scholarly journals Detection and Typing of Human Enteroviruses from Clinical Samples by Entire-Capsid Next Generation Sequencing

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 641
Author(s):  
Manasi Majumdar ◽  
Cristina Celma ◽  
Elaine Pegg ◽  
Krunal Polra ◽  
Jake Dunning ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sensitivity And Specificity ◽  
Severe Disease ◽  
Clinical Samples ◽  
Whole Genome ◽  
Next Generation ◽  
Coding Region ◽  
Diagnostic Laboratory ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

There are increasing concerns of infections by enteroviruses (EVs) causing severe disease in humans. EV diagnostic laboratory methods show differences in sensitivity and specificity as well as the level of genetic information provided. We examined a detection method for EVs based on next generation sequencing (NGS) analysis of amplicons covering the entire capsid coding region directly synthesized from clinical samples. One hundred and twelve clinical samples from England; previously shown to be positive for EVs, were analyzed. There was high concordance between the results obtained by the new NGS approach and those from the conventional Sanger method used originally with agreement in the serotypes identified in the 83 samples that were typed by both methods. The sensitivity and specificity of the NGS method compared to those of the conventional Sanger sequencing typing assay were 94.74% (95% confidence interval, 73.97% to 99.87%) and 97.85% (92.45% to 99.74%) for Enterovirus A, 93.75% (82.80% to 98.69%) and 89.06% (78.75% to 95.49%) for Enterovirus B, 100% (59.04% to 100%) and 98.10% (93.29% to 99.77%) for Enterovirus C, and 100% (75.29% to 100%) and 100% (96.34% to 100%) for Enterovirus D. The NGS method identified five EVs in previously untyped samples as well as additional viruses in some samples, indicating co-infection. This method can be easily expanded to generate whole-genome EV sequences as we show here for EV-D68. Information from capsid and whole-genome sequences is critical to help identifying the genetic basis for changes in viral properties and establishing accurate spatial-temporal associations between EV strains of public health relevance.

scholarly journals k-mer-Based Metagenomics Tools Provide a Fast and Sensitive Approach for the Detection of Viral Contaminants in Biopharmaceutical and Vaccine Manufacturing Applications Using Next-Generation Sequencing

mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Madolyn L. MacDonald ◽  
Shawn W. Polson ◽  
Kelvin H. Lee
Keyword(s):  
Next Generation Sequencing ◽  
Sensitivity And Specificity ◽  
Data Sets ◽  
Next Generation ◽  
Viral Detection ◽  
Read Alignment ◽  
Ensure Patient Safety ◽  
Hela Cell Lines ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

ABSTRACT Adventitious agent detection during the production of vaccines and biotechnology-based medicines is of critical importance to ensure the final product is free from any possible viral contamination. Increasing the speed and accuracy of viral detection is beneficial as a means to accelerate development timelines and to ensure patient safety. Here, several rapid viral metagenomics approaches were tested on simulated next-generation sequencing (NGS) data sets and existing data sets from virus spike-in studies done in CHO-K1 and HeLa cell lines. It was observed that these rapid methods had comparable sensitivity to full-read alignment methods used for NGS viral detection for these data sets, but their specificity could be improved. A method that first filters host reads using KrakenUniq and then selects the virus classification tool based on the number of remaining reads is suggested as the preferred approach among those tested to detect nonlatent and nonendogenous viruses. Such an approach shows reasonable sensitivity and specificity for the data sets examined and requires less time and memory as full-read alignment methods. IMPORTANCE Next-generation sequencing (NGS) has been proposed as a complementary method to detect adventitious viruses in the production of biotherapeutics and vaccines to current in vivo and in vitro methods. Before NGS can be established in industry as a main viral detection technology, further investigation into the various aspects of bioinformatics analyses required to identify and classify viral NGS reads is needed. In this study, the ability of rapid metagenomics tools to detect viruses in biopharmaceutical relevant samples is tested and compared to recommend an efficient approach. The results showed that KrakenUniq can quickly and accurately filter host sequences and classify viral reads and had comparable sensitivity and specificity to slower full read alignment approaches, such as BLASTn, for the data sets examined.

scholarly journals Whole genome sequencing suggests transmission of Corynebacterium diphtheriae-caused cutaneous diphtheria in two siblings, Germany, 2018

2019 ◽  
Vol 24 (2) ◽  
Author(s):  
Anja Berger ◽  
Alexandra Dangel ◽  
Tilmann Schober ◽  
Birgit Schmidbauer ◽  
Regina Konrad ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Corynebacterium Diphtheriae ◽  
Whole Genome ◽  
Next Generation ◽  
Insect Bites ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Generation Sequencing

In September 2018, a child who had returned from Somalia to Germany presented with cutaneous diphtheria by toxigenic Corynebacterium diphtheriae biovar mitis. The child’s sibling had superinfected insect bites harbouring also toxigenic C. diphtheriae. Next generation sequencing (NGS) revealed the same strain in both patients suggesting very recent human-to-human transmission. Epidemiological and NGS data suggest that the two cutaneous diphtheria cases constitute the first outbreak by toxigenic C. diphtheriae in Germany since the 1980s.

