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Asset Tag: Infectious disease

September 22, 2019

Rapid infectious disease identification by next-generation DNA sequencing.

Currently, there is a critical need to rapidly identify infectious organisms in clinical samples. Next-Generation Sequencing (NGS) could surmount the deficiencies of culture-based methods; however, there are no standardized, automated programs to process NGS data. To address this deficiency, we developed the Rapid Infectious Disease Identification (RIDI™) system. The system requires minimal guidance, which reduces operator errors. The system is compatible with the three major NGS platforms. It automatically interfaces with the sequencing system, detects their data format, configures the analysis type, applies appropriate quality control, and analyzes the results. Sequence information is characterized using both the NCBI database and RIDI™ specific databases. RIDI™ was designed to identify high probability sequence matches and more divergent matches that could represent different or novel species. We challenged the system using defined American Type Culture Collection (ATCC) reference standards of 27 species, both individually and in varying combinations. The system was able to rapidly detect known organisms in <12h with multi-sample throughput. The system accurately identifies 99.5% of the DNA sequence reads at the genus-level and 75.3% at the species-level in reference standards. It has a limit of detection of 146cells/ml in simulated clinical samples, and is also able to identify the components of polymicrobial samples with 16.9% discrepancy at the genus-level and 31.2% at the species-level. Thus, the system's effectiveness may exceed current methods, especially in situations where culture methods could produce false negatives or where rapid results would influence patient outcomes. Copyright © 2016 Elsevier B.V. All rights reserved.

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September 22, 2019

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

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September 22, 2019

The Epstein-Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis.

The Epstein-Barr virus (EBV) miR-BHRF1 microRNA (miRNA) cluster has been shown to facilitate B-cell transformation and promote the rapid growth of the resultant lymphoblastoid cell lines (LCLs). However, we find that expression of physiological levels of the miR-BHRF1 miRNAs in LCLs transformed with a miR-BHRF1 null mutant (?123) fails to increase their growth rate. We demonstrate that the pri-miR-BHRF1-2 and 1-3 stem-loops are present in the 3’UTR of transcripts encoding EBNA-LP and that excision of pre-miR-BHRF1-2 and 1-3 by Drosha destabilizes these mRNAs and reduces expression of the encoded protein. Therefore, mutational inactivation of pri-miR-BHRF1-2 and 1-3 in the ?123 mutant upregulates the expression of not only EBNA-LP but also EBNA-LP-regulated mRNAs and proteins, including LMP1. We hypothesize that this overexpression causes the reduced transformation capacity of the ?123 EBV mutant. Thus, in addition to regulating cellular mRNAs in trans, miR-BHRF1-2 and 1-3 also regulate EBNA-LP mRNA expression in cis. Copyright © 2017 Elsevier Inc. All rights reserved.

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September 22, 2019

SMRT-Cappable-seq reveals complex operon variants in bacteria.

Current methods for genome-wide analysis of gene expression require fragmentation of original transcripts into small fragments for short-read sequencing. In bacteria, the resulting fragmented information hides operon complexity. Additionally, in vivo processing of transcripts confounds the accurate identification of the 5′ and 3′ ends of operons. Here we develop a methodology called SMRT-Cappable-seq that combines the isolation of un-fragmented primary transcripts with single-molecule long read sequencing. Applied to E. coli, this technology results in an accurate definition of the transcriptome with 34% of known operons from RegulonDB being extended by at least one gene. Furthermore, 40% of transcription termination sites have read-through that alters the gene content of the operons. As a result, most of the bacterial genes are present in multiple operon variants reminiscent of eukaryotic splicing. By providing such granularity in the operon structure, this study represents an important resource for the study of prokaryotic gene network and regulation.

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September 22, 2019

Carbohydrate staple food modulates gut microbiota of Mongolians in China.

Gut microbiota is a determining factor in human physiological functions and health. It is commonly accepted that diet has a major influence on the gut microbial community, however, the effects of diet is not fully understood. The typical Mongolian diet is characterized by high and frequent consumption of fermented dairy products and red meat, and low level of carbohydrates. In this study, the gut microbiota profile of 26 Mongolians whom consumed wheat, rice and oat as the sole carbohydrate staple food for a week each consecutively was determined. It was observed that changes in staple carbohydrate rapidly (within a week) altered gut microbial community structure and metabolic pathway of the subjects. Wheat and oat favored bifidobacteria (Bifidobacterium catenulatum, Bifodobacteriumbifidum, Bifidobacterium adolescentis); whereas rice suppressed bifidobacteria (Bifidobacterium longum, Bifidobacterium adolescentis) and wheat suppresses Lactobaciilus, Ruminococcus and Bacteroides. The study exhibited two gut microbial clustering patterns with the preference of fucosyllactose utilization linking to fucosidase genes (glycoside hydrolase family classifications: GH95 and GH29) encoded by Bifidobacterium, and xylan and arabinoxylan utilization linking to xylanase and arabinoxylanase genes encoded by Bacteroides. There was also a correlation between Lactobacillus ruminis and sialidase, as well as Butyrivibrio crossotus and xylanase/xylosidase. Meanwhile, a strong concordance was found between the gastrointestinal bacterial microbiome and the intestinal virome. Present research will contribute to understanding the impacts of the dietary carbohydrate on human gut microbiome, which will ultimately help understand relationships between dietary factor, microbial populations, and the health of global humans.

