scholarly journals Towards quantitative viromics for both double-stranded and single-stranded DNA viruses

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2777 ◽  
Author(s):  
Simon Roux ◽  
Natalie E. Solonenko ◽  
Vinh T. Dang ◽  
Bonnie T. Poulos ◽  
Sarah M. Schwenck ◽  
...  
Keyword(s):  
Multiple Displacement Amplification ◽  
Ecosystem Functions ◽  
Library Preparation ◽  
Single Stranded Dna ◽  
Preparation Methods ◽  
Dna Viruses ◽  
Viral Genomes ◽  
Sequencing Library ◽  
Dsdna Viruses ◽  
Ssdna Viruses

BackgroundViruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation).MethodsHere we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses.ResultsMock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from theMicroviridaefamily, can be among the most abundant viral genomes in a sample.DiscussionTogether these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

scholarly journals Amplification of Uncultured Single-Stranded DNA Viruses from Rice Paddy Soil

2008 ◽  
Vol 74 (19) ◽  
pp. 5975-5985 ◽  
Author(s):  
Kyoung-Ho Kim ◽  
Ho-Won Chang ◽  
Young-Do Nam ◽  
Seong Woon Roh ◽  
Min-Soo Kim ◽  
...  
Keyword(s):  
Multiple Displacement Amplification ◽  
Rice Paddy ◽  
Viral Diversity ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Amplification Method ◽  
Metagenome Sequencing ◽  
Biological Entities ◽  
Related Proteins ◽  
Ssdna Viruses

ABSTRACT Viruses are known to be the most numerous biological entities in soil; however, little is known about their diversity in this environment. In order to explore the genetic diversity of soil viruses, we isolated viruses by centrifugation and sequential filtration before performing a metagenomic investigation. We adopted multiple-displacement amplification (MDA), an isothermal whole-genome amplification method with φ29 polymerase and random hexamers, to amplify viral DNA and construct clone libraries for metagenome sequencing. By the MDA method, the diversity of both single-stranded DNA (ssDNA) viruses and double-stranded DNA viruses could be investigated at the same time. On the contrary, by eliminating the denaturing step in the MDA reaction, only ssDNA viral diversity could be explored selectively. Irrespective of the denaturing step, more than 60% of the soil metagenome sequences did not show significant hits (E-value criterion, 0.001) with previously reported viral sequences. Those hits that were considered to be significant were also distantly related to known ssDNA viruses (average amino acid similarity, approximately 34%). Phylogenetic analysis showed that replication-related proteins (which were the most frequently detected proteins) related to those of ssDNA viruses obtained from the metagenomic sequences were diverse and novel. Putative circular genome components of ssDNA viruses that are unrelated to known viruses were assembled from the metagenomic sequences. In conclusion, ssDNA viral diversity in soil is more complex than previously thought. Soil is therefore a rich pool of previously unknown ssDNA viruses.

scholarly journals Single-Stranded DNA Viruses in Antarctic Cryoconite Holes

Viruses ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1022 ◽  
Author(s):  
Pacifica Sommers ◽  
Rafaela S. Fontenele ◽  
Tayele Kringen ◽  
Simona Kraberger ◽  
Dorota L. Porazinska ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Open Reading Frames ◽  
Pairwise Identity ◽  
Capsid Protein Gene ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Viral Genomes ◽  
Freshwater Pond ◽  
Dry Valley ◽  
Population Structures