scholarly journals Comparison of targeted next-generation sequencing for whole-genome sequencing of Hantaan orthohantavirus in Apodemus agrarius lung tissues

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jin Sun No ◽  
Won-Keun Kim ◽  
Seungchan Cho ◽  
Seung-Ho Lee ◽  
Jeong-Ah Kim ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Clinical Samples ◽  
Whole Genome ◽  
Target Enrichment ◽  
Next Generation ◽  
Apodemus Agrarius ◽  
Target Capture ◽  
Generation Sequencing

Abstract Orthohantaviruses, negative-sense single-strand tripartite RNA viruses, are a global public health threat. In humans, orthohantavirus infection causes hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Whole-genome sequencing of the virus helps in identification and characterization of emerging or re-emerging viruses. Next-generation sequencing (NGS) is a potent method to sequence the viral genome, using molecular enrichment methods, from clinical specimens containing low virus titers. Hence, a comparative study on the target enrichment NGS methods is required for whole-genome sequencing of orthohantavirus in clinical samples. In this study, we used the sequence-independent, single-primer amplification, target capture, and amplicon NGS for whole-genome sequencing of Hantaan orthohantavirus (HTNV) from rodent specimens. We analyzed the coverage of the HTNV genome based on the viral RNA copy number, which is quantified by real-time quantitative PCR. Target capture and amplicon NGS demonstrated a high coverage rate of HTNV in Apodemus agrarius lung tissues containing up to 103–104 copies/μL of HTNV RNA. Furthermore, the amplicon NGS showed a 10-fold (102 copies/μL) higher sensitivity than the target capture NGS. This report provides useful insights into target enrichment NGS for whole-genome sequencing of orthohantaviruses without cultivating the viruses.

scholarly journals Rapid Metagenomic Next-Generation Sequencing during an Investigation of Hospital-Acquired Human Parainfluenza Virus 3 Infections

2016 ◽  
Vol 55 (1) ◽  
pp. 177-182 ◽  
Author(s):  
Alexander L. Greninger ◽  
Danielle M. Zerr ◽  
Xuan Qin ◽  
Amanda L. Adler ◽  
Reigran Sampoleo ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Infection Prevention ◽  
Genomic Sequence ◽  
Clinical Samples ◽  
Whole Genome ◽  
Next Generation ◽  
Sequence Coverage ◽  
Medical Unit ◽  
Hospital Acquired ◽  
Generation Sequencing

ABSTRACT Metagenomic next-generation sequencing (mNGS) is increasingly used for the unbiased detection of viruses, bacteria, fungi, and eukaryotic parasites in clinical samples. Whole-genome sequencing (WGS) of clinical bacterial isolates has been shown to inform hospital infection prevention practices, but this technology has not been utilized during potential respiratory virus outbreaks. Here, we report on the use of mNGS to inform the real-time infection prevention response to a cluster of hospital-acquired human parainfluenza 3 virus (HPIV3) infections at a children's hospital. Samples from 3 patients with hospital-acquired HPIV3 identified over a 12-day period on a general medical unit and 10 temporally associated samples from patients with community-acquired HPIV3 were analyzed. Our sample-to-sequencer time was <24 h, while our sample-to-answer turnaround time was <60 h with a hands-on time of approximately 6 h. Eight (2 cases and 6 controls) of 13 samples had sufficient sequencing coverage to yield the whole genome for HPIV3, while 10 (2 cases and 8 controls) of 13 samples gave partial genomes and all 13 samples had >1 read for HPIV3. Phylogenetic clustering revealed the presence of identical HPIV3 genomic sequence in the two of the cases with hospital-acquired infection, consistent with the concern for recent transmission within the medical unit. Adequate sequence coverage was not recovered for the third case. This work demonstrates the promise of mNGS for providing rapid information for infection prevention in addition to microbial detection.

scholarly journals Utility of Whole-Genome Next-Generation Sequencing of Plasma in Identifying Opportunistic Infections in HIV/AIDS

2019 ◽  
Vol 13 (1) ◽  
pp. 7-11 ◽  
Author(s):  
Yang Zhou ◽  
Vagish Hemmige ◽  
Sudeb C. Dalai ◽  
David K. Hong ◽  
Kenneth Muldrew ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Opportunistic Infections ◽  
Case Series ◽  
Whole Genome ◽  
Next Generation ◽  
Invasive Procedures ◽  
First Case ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Hiv Aids

