scholarly journals Deep Impact of Random Amplification and Library Construction Methods on Viral Metagenomics Results

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 253
Author(s):  
Béatrice Regnault ◽  
Thomas Bigot ◽  
Laurence Ma ◽  
Philippe Pérot ◽  
Sarah Temmam ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Limit Of Detection ◽  
Viral Metagenomics ◽  
High Background ◽  
Deep Impact ◽  
Library Construction ◽  
Viral Genomes ◽  
Construction Methods ◽  
Random Amplification ◽  
Reference Virus

Clinical metagenomics is a broad-range agnostic detection method of pathogens, including novel microorganisms. A major limit is the low pathogen load compared to the high background of host nucleic acids. To overcome this issue, several solutions exist, such as applying a very high depth of sequencing, or performing a relative enrichment of viral genomes associated with capsids. At the end, the quantity of total nucleic acids is often below the concentrations recommended by the manufacturers of library kits, which necessitates to random amplify nucleic acids. Using a pool of 26 viruses representative of viral diversity, we observed a deep impact of the nature of sample (total nucleic acids versus RNA only), the reverse transcription, the random amplification and library construction method on virus recovery. We further optimized the two most promising methods and assessed their performance with fully characterized reference virus stocks. Good genome coverage and limit of detection lower than 100 or 1000 genome copies per mL of plasma, depending on the genome viral type, were obtained from a three million reads dataset. Our study reveals that optimized random amplification is a technique of choice when insufficient amounts of nucleic acid are available for direct libraries constructions.

scholarly journals Highly sensitive dual mode electrochemical platform for microRNA detection

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Pawan Jolly ◽  
Marina R. Batistuti ◽  
Anna Miodek ◽  
Pavel Zhurauski ◽  
Marcelo Mulato ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Dynamic Range ◽  
Electrochemical Impedance ◽  
Limit Of Detection ◽  
Dual Mode ◽  
Electrode Surfaces ◽  
Microrna Detection ◽  
Highly Sensitive ◽  
Detection Mode ◽  
Amplification Strategy

Abstract MicroRNAs (miRNAs) play crucial regulatory roles in various human diseases including cancer, making them promising biomarkers. However, given the low levels of miRNAs present in blood, their use as cancer biomarkers requires the development of simple and effective analytical methods. Herein, we report the development of a highly sensitive dual mode electrochemical platform for the detection of microRNAs. The platform was developed using peptide nucleic acids as probes on gold electrode surfaces to capture target miRNAs. A simple amplification strategy using gold nanoparticles has been employed exploiting the inherent charges of the nucleic acids. Electrochemical impedance spectroscopy was used to monitor the changes in capacitance upon any binding event, without the need for any redox markers. By using thiolated ferrocene, a complementary detection mode on the same sensor was developed where the increasing peaks of ferrocene were recorded using square wave voltammetry with increasing miRNA concentration. This dual-mode approach allows detection of miRNA with a limit of detection of 0.37 fM and a wide dynamic range from 1 fM to 100 nM along with clear distinction from mismatched target miRNA sequences. The electrochemical platform developed can be easily expanded to other miRNA/DNA detection along with the development of microarray platforms.

scholarly journals Multiple Diverse Circoviruses Infect Farm Animals and Are Commonly Found in Human and Chimpanzee Feces

2009 ◽  
Vol 84 (4) ◽  
pp. 1674-1682 ◽  
Author(s):  
Linlin Li ◽  
Amit Kapoor ◽  
Beth Slikas ◽  
Oderinde Soji Bamidele ◽  
Chunlin Wang ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Dna Sequences ◽  
The United States ◽  
Viral Metagenomics ◽  
Farm Animals ◽  
Tissue Samples ◽  
Viral Genomes ◽  
Wide Range ◽  
Stool Samples ◽  
Meat Samples

