scholarly journals Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing

2018 ◽  
Author(s):  
Zhihui Yang ◽  
Mark Mammel ◽  
Chris A. Whitehouse ◽  
Diana Ngo ◽  
Michael Kulka
Keyword(s):  
Next Generation Sequencing ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Rna Viruses ◽  
Sequence Variant ◽  
Single Individual ◽  
Next Generation ◽  
Source Attribution ◽  
Clinical Specimens ◽  
Generation Sequencing

The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as Hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

scholarly journals Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 619 ◽  
Author(s):  
Zhihui Yang ◽  
Mark Mammel ◽  
Chris Whitehouse ◽  
Diana Ngo ◽  
Michael Kulka
Keyword(s):  
Next Generation Sequencing ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Rna Viruses ◽  
Sequence Variant ◽  
Single Individual ◽  
Next Generation ◽  
Source Attribution ◽  
Clinical Specimens ◽  
Generation Sequencing

The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

scholarly journals Genotyping and Molecular Diagnosis of Hepatitis A Virus in Human Clinical Samples Using Multiplex PCR-Based Next-Generation Sequencing

2022 ◽  
Vol 10 (1) ◽  
pp. 100
Author(s):  
Geum-Young Lee ◽  
Won-Keun Kim ◽  
Seungchan Cho ◽  
Kyungmin Park ◽  
Jongwoo Kim ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Multiplex Pcr ◽  
Molecular Diagnosis ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Severe Illness ◽  
Whole Genome ◽  
Next Generation ◽  
Genome Sequences ◽  
Generation Sequencing

Hepatitis A virus (HAV) is a serious threat to public health worldwide. We used multiplex polymerase chain reaction (PCR)-based next-generation sequencing (NGS) to derive information on viral genetic diversity and conduct precise phylogenetic analysis. Four HAV genome sequences were obtained using multiplex PCR-based NGS. HAV whole-genome sequence of one sample was obtained by conventional Sanger sequencing. The HAV strains demonstrated a geographic cluster with sub-genotype IA strains in the Republic of Korea. The phylogenetic pattern of HAV viral protein (VP) 3 region showed no phylogenetic conflict between the whole-genome and partial-genome sequences. The VP3 region in serum and stool samples showed sensitive detection of HAV with differences of quantification that did not exceed <10 copies/μL than the consensus VP4 region using quantitative PCR (qPCR). In conclusion, multiplex PCR-based NGS was implemented to define HAV genotypes using nearly whole-genome sequences obtained directly from hepatitis A patients. The VP3 region might be a potential candidate for tracking the genotypic origin of emerging HAV outbreaks. VP3-specific qPCR was developed for the molecular diagnosis of HAV infection. This study may be useful to predict for the disease management and subsequent development of hepatitis A infection at high risk of severe illness.

Molecular marker information from de novo assembled transcriptomes of chilli pepper (Capsicum annuum L.) varieties based on next-generation sequencing technology

2014 ◽  
Vol 12 (S1) ◽  
pp. S83-S86 ◽  
Author(s):  
Yul-Kyun Ahn ◽  
Swati Tripathi ◽  
Young-Il Cho ◽  
Jeong-Ho Kim ◽  
Hye-Eun Lee ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Next Generation Sequencing ◽  
De Novo ◽  
Transcriptome Assembly ◽  
Sequence Variant ◽  
Nucleotide Polymorphisms ◽  
Next Generation ◽  
Chilli Pepper ◽  
Next Generation Sequencing Technology ◽  
Generation Sequencing

Next-generation sequencing technique has been known as a useful tool for de novo transcriptome assembly, functional annotation of genes and identification of molecular markers. This study was carried out to mine molecular markers from de novo assembled transcriptomes of four chilli pepper varieties, the highly pungent ‘Saengryeg 211’ and non-pungent ‘Saengryeg 213’ and variably pigmented ‘Mandarin’ and ‘Blackcluster’. Pyrosequencing of the complementary DNA library resulted in 361,671, 274,269, 279,221, and 316,357 raw reads, which were assembled in 23,607, 19,894, 18,340 and 20,357 contigs, for the four varieties, respectively. Detailed sequence variant analysis identified numerous potential single-nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) for all the varieties for which the primers were designed. The transcriptome information and SNP/SSR markers generated in this study provide valuable resources for high-density molecular genetic mapping in chilli pepper and Quantitative trait loci analysis related to fruit qualities. These markers for pepper will be highly valuable for marker-assisted breeding and other genetic studies.