Background:AIDS-associated Opportunistic Infections (OIs) have significant morbidity and mortality and can be diagnostically challenging, requiring invasive procedures as well as a combination of culture and targeted molecular approaches.Objective:We aimed to demonstrate the clinical utility of Next-generation Sequencing (NGS) in pathogen identification; NGS is a maturing technology enabling the detection of miniscule amounts of cell-free microbial DNA from the bloodstream.Methods:We utilized a novel Next-generation Sequencing (NGS) test on plasma samples to diagnose a series of HIV-associated OIs that were diagnostically confirmed through conventional microbial testing.Results:In all cases, NGS test results were available sooner than conventional testing. This is the first case series demonstrating the utility of whole-genome NGS testing to identify OIs from plasma in HIV/AIDS patients.Conclusion:NGS approaches present a clinically-actionable, comprehensive means of diagnosing OIs and other systemic infections while avoiding the labor, expense, and delays of multiple tests and invasive procedures.

scholarly journals Next-Generation Sequencing to Detect Pathogens in Pediatric Febrile Neutropenia: A Single-Center Retrospective Study of 112 Cases

2021 ◽  
Author(s):  
Kazuhiro Horiba ◽  
Yuka Torii ◽  
Toshihiko Okumura ◽  
Suguru Takeuchi ◽  
Takako Suzuki ◽  
...  
Keyword(s):  
Cluster Analysis ◽  
Next Generation Sequencing ◽  
Blood Culture ◽  
Febrile Neutropenia ◽  
Clinical Samples ◽  
Next Generation ◽  
Serum Samples ◽  
Blood Samples ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Background Febrile neutropenia (FN) is a frequent complication in immunocompromised patients. However, causative microorganisms are detected in only 10% of patients. This study aimed to detect the microorganisms that cause FN using next-generation sequencing (NGS) to idenjpgy the genome derived from pathogenic microorganisms in the bloodstream. Here, we implemented a metagenomic approach to comprehensively analyze microorganisms present in clinical samples from patients with FN. Methods FN is defined as 1) a neutrophil count &lt; 500/µL, and 2) fever ≥ 37.5 °C. Plasma/serum samples of 112 pediatric patients with FN, 10 patients with neutropenia without fever (NE), were sequenced by NGS and analyzed by a metagenomic pipeline PATHDET. Results The putative pathogens were detected by NGS in 5 of 10 patients with FN with positive for blood culture results, 15 of 87 patients (17%) with negative for blood culture results, and 3 of 8 patients with NE. Several bacteria that were common in the oral, skin, and gut flora were commonly detected in blood samples, suggesting translocation of the human microbiota to the bloodstream in the setting of neutropenia. The cluster analysis of the microbiota in blood samples using NGS demonstrated that the representative bacteria of each cluster was mostly consistent with the pathogens in each patient. Conclusions NGS technique has a great potential for detecting causative pathogens in patients with FN. Cluster analysis, which extracts characteristic microorganisms from a complex microbial population, may be effective to detect pathogens in minute quantities of microbiota, such as those from the bloodstream.

scholarly journals PEMANFAATAN TEKNOLOGI SEKUENSING GENOM UNTUK MEMPERCEPAT PROGRAM PEMULIAAN TANAMAN

2016 ◽  
Vol 34 (4) ◽  
pp. 159
Author(s):  
I Made Tasma
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Coding Region ◽  
Protein Coding ◽  
Snp Chip ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Sumber daya genetik (SDG) tanaman menyediakan materi dasar untuk program pemuliaan tanaman. Namun, baru sebagian kecil (&lt;1%) koleksi SDG yang dimanfaatkan untuk pemuliaan tanaman. Karakterisasi SDG sudah banyak dilakukan dengan menggunakan karakter morfologi, namun metode ini lambat, menyita waktu, dan memerlukan banyak tenaga. Teknologi sekuensing modern menghasilkan peta genom rujukan suatu spesies tanaman yang   dapat mempercepat karakterisasi SDG menggunakan teknik next generation sequencing (NGS). Tulisan ini mengulas pemanfaatan teknologi sekuensing genom untuk karakterisasi, proteksi, dan pemanfaatan SDG untuk mempercepat program pemuliaan tanaman. Di Indonesia, teknologi NGS telah dimanfaatkan sejak 2010 untuk resekuensing genom komoditas unggulan nasional seperti kedelai, kakao, jagung, dan cabai merah. Jutaan SNP dan Indel telah diidentifikasi pada setiap komoditas sebagai sumber daya pemuliaan yang bernilai tinggi. Sebagian kecil SNP/Indel tersebut berada pada protein coding region yang potensial untuk penemuan gen-gen unggul. Selain SNP yang diidentifikasi pada semua genotipe, ditemukan SNP pada genotipe tertentu (SNP unik). Koleksi SNP dalam jumlah besar ini digunakan untuk mensintesis SNP chip untuk genotyping SDG secara cepat dan komprehensif. Didukung data fenotipe, SNP chip bermanfaat untuk melabel gen-gen unggul. Marka SNP yang berpautan dengan karakter unggul digunakan untuk menyeleksi individu pembawa karakter unggul tersebut. Dengan teknologi NGS, perakitan VUB tanaman dapat dilakukan lebih cepat, akurat, dan efisien. Dengan demikian, teknologi NGS dapat memfasilitasi karakterisasi dan pemanfaatan SDG untuk mem-percepat program pemuliaan tanaman.

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