Whole genome sequencing based differentiation between re-infection and relapse in Indian patients with tuberculosis recurrence, with and without HIV co-infection.

INTRODUCTION: Differentiation between relapse and reinfection in cases with tuberculosis (TB) recurrence has important implications for public health, especially in patients with human immunodeficiency virus (HIV) co-infection. We compared Mycobacterial Interspersed Repeat Unit (MIRU) typing and spoligotyping with whole genome sequencing (WGS) to differentiate between relapse and reinfection in patients (HIV-positive and HIV-negative) with TB recurrence. We also assessed the value of WGS to track acquired drug resistance in those with relapse after successful treatment. METHOD: Forty-one paired M. tuberculosis isolates collected from 20 HIV-positive and 21 HIV-negative patients were subjected to WGS in addition to spoligotyping and MIRU typing. Phylogenetic and Single Nucleotide Substitution (SNP) clustering analyses were performed to determine whether recurrences were due to relapse or re-infection. RESULTS: Comparison of M. tuberculosis genomes indicated that 95% of TB recurrences in the HIV-negative cohort were due to relapse, while the majority of TB recurrences (75%) in the HIV-positive cohort was due to reinfection (P = 0.0001). New drug resistance mutations were acquired in 5/24 cases (20.8%) that experienced relapse. CONCLUSIONS: WGS provided increased resolution, but differentiation between relapse and reinfection was broadly consistent with MIRU and spoligotyping. The high contribution of reinfection among HIV infected patients experiencing TB recurrence warrants further study to explore risk factors for TB exposure.

Tracking a moving user in indoor environments using Bluetooth low energy beacons.

BACKGROUND: Bluetooth low energy (BLE) beacons have been used to track the locations of individuals in indoor environments for clinical applications such as workflow analysis and infectious disease modelling. Most current approaches use the received signal strength indicator (RSSI) to track locations. When using the RSSI to track indoor locations, devices need to be calibrated to account for complex interference patterns, which is a laborious process. Our aim was to investigate an alternative method for indoor location tracking of a moving user using BLE beacons in dynamic indoor environments. METHODS AND MATERIALS: We developed a new method based on the received number of signals indicator (RNSI) and compared it to a standard RSSI-based method for predicting a user's location. Experiments were performed in an office environment and a tertiary hospital. Both RNSI and RSSI were compared at various distances from BLE beacons. In moving user experiments, a user wearing a beacon walked from one location to another based on a pre-defined route. Performance in predicting user locations was measured based on accuracy. RESULTS: RNSI values decreased substantially with distance from the BLE beacon than RSSI values. Moving user experiments in the office environment demonstrated that the RNSI-based method produced higher accuracy (80.0%) than the RSSI-based method (76.2%). In the hospital, where the environment may introduce signal quality problems due to increased signal interference, the RNSI-based method still outperformed (83.3%) the RSSI-based method (51.9%). CONCLUSIONS: Our results suggest that the RNSI-based method could be useful to track the locations of a moving user without involving complex calibration, especially when deploying within a new environment. RNSI has the potential to be used together with other methods in more robust indoor positioning systems.

Mycobacterium tuberculosis Drug Resistance and Transmission among Human Immunodeficiency Virus-Infected Patients in Ho Chi Minh City, Vietnam.

Vietnam has a high burden of tuberculosis (TB) and multidrug-resistant (MDR) TB, but drug resistance patterns and TB transmission dynamics among TB/human immunodeficiency virus (HIV) coinfected patients are not well described. We characterized 200 Mycobacterium tuberculosis isolates from TB/HIV coinfected patients diagnosed at the main TB referral hospital in Ho Chi Minh City, Vietnam. Phenotypic drug susceptibility testing (DST) for first-line drugs, spoligotyping, and 24-locus mycobacterial interspersed repetitive unit (MIRU-24) analysis was performed on all isolates. The 24-locus mycobacterial interspersed repetitive unit clusters and MDR isolates were subjected to whole genome sequencing (WGS). Most of the TB/HIV coinfected patients were young (162/174; 93.1% aged < 45 years) males (173; 86.5% male). Beijing (98; 49.0%) and Indo-Oceanic (70; 35.0%) lineage strains were most common. Phenotypic drug resistance was detected in 84 (42.0%) isolates, of which 17 (8.5%) were MDR; three additional MDR strains were identified on WGS. Strain clustering was reduced from 84.0% with spoligotyping to 20.0% with MIRU-24 typing and to 13.5% with WGS. Whole genome sequencing identified five additional clusters, or members of clusters, not recognized by MIRU-24. In total, 13 small (two to three member) WGS clusters were identified, with less clustering among drug susceptible (2/27; 7.4%) than among drug-resistant strains (25/27; 92.6%). On phylogenetic analysis, strains from TB/HIV coinfected patients were interspersed among strains from the general community; no major clusters indicating transmission among people living with HIV were detected. Tuberculosis/HIV coinfection in Vietnam was associated with high rates of drug resistance and limited genomic evidence of ongoing M. tuberculosis transmission among HIV-infected patients.