scholarly journals A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

mSystems ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Lindsey A. Moser ◽  
Lisbeth Ramirez-Carvajal ◽  
Vinita Puri ◽  
Steven J. Pauszek ◽  
Krystal Matthews ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Operating Procedure ◽  
Rna Viruses ◽  
Standard Operating Procedure ◽  
Next Generation ◽  
Standard Operating ◽  
Level 3 ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Biosafety Level

ABSTRACT This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories. Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories.

scholarly journals Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses

Viruses ◽  
2016 ◽  
Vol 8 (3) ◽  
pp. 70 ◽  
Author(s):  
Hironobu Yanagisawa ◽  
Reiko Tomita ◽  
Koji Katsu ◽  
Takuya Uehara ◽  
Go Atsumi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rna Viruses ◽  
Next Generation ◽  
Efficient Detection ◽  
Generation Sequencing

scholarly journals Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

2016 ◽  
Vol 54 (4) ◽  
pp. 919-927 ◽  
Author(s):  
Mohammad R. Hasan ◽  
Arun Rawat ◽  
Patrick Tang ◽  
Puthen V. Jithesh ◽  
Eva Thomas ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Pathogen Detection ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Next Generation ◽  
Simple Method ◽  
Clinical Specimens ◽  
Human Dna ◽  
Pathogen Dna ◽  
Generation Sequencing

Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%;P< 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome (P< 0.05) and improved the sensitivity of pathogen detection (P< 0.01), with a 20- to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples.

scholarly journals 665. Clinical and Financial Impact of Next Generation Sequencing (NGS) in addition to Conventional Microbiology Testing in our Urban Referral Health Center

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S434-S435
Author(s):  
Vikram Saini ◽  
Tariq Jaber ◽  
James D Como ◽  
Rasha Abdulmassih ◽  
Zaw Min ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Financial Burden ◽  
Tissue Fluid ◽  
Next Generation ◽  
Financial Cost ◽  
Clinical Specimens ◽  
Microbial Identification ◽  
Soc Testing ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Background Clinical microbiology traditionally relies on culture methodology and serological testing, that have inherent limitations. Newer diagnostic techniques such as Next Generation Sequencing (NGS) have shown promise to improve microbial identification. In select scenarios, we send clinical specimens to reference laboratories for NGS testing in addition to current standard of care (SOC) diagnostics. We wanted to determine how this diagnostic approach has impacted patient care. We also wanted to review the financial burden through cost-benefit analysis for these ‘send-out’ tests. Methods We performed a retrospective chart review of all cases over a 3-year period in which NGS was submitted. Data, including demographics, comorbidities, antimicrobial use, and diagnosis (by SOC and NGS) were gathered. We delineated how often there was concordance or discordance between SOC and NGS. We also obtained information on financial cost (direct and indirect) and turnaround time (TAT) for NGS results. Results A total of 33 clinical specimens from 25 patients were sent for NGS. The majority of specimens comprised joint tissue/fluid, organ tissue and CSF. Concordance occurred between SOC and NGS testing in 75.8% (25/33) of samples; of those, 88% excluded infection. NGS identified a pathogen in 20% (5/25) patients in which concomitant SOC testing was negative. A subsequent change in antimicrobial management occurred in 16% (4/25) of patients. The mean TAT was 14 days and average cost per specimen was &821.52 (range: &573-&1590). Table 1. Pathogens identified by NGS with negative traditional microbiological test results Figure 1. Distribution of specimen site (in %) sent for NGS Conclusion NGS can provide additional diagnostic sensitivity in infectious diseases, which at our institution identified a new pathogen in 20% and a resultant treatment change in 16% of our patients. This testing may also allow physicians to reaffirm the absence of an infection diagnosis. A larger NGS testing population may reveal more significant benefits. While the attributable cost of NGS was substantial, it should be measured against the costs of administration of unnecessary antibiotics, inaccurate diagnosis, and adverse patient outcomes that may result from SOC testing alone. Considering its financial cost and extended TAT, in-house NGS testing may be warranted to facilitate a higher volume of testing. Disclosures All Authors: No reported disclosures

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