in immunisation. No immunomodulatory effects were observed when Opa was contained within meningococcal outer membrane vesicles (OMVs), compared to Opa-negative OMVs. These observations support the incorporation of Opa in meningococcal vaccines.

Opa protein repertoires of disease-causing and carried meningococci.

The meningococcal Opa proteins play an important role in pathogenesis by mediating invasion of human cells. The aim of this investigation was to determine whether carried and disease-associated meningococci possess different Opa repertoires and whether the diversity of these proteins is associated with clinical severity of disease. Opa repertoires in 227 disease-associated meningococci, isolated in the United Kingdom over a period of 6 years, were compared to the repertoires in 190 asymptomatically carried meningococci isolated in the United Kingdom from a contemporary, nonepidemic period. Multidimensional scaling (MDS) was employed to investigate the association between Opa repertoires and multilocus sequence typing (MLST) genotypes. Associations with clinical severity were also analyzed statistically. High levels of diversity were observed in opa alleles, variable regions, and repertoires, and MDS revealed that MLST genotypes were strongly associated with particular Opa repertoires. Individual Opa proteins or repertoires were not associated with clinical severity, though there was a trend toward an association with the opaD locus. Meningococcal Opa repertoire is strongly linked to MLST genotype irrespective of epidemiological sampling and therefore correlates with invasiveness. It is not, however, strongly associated with severity of meningococcal disease.

Variation in the Neisseria lactamica porin, and its relationship to meningococcal PorB.

One potential vaccine strategy in the fight against meningococcal disease involves the exploitation of outer-membrane components of Neisseria lactamica, a commensal bacterium closely related to the meningococcus, Neisseria meningitidis. Although N. lactamica shares many surface structures with the meningococcus, little is known about the antigenic diversity of this commensal bacterium or the antigenic relationships between N. lactamica and N. meningitidis. Here, the N. lactamica porin protein (Por) was examined and compared to the related PorB antigens of N. meningitidis, to investigate potential involvement in anti-meningococcal immunity. Relationships among porin sequences were determined using distance-based methods and F(ST), and maximum-likelihood analyses were used to compare the selection pressures acting on the encoded proteins. These analyses demonstrated that the N. lactamica porin was less diverse than meningococcal PorB and although it was subject to positive selection, this was not as strong as the positive selection pressures acting on the meningococcal porin. In addition, the N. lactamica porin gene sequences and the protein sequences of the loop regions predicted to be exposed to the human immune system were dissimilar to the corresponding sequences in the meningococcus. This suggests that N. lactamica Por, contrary to previous suggestions, may have limited involvement in the development of natural immunity to meningococcal disease and might not be effective as a meningococcal vaccine component.

The effect of immune selection on the structure of the meningococcal opa protein repertoire.

The opa genes of the Gram negative bacterium Neisseria meningitidis encode Opacity-associated outer membrane proteins whose role is to promote adhesion to the human host tissue during colonisation and invasion. Each meningococcus contains 3-4 opa loci, each of which may be occupied by one of a large number of alleles. We analysed the Opa repertoire structure in a large, well-characterised collection of asymptomatically carried meningococci. Our data show an association between Opa repertoire and meningococcal lineages similar to that observed previously for meningococci isolated from cases of invasive disease. Furthermore, these Opa repertoires exhibit discrete, non-overlapping structure at a population level, and yet low within-repertoire diversity. These data are consistent with the predictions of a mathematical model of strong immune selection upon a system where identical alleles may occupy different loci.

Biliary cirrhosis in a child with inherited interleukin-12 deficiency.