Proteogenomic analysis prioritises functional single nucleotide variants in cancer samples.

Massively parallel DNA sequencing enables the detection of thousands of germline and somatic single nucleotide variants (SNVs) in cancer samples. The functional analysis of these mutations is often carried out through in silico predictions, with further downstream experimental validation rarely performed. Here, we examine the potential of using mass spectrometry-based proteomics data to further annotate the function of SNVs in cancer samples. RNA-seq and whole genome sequencing (WGS) data from Jurkat cells were used to construct a custom database of single amino acid variant (SAAV) containing peptides and identified over 1,000 such peptides in two Jurkat proteomics datasets. The analysis enabled the detection of a truncated form of splicing regulator YTHDC1 at the protein level. To extend the functional annotation further, a Jurkat phosphoproteomics dataset was analysed, identifying 463 SAAV containing phosphopeptides. Of these phosphopeptides, 24 SAAVs were found to directly impact the phosphorylation event through the creation of either a phosphorylation site or a kinase recognition motif. We identified a novel phosphorylation site created by a SAAV in splicing factor SF3B1, a protein that is frequently mutated in leukaemia. To our knowledge, this is the first study to use phosphoproteomics data to directly identify novel phosphorylation events arising from the creation of phosphorylation sites by SAAVs. Our study reveals multiple functional mutations impacting the splicing pathway in Jurkat cells and demonstrates potential benefits of an integrative proteogenomics analysis for high-throughput functional annotation of SNVs in cancer.

TranSeqAnnotator: large-scale analysis of transcriptomic data.

BACKGROUND: The transcriptome of an organism can be studied with the analysis of expressed sequence tag (EST) data sets that offers a rapid and cost effective approach with several new and updated bioinformatics approaches and tools for assembly and annotation. The comprehensive analyses comprehend an organism along with the genome and proteome analysis. With the advent of large-scale sequencing projects and generation of sequence data at protein and cDNA levels, automated analysis pipeline is necessary to store, organize and annotate ESTs. RESULTS: TranSeqAnnotator is a workflow for large-scale analysis of transcriptomic data with the most appropriate bioinformatics tools for data management and analysis. The pipeline automatically cleans, clusters, assembles and generates consensus sequences, conceptually translates these into possible protein products and assigns putative function based on various DNA and protein similarity searches. Excretory/secretory (ES) proteins inferred from ESTs/short reads are also identified. The TranSeqAnnotator accepts FASTA format raw and quality ESTs along with protein and short read sequences and are analysed with user selected programs. After pre-processing and assembly, the dataset is annotated at the nucleotide, protein and ES protein levels. CONCLUSION: TranSeqAnnotator has been developed in a Linux cluster, to perform an exhaustive and reliable analysis and provide detailed annotation. TranSeqAnnotator outputs gene ontologies, protein functional identifications in terms of mapping to protein domains and metabolic pathways. The pipeline is applied to annotate large EST datasets to identify several novel and known genes with therapeutic experimental validations and could serve as potential targets for parasite intervention. TransSeqAnnotator is freely available for the scientific community at http://estexplorer.biolinfo.org/TranSeqAnnotator/.

An analysis of the transcriptome of Teladorsagia circumcincta: its biological and biotechnological implications.

