Correction: Specific Capture and Whole-Genome Sequencing of Viruses from Clinical Samples
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PROVIDER: S-EPMC3267775 | biostudies-literature | 2012 Jan
REPOSITORIES: biostudies-literature
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Project description:Whole genome sequencing of viruses directly from clinical samples is integral for understanding the genetics of host-virus interactions. Here, we report the use of sample sparing target enrichment (by hybridisation) for viral nucleic acid separation and deep-sequencing of herpesvirus genomes directly from a range of clinical samples including saliva, blood, virus vesicles, cerebrospinal fluid, and tumour cell lines. We demonstrate the effectiveness of the method by deep-sequencing 13 highly cell-associated human herpesvirus genomes and generating full length genome alignments at high read depth. Moreover, we show the specificity of the method enables the study of viral population structures and their diversity within a range of clinical samples types.
| S-EPMC3220689 | biostudies-literature
Project description:Mycobacterium tuberculosis whole-genome sequencing (WGS) is a powerful tool as it can provide data on population diversity, drug resistance, disease transmission, and mixed infections. Successful WGS is still reliant on high concentrations of DNA obtained through M. tuberculosis culture. Microfluidics technology plays a valuable role in single-cell research but has not yet been assessed as a bacterial enrichment strategy for culture-free WGS of M. tuberculosis. In a proof-of-principle study, we evaluated the use of Capture-XT, a microfluidic lab-on-chip cleanup and pathogen concentration platform to enrich M. tuberculosis bacilli from clinical sputum specimens for downstream DNA extraction and WGS. Three of the four (75%) samples processed by the microfluidics application passed the library preparation quality control, compared to only one of the four (25%) samples not enriched by the microfluidics M. tuberculosis capture application. WGS data were of sufficient quality, with mapping depth of ≥25× and 9 to 27% of reads mapping to the reference genome. These results suggest that microfluidics-based M. tuberculosis cell capture might be a promising method for M. tuberculosis enrichment in clinical sputum samples, which could facilitate culture-free M. tuberculosis WGS. IMPORTANCE Diagnosis of tuberculosis is effective using molecular methods; however, a comprehensive characterization of the resistance profile of Mycobacterium tuberculosis often requires culturing and phenotypic drug susceptibility testing or culturing followed by whole-genome sequencing (WGS). The phenotypic route can take anywhere from 1 to >3 months to result, by which point the patient may have acquired additional drug resistance. The WGS route is a very attractive option; however, culturing is the rate-limiting step. In this original article, we provide proof-of-principle evidence that microfluidics-based cell capture can be used on high-bacillary-load clinical samples for culture-free WGS.
| S-EPMC10433858 | biostudies-literature
Project description:Metagenomic shotgun sequencing (MSS) is a revolutionary approach to viral diagnostic testing that allows simultaneous detection of a broad range of viruses, detailed taxonomic assignment, and detection of mutations associated with antiviral drug resistance. To enhance sensitivity for virus detection, we previously developed ViroCap, a targeted sequence capture panel designed to enrich nucleic acid from a comprehensive set of eukaryotic viruses prior to sequencing. To demonstrate the utility of MSS with targeted sequence capture for detecting clinically important viruses and characterizing clinically important viral features, we used ViroCap to analyze clinical samples from a diagnostic virology laboratory containing a broad range of medically relevant viruses. From 26 samples, MSS with ViroCap detected all of the expected viruses and 30 additional viruses. Comparing sequencing after capture enrichment with standard MSS, we detected 13 viruses only with capture enrichment and observed a consistent increase in the number and percentage of viral sequence reads as well as the breadth and depth of coverage of the viral genomes. Compared with clinical testing, MSS enhanced taxonomic assignment for 15 viruses, and codons associated with antiviral drug resistance in influenza A virus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), and hepatitis C virus (HCV) could be analyzed. Overall, in clinical samples, MSS with targeted sequence capture provides enhanced virus detection and information of clinical and epidemiologic relevance compared with clinical testing and MSS without targeted sequence capture.
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Project description:BackgroundThe life-threatening pathogen Leptospira interrogans is the most common agent of leptospirosis, an emerging zoonotic disease. However, little is known about the strains that are currently circulating worldwide due to the fastidious nature of the bacteria and the difficulty to isolate cultures. In addition, the paucity of bacteria in blood and other clinical samples has proven to be a considerable challenge for directly genotyping the agent of leptospirosis directly from patient material. Our understanding of the genetic diversity of strains during human infection is therefore limited.MethodsHere, we carried out hybridization capture followed by Illumina sequencing of the core genome directly from 20 clinical samples that were PCR positive for pathogenic Leptospira to elucidate the genetic diversity of currently circulating Leptospira strains in mainland France.ResultsCapture with RNA probes covering the L. interrogans core genome resulted in a 72 to 13,000-fold increase in pathogen reads relative to standard sequencing without capture. Variant analysis of the genomes sequenced from the biological samples using 273 Leptospira reference genomes was then carried out to determine the genotype of the infecting strain. For samples with sufficient coverage (19/20 samples with coverage > 8×), we could unambiguously identify L. interrogans serovars Icterohaemorrhagiae and Copenhageni (14 samples), L. kirschneri serovar Grippotyphosa (4 samples), and L. interrogans serovar Pyrogenes (1 sample) as the infecting strains.ConclusionsWe obtained high-quality genomic data with suitable coverage for confident core genome genotyping of the agent of leptospirosis for most of our clinical samples. The recovery of the genome of the serovars Icterohaemorrhagiae and Copenhageni directly from multiple clinical samples revealed low adaptive diversification of the core genes during human infection. The ability to generate culture-free genomic data opens new opportunities for better understanding of the epidemiology of this fastidious pathogen and pathogenesis of this neglected disease.