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September 22, 2019

16S rRNA long-read sequencing of the granulation tissue from nonsmokers and smokers-severe chronic periodontitis patients

Smoking has been associated with increased risk of periodontitis. The aim of the present study was to compare the periodontal disease severity among smokers and nonsmokers which may help in better understanding of predisposition to this chronic inflammation mediated diseases. We selected deep-seated infected granulation tissue removed during periodontal flap surgery procedures for identification and differential abundance of residential bacterial species among smokers and nonsmokers through long-read sequencing technology targeting full-length 16S rRNA gene. A total of 8 phyla were identified among which Firmicutes and Bacteroidetes were most dominating. Differential abundance analysis of OTUs through PICRUST showed significant (p>0.05) abundance of Phyla-Fusobacteria (Streptobacillus moniliformis); Phyla-Firmicutes (Streptococcus equi), and Phyla Proteobacteria (Enhydrobacter aerosaccus) in nonsmokers compared to smokers. The differential abundance of oral metagenomes in smokers showed significant enrichment of host genes modulating pathways involving primary immunodeficiency, citrate cycle, streptomycin biosynthesis, vitamin B6 metabolism, butanoate metabolism, glycine, serine, and threonine metabolism pathways. While thiamine metabolism, amino acid metabolism, homologous recombination, epithelial cell signaling, aminoacyl-tRNA biosynthesis, phosphonate/phosphinate metabolism, polycyclic aromatic hydrocarbon degradation, synthesis and degradation of ketone bodies, translation factors, Ascorbate and aldarate metabolism, and DNA replication pathways were significantly enriched in nonsmokers, modulation of these pathways in oral cavities due to differential enrichment of metagenomes in smokers may lead to an increased susceptibility to infections and/or higher formation of DNA adducts, which may increase the risk of carcinogenesis.

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September 22, 2019

Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing.

Alternative RNA splicing greatly expands the repertoire of proteins encoded by genomes. Next-generation sequencing (NGS) is attractive for studying alternative splicing because of the efficiency and low cost per base, but short reads typical of NGS only report mRNA fragments containing one or few splice junctions. Here, we used single-molecule amplification and long-read sequencing to study the HIV-1 provirus, which is only 9700 bp in length, but encodes nine major proteins via alternative splicing. Our data showed that the clinical isolate HIV-1(89.6) produces at least 109 different spliced RNAs, including a previously unappreciated ~1 kb class of messages, two of which encode new proteins. HIV-1 message populations differed between cell types, longitudinally during infection, and among T cells from different human donors. These findings open a new window on a little studied aspect of HIV-1 replication, suggest therapeutic opportunities and provide advanced tools for the study of alternative splicing.

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September 22, 2019

Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation.

Shotgun metagenomics methods enable characterization of microbial communities in human microbiome and environmental samples. Assembly of metagenome sequences does not output whole genomes, so computational binning methods have been developed to cluster sequences into genome ‘bins’. These methods exploit sequence composition, species abundance, or chromosome organization but cannot fully distinguish closely related species and strains. We present a binning method that incorporates bacterial DNA methylation signatures, which are detected using single-molecule real-time sequencing. Our method takes advantage of these endogenous epigenetic barcodes to resolve individual reads and assembled contigs into species- and strain-level bins. We validate our method using synthetic and real microbiome sequences. In addition to genome binning, we show that our method links plasmids and other mobile genetic elements to their host species in a real microbiome sample. Incorporation of DNA methylation information into shotgun metagenomics analyses will complement existing methods to enable more accurate sequence binning.