BACKGROUND: Teladorsagia circumcincta (order Strongylida) is an economically important parasitic nematode of small ruminants (including sheep and goats) in temperate climatic regions of the world. Improved insights into the molecular biology of this parasite could underpin alternative methods required to control this and related parasites, in order to circumvent major problems associated with anthelmintic resistance. The aims of the present study were to define the transcriptome of the adult stage of T. circumcincta and to infer the main pathways linked to molecules known to be expressed in this nematode. Since sheep develop acquired immunity against T. circumcincta, there is some potential for the development of a vaccine against this parasite. Hence, we infer excretory/secretory molecules for T. circumcincta as possible immunogens and vaccine candidates. RESULTS: A total of 407,357 ESTs were assembled yielding 39,852 putative gene sequences. Conceptual translation predicted 24,013 proteins, which were then subjected to detailed annotation which included pathway mapping of predicted proteins (including 112 excreted/secreted [ES] and 226 transmembrane peptides), domain analysis and GO annotation was carried out using InterProScan along with BLAST2GO. Further analysis was carried out for secretory signal peptides using SignalP and non-classical sec pathway using SecretomeP tools. For ES proteins, key pathways, including Fc epsilon RI, T cell receptor, and chemokine signalling as well as leukocyte transendothelial migration were inferred to be linked to immune responses, along with other pathways related to neurodegenerative diseases and infectious diseases, which warrant detailed future studies. KAAS could identify new and updated pathways like phagosome and protein processing in endoplasmic reticulum. Domain analysis for the assembled dataset revealed families of serine, cysteine and proteinase inhibitors which might represent targets for parasite intervention. InterProScan could identify GO terms pertaining to the extracellular region. Some of the important domain families identified included the SCP-like extracellular proteins which belong to the pathogenesis-related proteins (PRPs) superfamily along with C-type lectin, saposin-like proteins. The 'extracellular region' that corresponds to allergen V5/Tpx-1 related, considered important in parasite-host interactions, was also identified. Six cysteine motif (SXC1) proteins, transthyretin proteins, C-type lectins, activation-associated secreted proteins (ASPs), which could represent potential candidates for developing novel anthelmintics or vaccines were few other important findings. Of these, SXC1, protein kinase domain-containing protein, trypsin family protein, trypsin-like protease family member (TRY-1), putative major allergen and putative lipid binding protein were identified which have not been reported in the published T. circumcincta proteomics analysis. Detailed analysis of 6,058 raw EST sequences from dbEST revealed 315 putatively secreted proteins. Amongst them, C-type single domain activation associated secreted protein ASP3 precursor, activation-associated secreted proteins (ASP-like protein), cathepsin B-like cysteine protease, cathepsin L cysteine protease, cysteine protease, TransThyretin-Related and Venom-Allergen-like proteins were the key findings. CONCLUSIONS: We have annotated a large dataset ESTs of T. circumcincta and undertaken detailed comparative bioinformatics analyses. The results provide a comprehensive insight into the molecular biology of this parasite and disease manifestation which provides potential focal point for future research. We identified a number of pathways responsible for immune response. This type of large-scale computational scanning could be coupled with proteomic and metabolomic studies of this parasite leading to novel therapeutic intervention and disease control strategies. We have also successfully affirmed the use of bioinformatics tools, for the study of ESTs, which could now serve as a benchmark for the development of new computational EST analysis pipelines.

An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host.

To infect their mammalian hosts, Fasciola hepatica larvae must penetrate and traverse the intestinal wall of the duodenum, move through the peritoneum, and penetrate the liver. After migrating through and feeding on the liver, causing extensive tissue damage, the parasites move to their final niche in the bile ducts where they mature and produce eggs. Here we integrated a transcriptomics and proteomics approach to profile Fasciola secretory proteins that are involved in host-pathogen interactions and to correlate changes in their expression with the migration of the parasite. Prediction of F. hepatica secretory proteins from 14,031 expressed sequence tags (ESTs) available from the Wellcome Trust Sanger Centre using the semiautomated EST2Secretome pipeline showed that the major components of adult parasite secretions are proteolytic enzymes including cathepsin L, cathepsin B, and asparaginyl endopeptidase cysteine proteases as well as novel trypsin-like serine proteases and carboxypeptidases. Proteomics analysis of proteins secreted by infective larvae, immature flukes, and adult F. hepatica showed that these proteases are developmentally regulated and correlate with the passage of the parasite through host tissues and its encounters with different host macromolecules. Proteases such as FhCL3 and cathepsin B have specific functions in larvae activation and intestinal wall penetration, whereas FhCL1, FhCL2, and FhCL5 are required for liver penetration and tissue and blood feeding. Besides proteases, the parasites secrete an array of antioxidants that are also highly regulated according to their migration through host tissues. However, whereas the proteases of F. hepatica are secreted into the parasite gut via a classical endoplasmic reticulum/Golgi pathway, we speculate that the antioxidants, which all lack a signal sequence, are released via a non-classical trans-tegumental pathway.

Advanced in silico analysis of expressed sequence tag (EST) data for parasitic nematodes of major socio-economic importance--fundamental insights toward biotechnological outcomes.

Parasitic nematodes infect humans, other animals and plants, and impose a significant public health and economic burden worldwide due to the diseases that they cause. A better understanding of parasite genomes, host-parasite relationships and the molecular biology of parasites themselves will enable the rational development of diagnostic tests and/or safe anti-parasitic compounds, following the functional annotation of parasite genomic sequences. With only a few completely sequenced nematode genomes, expressed sequence tag (EST) datasets provide a low-cost alternative ("poor man's genome") to whole genome sequences and a glimpse of the transcriptome of an organism. EST data require a number of computational methods for their pre-processing, clustering, assembly and annotation to yield biologically relevant information. In this article, we review the steps involved in EST data analysis, the development of new semi-automated bioinformatic pipelines and their application to parasitic nematodes of major socio-economic significance, focused on identifying molecules involved in key biological processes or pathways that might serve as targets for new drugs or vaccines.