Interleukin-12 (IL-12) is a key cytokine in the defense against intracellular bacteria notably Mycobacteria and Salmonella species. We report a case of disseminated mycobacterial infection, following BCG vaccination, in a child who later developed tuberculosis. Functional tests and a novel diagnostic polymerase chain reaction (PCR) assay, revealed a loss-of-function deletion in the IL12 gene. Analysis of samples from the parents and siblings of the patient indicated an autosomal recessive inheritance pattern with varying degrees of phenotypic expression in identical genotypes. Interferon-gamma (IFN-gamma) therapy was associated with marked clinical improvement. Biliary cirrhosis, a hitherto unreported complication of IL-12 deficiency, developed later and required liver transplantation. A defect in the IL-12-IFN-gamma pathway should be suspected in patients presenting with multiple, repeated or persistent infection with intracellular bacteria. The diagnostic work-up and the immuno-genetic assay described here can aid in the quick and reliable diagnosis of IL-12 deficiency resulting from genetic defects and its subsequent management.

High allelic diversity in the methyltransferase gene of a phase variable type III restriction-modification system has implications for the fitness of Haemophilus influenzae.

Phase variable restriction-modification (R-M) systems are widespread in Eubacteria. Haemophilus influenzae encodes a phase variable homolog of Type III R-M systems. Sequence analysis of this system in 22 non-typeable H.influenzae isolates revealed a hypervariable region in the central portion of the mod gene whereas the res gene was conserved. Maximum likelihood (ML) analysis indicated that most sites outside this hypervariable region experienced strong negative selection but evidence of positive selection for a few sites in adjacent regions. A phylogenetic analysis of 61 Type III mod genes revealed clustering of these H.influenzae mod alleles with mod genes from pathogenic Neisseriae and, based on sequence analysis, horizontal transfer of the mod-res complex between these species. Neisserial mod alleles also contained a hypervariable region and all mod alleles exhibited variability in the repeat tract. We propose that this hypervariable region encodes the target recognition domain (TRD) of the Mod protein and that variability results in alterations to the recognition sequence of this R-M system. We argue that the high allelic diversity and phase variable nature of this R-M system have arisen due to selective pressures exerted by diversity in bacteriophage populations but also have implications for other fitness attributes of these bacterial species.

Opacity-associated adhesin repertoire in hyperinvasive Neisseria meningitidis.

The opacity (Opa) proteins mediate a variety of interactions between the bacterium Neisseria meningitidis and its human host. These interactions are thought to be of central importance in both the asymptomatic colonization of the nasopharynx and the sporadic occurrence of meningococcal disease. The receptor specificities of a limited number of Opa protein variants have been explored, but the high level of amino acid sequence diversity among variants has complicated the assignment of specific roles to individual Opa variants or combinations of variants. In addition, the distribution of Opa protein variants among diverse meningococci, information that is potentially informative for studies of Opa function, is poorly understood. A systematic survey of the genetic diversity in the four opa gene loci in each of 77 meningococcal isolates was undertaken. These isolates were representative of the seven hyperinvasive meningococcal clonal complexes that caused the majority of meningococcal disease over the last 50 years. Consistent with previous studies, a high level of sequence diversity was observed among the opa genes and the proteins that they encoded; however, particular sets of Opa protein variants were consistently associated with each of the clonal complexes over time periods often spanning decades and during global spread. These observations were consistent with the postulate that particular combinations of Opa proteins confer fitness advantages to individual clonal complexes and have implications for studies of Opa function and the inclusion of Opa proteins in novel meningococcal vaccines.

Disease susceptibility to ST11 complex meningococci bearing serogroup C or W135 polysaccharide capsules, North America.

Clusters of meningococcal disease caused by a hyperinvasive lineage of Neisseria meningitidis, the ST11 complex, bearing a serogroup C polysaccharide capsule, have been prominent in Europe and North America since the early 1990s. This situation has led to expensive public health measures for outbreak control and, finally, to the introduction of a serogroup C glyconjugate vaccine into the primary immunization schedule in the United Kingdom and elsewhere. ST11 complex meningococci may also express serogroup W135 polysaccharide capsules. We investigated the level of population immunity to this hyperinvasive clone in association with the appearance of outbreaks of meningococcal disease in southern British Columbia. We found that most adults and almost all children were apparently susceptible to infection with ST11 complex meningococci bearing both C and W135 polysaccharide capsules, which suggests that a vaccine program directed against only serogroup C meningococci may be insufficient to prevent hyperinvasive ST11 disease.