ABSTRACT Circoviruses are known to infect birds and pigs and can cause a wide range of severe symptoms with significant economic impact. Using viral metagenomics, we identified circovirus-like DNA sequences and characterized 15 circular viral DNA genomes in stool samples from humans in Pakistan, Nigeria, Tunisia, and the United States and from wild chimpanzees. Distinct genomic features and phylogenetic analysis indicate that some viral genomes were part of a previously unrecognized genus in the Circoviridae family we tentatively named “Cyclovirus” whose genetic diversity is comparable to that of all the known species in the Circovirus genus. Circoviridae detection in the stools of U.S. adults was limited to porcine circoviruses which were also found in most U.S. pork products. To determine whether the divergent cycloviruses found in non-U.S. human stools were of dietary origin, we genetically compared them to the cycloviruses in muscle tissue samples of commonly eaten farm animals in Pakistan and Nigeria. Limited genetic overlap between cycloviruses in human stool samples and local cow, goat, sheep, camel, and chicken meat samples indicated that the majority of the 25 Cyclovirus species identified might be human viruses. We show that the genetic diversity of small circular DNA viral genomes in various mammals, including humans, is significantly larger than previously recognized, and frequent exposure through meat consumption and contact with animal or human feces provides ample opportunities for cyclovirus transmission. Determining the role of cycloviruses, found in 7 to 17% of non-U.S. human stools and 3 to 55% of non-U.S. meat samples tested, in both human and animal diseases is now facilitated by knowledge of their genomes.

scholarly journals Multiple known and a novel parvovirus associated with an outbreak of feline diarrhea and vomiting

2020 ◽  
Author(s):  
Yanpeng Li ◽  
Emilia Gordon ◽  
Amanda Idle ◽  
Eda Altan ◽  
M. Alexis Seguin ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Bacterial Pathogens ◽  
Food Products ◽  
Viral Metagenomics ◽  
Epidemiological Investigation ◽  
Fecal Samples ◽  
Attenuated Vaccine

AbstractAn unexplained outbreak of feline diarrhea and vomiting, negative for common enteric viral and bacterial pathogens, was subjected to viral metagenomics and PCR. We characterized from fecal samples the genome of a novel chapparvovirus we named fechavirus that was shed by 8/17 affected cats and different feline bocaviruses shed by 9/17 cats. Also detected were nucleic acids from attenuated vaccine viruses, members of the normal feline virome, viruses found in only one or two cases, and viruses likely derived from ingested food products. Epidemiological investigation of disease signs, time of onset, and transfers of affected cats between three facilities support a possible role for this new chapparvovirus in a highly contagious feline diarrhea and vomiting disease.

scholarly journals VIBRANT: Automated recovery, annotation and curation of microbial viruses, and evaluation of virome function from genomic sequences

2019 ◽  
Author(s):  
Kristopher Kieft ◽  
Zhichao Zhou ◽  
Karthik Anantharaman
Keyword(s):  
Microbial Community ◽  
Ecosystem Dynamics ◽  
Superior Performance ◽  
Natural Environments ◽  
Virus Identification ◽  
Viral Genomes ◽  
Reference Virus ◽  
Microbe Interactions ◽  
Host Microbe Interactions ◽  
Viral Sequences