Antarctic cryoconite holes, or small melt-holes in the surfaces of glaciers, create habitable oases for isolated microbial communities with tightly linked microbial population structures. Viruses may influence the dynamics of polar microbial communities, but the viromes of the Antarctic cryoconite holes have yet to be characterized. We characterize single-stranded DNA (ssDNA) viruses from three cryoconite holes in the Taylor Valley, Antarctica, using metagenomics. Half of the assembled metagenomes cluster with those in the viral family Microviridae (n = 7), and the rest with unclassified circular replication associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses (n = 7). An additional 18 virus-like circular molecules encoding either a Rep, a capsid protein gene, or other unidentified but viral-like open reading frames were identified. The samples from which the genomes were identified show a strong gradient in microbial diversity and abundances, and the number of viral genomes detected in each sample mirror that gradient. Additionally, one of the CRESS genomes assembled here shares ~90% genome-wide pairwise identity with a virus identified from a freshwater pond on the McMurdo Ice Shelf (Antarctica). Otherwise, the similarity of these viruses to those previously identified is relatively low. Together, these patterns are consistent with the presence of a unique regional virome present in fresh water host populations of the McMurdo Dry Valley region.

scholarly journals Comparing library preparation methods for SARS-CoV-2 multiplex amplicon sequencing on the Illumina MiSeq platform

2020 ◽  
Author(s):  
Elizabeth M. Batty ◽  
Theerarat Kochakarn ◽  
Arporn Wangwiwatsin ◽  
Khajohn Joonlasak ◽  
Angkana T. Huang ◽  
...  
Keyword(s):  
Variant Calling ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Library Preparation ◽  
Preparation Methods ◽  
Base Calling ◽  
Sequencing Library ◽  
Miseq Platform ◽  
Downstream Analysis ◽  
Sequencing Library Preparation

AbstractGenomic surveillance has a key role in tracking the ongoing COVID-19 pandemic, but information on how different sequencing library preparation approaches affect the data produced are lacking. We compared three library preparation methods using both tagmentation (Nextera XT and Nextera Flex) and ligation-based (KAPA HyperPrep) approaches on both positive and negative samples to provide insights into any methodological differences between the methods, and validate their use in SARS-CoV-2 amplicon sequencing. We show that all three library preparation methods allow us to recover near-complete SARS-CoV-2 genomes with identical SNP calls. The Nextera Flex and KAPA library preparation methods gave better coverage than libraries prepared with Nextera XT, which required more reads to call the same number of genomic positions. The KAPA ligation-based approach shows the lowest levels of human contamination, but contaminating reads had no effect on the downstream analysis. We found some examples of library preparation-specific differences in minority variant calling. Overall our data shows that the choice of Illumina library preparation method has minimal effects on consensus base calling and downstream phylogenetic analysis, and suggests that all methods would be suitable for use if specific reagents are difficult to obtain.

A variety of highly divergent eukaryotic ssDNA viruses in sera of pigs

2021 ◽  
Vol 102 (12) ◽  
Author(s):  
Caroline Tochetto ◽  
Samuel Paulo Cibulski ◽  
Ana Paula Muterle Varela ◽  
Cristine Cerva ◽  
Diane Alves de Lima ◽  
...  
Keyword(s):  
Potential Role ◽  
Clinical Signs ◽  
Viral Metagenomics ◽  
Dna Viruses ◽  
Circular Dna ◽  
Viral Genomes ◽  
The Family ◽  
Circular Ssdna ◽  
Ssdna Viruses ◽  
Targeted Approach

Over the last decade, viral metagenomics has been established as a non-targeted approach for identifying viruses in stock animals, including pigs. This has led to the identification of a vast diversity of small circular ssDNA viruses. The present study focuses on the investigation of eukaryotic circular Rep-encoding single-stranded (CRESS) DNA viral genomes present in serum of commercially reared pigs from southern Brazil. Several CRESS DNA viral genomes were detected, including representatives of the families Smacoviridae (n=5), Genomoviridae (n=3), Redondoviridae (n=1), Nenyaviridae (n=1) and other yet unclassified genomes (n=9), plus a circular DNA molecule, which probably belongs to the phylum Cressdnaviricota. A novel genus within the family Smacoviridae, tentatively named ‘Suismacovirus’, comprising 21 potential new species, is proposed. Although the reported genomes were recovered from pigs with clinical signs of respiratory disease, further studies should examine their potential role as pathogens. Nonetheless, these findings highlight the diversity of circular ssDNA viruses in serum of domestic pigs, expand the knowledge on CRESS DNA viruses’ genetic diversity and distribution and contribute to the global picture of the virome of commercially reared pigs.