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September 22, 2019

Profiling of oral microbiota in early childhood caries using Single-Molecule Real-Time Sequencing

Background: Alterations of oral microbiota are the main cause of the progression of caries. The goal of this study was to characterize the oral microbiota in childhood caries based on single-molecule real-time sequencing. Methods: A total of 21 preschoolers, aged 3-5 years old with severe early childhood caries, and 20 age-matched, caries-free children as controls were recruited. Saliva samples were collected, followed by DNA extraction, Pacbio sequencing and phylogenetic analyses of the oral microbial communities. Results: 876 species derived from 13 known bacterial phyla and 110 genera were detected from 41 children using Pacbio sequencing. At the species level, 38 species, including Veillonella spp., Streptococcus spp., Prevotella spp. and Lactobacillus spp., showed higher abundance in the caries group compared to the caries-free group (p<0.05). The core microbiota at the genus and species levels was more stable in the caries-free micro-ecological niche. At follow-up, oral examinations 6 months after sample collection, development of new dental caries was observed in 5 children (the transitional group) among the 21 caries free children. Compared with the caries-free children, in the transitional and caries groups, 6 species, which were more abundant in the caries-free group, exhibited a relatively low abundance in both the caries group and the transitional group (p<0.05). We conclude that Abiotrophia spp., Neisseria spp. and Veillonella spp., are essential for maintaining a healthy oral microbial ecosystem. Prevotella spp., Lactobacillus spp., Dialister spp. and Filifactor spp. may be related to the pathogenesis and progression of dental caries.

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September 22, 2019

Exploring the genome and transcriptome of the cave nectar bat Eonycteris spelaea with PacBio long-read sequencing.

In the past two decades, bats have emerged as an important model system to study host-pathogen interactions. More recently, it has been shown that bats may also serve as a new and excellent model to study aging, inflammation, and cancer, among other important biological processes. The cave nectar bat or lesser dawn bat (Eonycteris spelaea) is known to be a reservoir for several viruses and intracellular bacteria. It is widely distributed throughout the tropics and subtropics from India to Southeast Asia and pollinates several plant species, including the culturally and economically important durian in the region. Here, we report the whole-genome and transcriptome sequencing, followed by subsequent de novo assembly, of the E. spelaea genome solely using the Pacific Biosciences (PacBio) long-read sequencing platform.The newly assembled E. spelaea genome is 1.97 Gb in length and consists of 4,470 sequences with a contig N50 of 8.0 Mb. Identified repeat elements covered 34.65% of the genome, and 20,640 unique protein-coding genes with 39,526 transcripts were annotated.We demonstrated that the PacBio long-read sequencing platform alone is sufficient to generate a comprehensive de novo assembled genome and transcriptome of an important bat species. These results will provide useful insights and act as a resource to expand our understanding of bat evolution, ecology, physiology, immunology, viral infection, and transmission dynamics.

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September 22, 2019

Clinical PathoScope: rapid alignment and filtration for accurate pathogen identification in clinical samples using unassembled sequencing data.

The use of sequencing technologies to investigate the microbiome of a sample can positively impact patient healthcare by providing therapeutic targets for personalized disease treatment. However, these samples contain genomic sequences from various sources that complicate the identification of pathogens.Here we present Clinical PathoScope, a pipeline to rapidly and accurately remove host contamination, isolate microbial reads, and identify potential disease-causing pathogens. We have accomplished three essential tasks in the development of Clinical PathoScope. First, we developed an optimized framework for pathogen identification using a computational subtraction methodology in concordance with read trimming and ambiguous read reassignment. Second, we have demonstrated the ability of our approach to identify multiple pathogens in a single clinical sample, accurately identify pathogens at the subspecies level, and determine the nearest phylogenetic neighbor of novel or highly mutated pathogens using real clinical sequencing data. Finally, we have shown that Clinical PathoScope outperforms previously published pathogen identification methods with regard to computational speed, sensitivity, and specificity.Clinical PathoScope is the only pathogen identification method currently available that can identify multiple pathogens from mixed samples and distinguish between very closely related species and strains in samples with very few reads per pathogen. Furthermore, Clinical PathoScope does not rely on genome assembly and thus can more rapidly complete the analysis of a clinical sample when compared with current assembly-based methods. Clinical PathoScope is freely available at: http://sourceforge.net/projects/pathoscope/.

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September 22, 2019

Identification of Burkholderia fungorum in the urine of an individual with spinal cord injury and augmentation cystoplasty using 16S sequencing: copathogen or innocent bystander?

People with neuropathic bladder (NB) secondary to spinal cord injury (SCI) are at risk for multiple genitourinary complications, the most frequent of which is urinary tract infection (UTI). Despite the high frequency with which UTI occurs, our understanding of the role of urinary microbes in health and disease is limited. In this paper, we present the first prospective case study integrating symptom reporting, urinalysis, urine cultivation, and 16S ribosomal ribonucleic acid (rRNA) sequencing of the urine microbiome.A 55-year-old male with NB secondary to SCI contributed 12 urine samples over an 8-month period during asymptomatic, symptomatic, and postantibiotic periods. All bacteria identified on culture were present on 16S rRNA sequencing, however, 16S rRNA sequencing revealed the presence of bacteria not isolated on culture. In particular, Burkholderia fungorum was present in three samples during both asymptomatic and symptomatic periods. White blood cells of =5-10/high power field and leukocyte esterase =2 on urinalysis was associated with the presence of symptoms.In this patient, there was a predominance of pathogenic bacteria and a lack of putative probiotic bacteria during both symptomatic and asymptomatic states. Urinalysis-defined inflammatory markers were present to a greater extent during symptomatic periods compared to the asymptomatic state, which may underscore a role for urinalysis or other inflammatory markers in differentiating asymptomatic bacteriuria from UTI in patients with NB. The finding of potentially pathogenic bacteria identified by sequencing but not cultivation, suggests a need for greater understanding of the relationships amongst bacterial species in the bacteriuric neuropathic bladder.