Abstract Background Viruses are central to microbial community structure in all environments. The ability to generate large metagenomic assemblies of mixed microbial and viral sequences provides the opportunity to tease apart complex microbiome dynamics, but these analyses are currently limited by the tools available for analyses of viral genomes and assessing their metabolic impacts on microbiomes. Design Here we present VIBRANT, the first method to utilize a hybrid machine learning and protein similarity approach that is not reliant on sequence features for automated recovery and annotation of viruses, determination of genome quality and completeness, and characterization of virome function from metagenomic assemblies. VIBRANT uses neural networks of protein signatures and a novel v-score metric that circumvents traditional boundaries to maximize identification of lytic viral genomes and integrated proviruses, including highly diverse viruses. VIBRANT highlights viral auxiliary metabolic genes and metabolic pathways, thereby serving as a user-friendly platform for evaluating virome function. VIBRANT was trained and validated on reference virus datasets as well as microbiome and virome data. Results VIBRANT showed superior performance in recovering higher quality viruses and concurrently reduced the false identification of non-viral genome fragments in comparison to other virus identification programs, specifically VirSorter and VirFinder. When applied to 120,834 metagenomically derived viral sequences representing several human and natural environments, VIBRANT recovered an average of 94.5% of the viruses, whereas VirFinder and VirSorter achieved less powerful performance, averaging 48.1% and 56.0%, respectively. Similarly, VIBRANT identified more total viral sequence and proteins when applied to real metagenomes. When compared to PHASTER and Prophage Hunter for the ability to extract integrated provirus regions from host scaffolds, VIBRANT performed comparably and even identified proviruses that the other programs did not. To demonstrate applications of VIBRANT, we studied viromes associated with Crohn’s Disease to show that specific viral groups, namely Enterobacteriales-like viruses, as well as putative dysbiosis associated viral proteins are more abundant compared to healthy individuals, providing a possible viral link to maintenance of diseased states. Conclusions The ability to accurately recover viruses and explore viral impacts on microbial community metabolism will greatly advance our understanding of microbiomes, host-microbe interactions and ecosystem dynamics.

Feasibility of Immunodiagnostic Devices for the Detection of Ricin, Amanitin, and T-2 Toxin in Food

2005 ◽  
Vol 68 (6) ◽  
pp. 1294-1301 ◽  
Author(s):  
ERIC A. E. GARBER ◽  
ROBERT M. EPPLEY ◽  
MICHAEL E. STACK ◽  
MICHAEL A. McLAUGHLIN ◽  
DOUGLAS L. PARK
Keyword(s):  
High Throughput Screening ◽  
Limit Of Detection ◽  
Extraction Methods ◽  
Food Samples ◽  
Lateral Flow ◽  
Health Concern ◽  
High Background ◽  
Baked Goods ◽  
Simple Extraction ◽  
Flow Devices

Qualitative and quantitative comparisons were conducted of commercially available immunodiagnostic devices for the detection of three select agents with oral LD50 values ≥0.1 mg/kg of body weight. Ricin (oral LD50 > 1 mg/kg), amanitin (oral LD50 approximately 0.1 mg/kg), and T-2 toxin (oral LD50 > 1 mg/kg) were spiked into beverages, produce, dairy, and baked goods and assayed using commercially available enzyme-linked immunosorbent assays (ELISAs) and lateral flow devices. In all cases, the commercial diagnostic kits successfully detected all three select agents at concentrations below what might be a health concern. The considerable difference between the limit of detection of the immunodiagnostic devices employed (typically ≤0.020 μg/g) and the amount of the select agent necessary to pose a health threat in a single serving of food facilitated the design of protocols for the high throughput screening of food samples. These protocols entailed simple extraction methods followed by sample dilution. Lateral flow devices and sandwich ELISAs for the detection of ricin had no significant background problems due to the food matrices. Competitive ELISAs, which typically have unacceptably high background reactions with food samples, successfully detected amanitin and T-2 toxin.

scholarly journals Bat Guano Virome: Predominance of Dietary Viruses from Insects and Plants plus Novel Mammalian Viruses

2010 ◽  
Vol 84 (14) ◽  
pp. 6955-6965 ◽  
Author(s):  
Linlin Li ◽  
Joseph G. Victoria ◽  
Chunlin Wang ◽  
Morris Jones ◽  
Gary M. Fellers ◽  
...  
Keyword(s):  
Disease Outbreaks ◽  
Viral Metagenomics ◽  
Close Relative ◽  
Viral Pathogens ◽  
Wild Mammals ◽  
Viral Genomes ◽  
Second Generation Sequencing ◽  
Taxonomic Groups ◽  
Bat Guano