scholarly journals Diverse circular ssDNA viruses discovered in dragonflies (Odonata: Epiprocta)

2012 ◽  
Vol 93 (12) ◽  
pp. 2668-2681 ◽  
Author(s):  
Karyna Rosario ◽  
Anisha Dayaram ◽  
Milen Marinov ◽  
Jessica Ware ◽  
Simona Kraberger ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Molecular Techniques ◽  
Limited Information ◽  
Dna Viruses ◽  
Rolling Circle ◽  
Viral Genomes ◽  
Initiator Protein ◽  
Circular Ssdna ◽  
Insect Populations ◽  
Ssdna Viruses

Viruses with circular ssDNA genomes that encode a replication initiator protein (Rep) are among the smallest viruses known to infect both eukaryotic and prokaryotic organisms. In the past few years an overwhelming diversity of novel circular Rep-encoding ssDNA (CRESS-DNA) viruses has been unearthed from various hosts and environmental sources. Since there is limited information regarding CRESS-DNA viruses in invertebrates, this study explored the diversity of CRESS-DNA viruses circulating among insect populations by targeting dragonflies (Epiprocta), top insect predators that accumulate viruses from their insect prey over space and time. Using degenerate PCR and rolling circle amplification coupled with restriction digestion, 17 CRESS-DNA viral genomes were recovered from eight different dragonfly species collected in tropical and temperate regions. Nine of the genomes are similar to cycloviruses and represent five species within this genus, suggesting that cycloviruses are commonly associated with insects. Three of the CRESS-DNA viruses share conserved genomic features with recently described viruses similar to the mycovirus Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, leading to the proposal of the genus Gemycircularvirus. The remaining viruses are divergent species representing four novel CRESS-DNA viral genera, including a gokushovirus-like prokaryotic virus (microphage) and three eukaryotic viruses with Reps similar to circoviruses. The novelty of CRESS-DNA viruses identified in dragonflies using simple molecular techniques indicates that there is an unprecedented diversity of ssDNA viruses among insect populations.

scholarly journals Diversity and Abundance of Single-Stranded DNA Viruses in Human Feces

2011 ◽  
Vol 77 (22) ◽  
pp. 8062-8070 ◽  
Author(s):  
Min-Soo Kim ◽  
Eun-Jin Park ◽  
Seong Woon Roh ◽  
Jin-Woo Bae
Keyword(s):  
Capsid Protein ◽  
Protein Sequences ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Content Type ◽  
Double Stranded Dna ◽  
Abundance And Diversity ◽  
Ssdna Viruses ◽  
Virus Abundance ◽  
Large Contigs

ABSTRACTIn this study, we investigated the abundance and diversity of single-stranded DNA (ssDNA) viruses in fecal samples from five healthy individuals through a combination of serial filtration and CsCl gradient ultracentrifugation. Virus abundance ranged from 108to 109per gram of feces, and virus-to-bacterium ratios were much lower (less than 0.1) than those observed in aquatic environments (5 to 10). Viral DNA was extracted and randomly amplified using phi29 polymerase and analyzed through high-throughput 454 pyrosequencing. Among 400,133 sequences, an average of 86.2% viromes were previously uncharacterized in public databases. Among previously known viruses, double-stranded DNA podophages (52 to 74%), siphophages (11 to 30%), myophages (1 to 4%), and ssDNA microphages (3 to 9%) were major constituents of human fecal viromes. A phylogenetic analysis of 24 large contigs of microphages based on conserved capsid protein sequences revealed five distinct newly discovered evolutionary microphage groups that were distantly related to previously known microphages. Moreover, putative capsid protein sequences of five contigs were closely related to prophage-like sequences in the genomes of threeBacteroidesand threePrevotellastrains, suggesting thatBacteroidesandPrevotellaare the sources of infecting microphages in their hosts.