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September 22, 2019

Survey of Ixodes pacificus ticks in California reveals a diversity of microorganisms and a novel and widespread Anaplasmataceae species.

Ixodes pacificus ticks can harbor a wide range of human and animal pathogens. To survey the prevalence of tick-borne known and putative pathogens, we tested 982 individual adult and nymphal I. pacificus ticks collected throughout California between 2007 and 2009 using a broad-range PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to detect a wide range of tick-borne microorganisms. Overall, 1.4% of the ticks were found to be infected with Borrelia burgdorferi, 2.0% were infected with Borrelia miyamotoi and 0.3% were infected with Anaplasma phagocytophilum. In addition, 3.0% were infected with Babesia odocoilei. About 1.2% of the ticks were co-infected with more than one pathogen or putative pathogen. In addition, we identified a novel Anaplasmataceae species that we characterized by sequencing of its 16S rRNA, groEL, gltA, and rpoB genes. Sequence analysis indicated that this organism is phylogenetically distinct from known Anaplasma species with its closest genetic near neighbors coming from Asia. The prevalence of this novel Anaplasmataceae species was as high as 21% at one site, and it was detected in 4.9% of ticks tested statewide. Based upon this genetic characterization we propose that this organism be called ‘Candidatus Cryptoplasma californiense’. Knowledge of this novel microbe will provide awareness for the community about the breadth of the I. pacificus microbiome, the concept that this bacterium could be more widely spread; and an opportunity to explore whether this bacterium also contributes to human or animal disease burden.

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September 22, 2019

Improved OTU-picking using long-read 16S rRNA gene amplicon sequencing and generic hierarchical clustering

BACKGROUND: High-throughput bacterial 16S rRNA gene sequencing followed by clustering of short sequences into operational taxonomic units (OTUs) is widely used for microbiome profiling. However, clustering of short 16S rRNA gene reads into biologically meaningful OTUs is challenging, in part because nucleotide variation along the 16S rRNA gene is only partially captured by short reads. The recent emergence of long-read platforms, such as single-molecule real-time (SMRT) sequencing from Pacific Biosciences, offers the potential for improved taxonomic and phylogenetic profiling. Here, we evaluate the performance of long- and short-read 16S rRNA gene sequencing using simulated and experimental data, followed by OTU inference using computational pipelines based on heuristic and complete-linkage hierarchical clustering. RESULTS: In simulated data, long-read sequencing was shown to improve OTU quality and decrease variance. We then profiled 40 human gut microbiome samples using a combination of Illumina MiSeq and Blautia-specific SMRT sequencing, further supporting the notion that long reads can identify additional OTUs. We implemented a complete-linkage hierarchical clustering strategy using a flexible computational pipeline, tailored specifically for PacBio circular consensus sequencing (CCS) data that outperforms heuristic methods in most settings: https://github.com/oscar-franzen/oclust/. CONCLUSION: Our data demonstrate that long reads can improve OTU inference; however, the choice of clustering algorithm and associated clustering thresholds has significant impact on performance.

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September 22, 2019

Discovery of a divergent HPIV4 from respiratory secretions using second and third generation metagenomic sequencing.

Molecular detection of viruses has been aided by high-throughput sequencing, permitting the genomic characterization of emerging strains. In this study, we comprehensively screened 500 respiratory secretions from children with upper and/or lower respiratory tract infections for viral pathogens. The viruses detected are described, including a divergent human parainfluenza virus type 4 from GS FLX pyrosequencing of 92 specimens. Complete full-genome characterization of the virus followed, using Single Molecule, Real-Time (SMRT) sequencing. Subsequent “primer walking” combined with Sanger sequencing validated the RS platform’s utility in viral sequencing from complex clinical samples. Comparative genomics reveals the divergent strain clusters with the only completely sequenced HPIV4a subtype. However, it also exhibits various structural features present in one of the HPIV4b reference strains, opening questions regarding their lifecycle and evolutionary relationships among these viruses. Clinical data from patients infected with the strain, as well as viral prevalence estimates using real-time PCR, is also described.

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