ABSTRACT Bats are hosts to a variety of viruses capable of zoonotic transmissions. Because of increased contact between bats, humans, and other animal species, the possibility exists for further cross-species transmissions and ensuing disease outbreaks. We describe here full and partial viral genomes identified using metagenomics in the guano of bats from California and Texas. A total of 34% and 58% of 390,000 sequence reads from bat guano in California and Texas, respectively, were related to eukaryotic viruses, and the largest proportion of those infect insects, reflecting the diet of these insectivorous bats, including members of the viral families Dicistroviridae, Iflaviridae, Tetraviridae, and Nodaviridae and the subfamily Densovirinae. The second largest proportion of virus-related sequences infects plants and fungi, likely reflecting the diet of ingested insects, including members of the viral families Luteoviridae, Secoviridae, Tymoviridae, and Partitiviridae and the genus Sobemovirus. Bat guano viruses related to those infecting mammals comprised the third largest group, including members of the viral families Parvoviridae, Circoviridae, Picornaviridae, Adenoviridae, Poxviridae, Astroviridae, and Coronaviridae. No close relative of known human viral pathogens was identified in these bat populations. Phylogenetic analysis was used to clarify the relationship to known viral taxa of novel sequences detected in bat guano samples, showing that some guano viral sequences fall outside existing taxonomic groups. This initial characterization of the bat guano virome, the first metagenomic analysis of viruses in wild mammals using second-generation sequencing, therefore showed the presence of previously unidentified viral species, genera, and possibly families. Viral metagenomics is a useful tool for genetically characterizing viruses present in animals with the known capability of direct or indirect viral zoonosis to humans.

scholarly journals Deep viral blood metagenomics reveals extensive anellovirus diversity in healthy humans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
María Cebriá-Mendoza ◽  
Cristina Arbona ◽  
Luís Larrea ◽  
Wladimiro Díaz ◽  
Vicente Arnau ◽  
...  
Keyword(s):  
Human Blood ◽  
High Speed ◽  
Viral Genomes ◽  
Healthy Humans ◽  
Healthy Donors ◽  
Random Amplification ◽  
Different Types ◽  
Rna And Dna ◽  
Apparently Healthy

AbstractHuman blood metagenomics has revealed the presence of different types of viruses in apparently healthy subjects. By far, anelloviruses constitute the viral family that is more frequently found in human blood, although amplification biases and contaminations pose a major challenge in this field. To investigate this further, we subjected pooled plasma samples from 120 healthy donors in Spain to high-speed centrifugation, RNA and DNA extraction, random amplification, and massive parallel sequencing. Our results confirm the extensive presence of anelloviruses in such samples, which represented nearly 97% of the total viral sequence reads obtained. We assembled 114 different viral genomes belonging to this family, revealing remarkable diversity. Phylogenetic analysis of ORF1 suggested 28 potentially novel anellovirus species, 24 of which were validated by Sanger sequencing to discard artifacts. These findings underscore the importance of implementing more efficient purification procedures that enrich the viral fraction as an essential step in virome studies and question the suggested pathological role of anelloviruses.

scholarly journals Comparison of double-stranded and single-stranded cfDNA library construction methods for targeted genome and methylation sequencing

2022 ◽  
Author(s):  
Jianchao Zheng ◽  
Zhilong Li ◽  
Xiuqing Zhang ◽  
Hongyun Zhang ◽  
Shida Zhu ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Mutation Detection ◽  
Therapy Response ◽  
Utilization Efficiency ◽  
Response Monitoring ◽  
Library Construction ◽  
Construction Methods ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Methylation Profiling

Cell-free DNA (cfDNA) profiling by deep sequencing (i.e., by next generation sequencing (NGS)) has wide applications in cancer diagnosis, prognosis, and therapy response monitoring. One key step of cfDNA deep sequencing workflow is NGS library construction, whose efficiency significantly affects the utilization efficiency of cfDNA molecules, and eventually determines effective sequencing depth and sequencing accuracy. In this study, we compared two different types of cfDNA library construction methods, namely double-stranded library (dsLib, the conventional method which captures dsDNA molecules) and single-stranded library (ssLib) preparation, which captures ssDNA molecules, for the applications of mutation detection and methylation profiling, respectively. Our results suggest that the dsLib method was suitable for mutation detection while the ssLib method proved more efficient for methylation analysis. Our findings could help researchers choose the more appropriate library construction method for corresponding downstream applications of cfDNA sequencing.

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