| S-EPMC10012794 | biostudies-literature
Project description:BackgroundMalaria caused by Plasmodium vivax is an experimentally neglected severe disease with a substantial burden on human health. Because of technical limitations, little is known about the biology of this important human pathogen. Whole genome analysis methods on patient-derived material are thus likely to have a substantial impact on our understanding of P. vivax pathogenesis and epidemiology. For example, it will allow study of the evolution and population biology of the parasite, allow parasite transmission patterns to be characterized, and may facilitate the identification of new drug resistance genes. Because parasitemias are typically low and the parasite cannot be readily cultured, on-site leukocyte depletion of blood samples is typically needed to remove human DNA that may be 1000X more abundant than parasite DNA. These features have precluded the analysis of archived blood samples and require the presence of laboratories in close proximity to the collection of field samples for optimal pre-cryopreservation sample preparation.ResultsHere we show that in-solution hybridization capture can be used to extract P. vivax DNA from human contaminating DNA in the laboratory without the need for on-site leukocyte filtration. Using a whole genome capture method, we were able to enrich P. vivax DNA from bulk genomic DNA from less than 0.5% to a median of 55% (range 20%-80%). This level of enrichment allows for efficient analysis of the samples by whole genome sequencing and does not introduce any gross biases into the data. With this method, we obtained greater than 5X coverage across 93% of the P. vivax genome for four P. vivax strains from Iquitos, Peru, which is similar to our results using leukocyte filtration (greater than 5X coverage across 96% ).ConclusionThe whole genome capture technique will enable more efficient whole genome analysis of P. vivax from a larger geographic region and from valuable archived sample collections.
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Project description:Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains.
| S-EPMC7472277 | biostudies-literature
Project description:Dengue virus (DENV) is the most globally prevalent member of the genus Flavivirus in the family Flaviviridae, which can be classified into four serotypes. Historically, molecular epidemiological studies of DENV depended on E gene sequencing. The development of next-generation sequencing (NGS) allowed its application to viral whole-genome sequencing (WGS). In this study, we report the improvement of the existing WGS process for DENV by optimizing the primer design procedure, designing serotype-specific primer panels and reducing the sizes of amplicons. A total of 31 DENV-positive serum samples belonging to 4 serotypes and 9 genotypes of DENV were involved in the validation of the primer panels. The threshold cycle (CT) values of these samples ranged from 23.91 to 35.11. The validation results showed that the length of consensus sequences generated at a coverage depth of 20× or more ranged from 10,370 to 10,672 bp, with 100.00% coverage of the open reading frames and 97.34% to 99.52% coverage of the DENV genome. The amplification efficiency varied across amplicons, genotypes, and serotypes of DENVs. These results indicate that the serotype-specific primer panels allow users to obtain the whole genome of DENV directly from clinical samples, providing a universal, rapid, and effective tool for the integration of genomics with dengue surveillance. IMPORTANCE Dengue virus (DENV) is becoming the most globally prevalent arbovirus. The number of people living under the threat of DENV is increasing year by year. With the development of next-generation sequencing (NGS) technology, whole-genome sequencing (WGS) has been more and more widely used in infectious disease surveillance and molecular epidemiological studies. DENV population sequencing by NGS can increase our understanding of the changing epidemiology and evolution of the DENV genome at the molecular level, which demands universal primer panels and combination with NGS platforms. Multiplex PCR with a short-amplicon approach proved superior for amplifying viral genomes from clinical samples, particularly when the viral RNA was present at low concentrations. Additionally, DENV are known for their genetic diversity within serotype groups and geographical regions, so the primer panels we designed focused on universality, which would be useful in future local DENV outbreaks.
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Project description:BackgroundThe recent global emergence and re-emergence of arboviruses has caused significant human disease. Common vectors, symptoms and geographical distribution make differential diagnosis both important and challenging. AimTo investigate the feasibility of metagenomic sequencing for recovering whole genome sequences of chikungunya and dengue viruses from clinical samples.MethodsWe performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION on clinical samples which were real-time reverse transcription-PCR (qRT-PCR) positive for chikungunya (CHIKV) or dengue virus (DENV), two of the most important arboviruses. A total of 26 samples with a range of representative clinical Ct values were included in the study.ResultsDirect metagenomic sequencing of nucleic acid extracts from serum or plasma without viral enrichment allowed for virus identification, subtype determination and elucidated complete or near-complete genomes adequate for phylogenetic analysis. One PCR-positive CHIKV sample was also found to be coinfected with DENV. ConclusionsThis work demonstrates that metagenomic whole genome sequencing is feasible for the majority of CHIKV and DENV PCR-positive patient serum or plasma samples. Additionally, it explores the use of Nanopore metagenomic sequencing for DENV and CHIKV, which can likely be applied to other RNA viruses, highlighting the applicability of this approach to front-line public health and potential portable applications using the MinION.
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Project description:Identifying the microbiome composition from primary tissues directly affords an opportunity to study the causative relationships between the host microbiome and disease. However, this is challenging due the low abundance of microbial DNA relative to the host. We present a systematic evaluation of microbiome profiling directly from endoscopic biopsies by whole genome sequencing. We compared our methods with other approaches on datasets with previously identified microbial composition. We applied this approach to identify the microbiome from 27 stomach biopsies, and validated the presence of Helicobacter pylori by quantitative PCR. Finally, we profiled the microbial composition in The Cancer Genome Atlas gastric adenocarcinoma cohort.
| S-EPMC4661937 | biostudies-literature
Project description:The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.
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