scholarly journals A comparative study of ChIP-seq sequencing library preparation methods

BMC Genomics ◽  
2016 ◽  
Vol 17 (1) ◽  
Author(s):  
Arvind Y. M. Sundaram ◽  
Timothy Hughes ◽  
Shea Biondi ◽  
Nathalie Bolduc ◽  
Sarah K. Bowman ◽  
...  
Keyword(s):  
Comparative Study ◽  
Library Preparation ◽  
Preparation Methods ◽  
Sequencing Library ◽  
Sequencing Library Preparation

scholarly journals Distinct Circular Single-Stranded DNA Viruses Exist in Different Soil Types

2015 ◽  
Vol 81 (12) ◽  
pp. 3934-3945 ◽  
Author(s):  
Brian Reavy ◽  
Maud M. Swanson ◽  
Peter J. A. Cock ◽  
Lorna Dawson ◽  
Thomas E. Freitag ◽  
...  
Keyword(s):  
Soil Types ◽  
Metagenomic Sequencing ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Unique Type ◽  
Content Type ◽  
Virus Particles ◽  
Intracellular Parasites ◽  
Ssdna Viruses ◽  
Virus Genomes

ABSTRACTThe potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the familyMicroviridae(icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis ofMicroviridaemajor coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamilyGokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar familyCircoviridaein BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in theCircoviridaeshowed that they are a novel clade ofCircoviridae-related CRESS-DNA viruses distinct from knownCircoviridaegenera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors.

scholarly journals Viral recombination blurs taxonomic lines: examination of single-stranded DNA viruses in a wastewater treatment plant

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2585 ◽  
Author(s):  
Victoria M. Pearson ◽  
S. Brian Caudle ◽  
Darin R. Rokyta
Keyword(s):  
Wastewater Treatment ◽  
Microbial Communities ◽  
Rolling Circle Amplification ◽  
Treatment Plant ◽  
Microbial Pathogens ◽  
Model Organisms ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Depth Analysis ◽  
Ssdna Viruses

Understanding the structure and dynamics of microbial communities, especially those of economic concern, is of paramount importance to maintaining healthy and efficient microbial communities at agricultural sites and large industrial cultures, including bioprocessors. Wastewater treatment plants are large bioprocessors which receive water from multiple sources, becoming reservoirs for the collection of many viral families that infect a broad range of hosts. To examine this complex collection of viruses, full-length genomes of circular ssDNA viruses were isolated from a wastewater treatment facility using a combination of sucrose-gradient size selection and rolling-circle amplification and sequenced on an Illumina MiSeq. Single-stranded DNA viruses are among the least understood groups of microbial pathogens due to genomic biases and culturing difficulties, particularly compared to the larger, more often studied dsDNA viruses. However, the group contains several notable well-studied examples, including agricultural pathogens which infect both livestock and crops (CircoviridaeandGeminiviridae), and model organisms for genetics and evolution studies (Microviridae). Examination of the collected viral DNA provided evidence for 83 unique genotypic groupings, which were genetically dissimilar to known viral types and exhibited broad diversity within the community. Furthermore, although these genomes express similarities to known viral families, such asCircoviridae,Geminiviridae, andMicroviridae, many are so divergent that they may represent new taxonomic groups. This study demonstrated the efficacy of the protocol for separating bacteria and large viruses from the sought after ssDNA viruses and the ability to use this protocol to obtain an in-depth analysis of the diversity within this group.

scholarly journals A Microfluidics Workflow for Sample Preparation for Next-Generation DNA Sequencing

2018 ◽  
Vol 24 (2) ◽  
pp. 196-208 ◽  
Author(s):  
Adam Snider ◽  
Michael Nilsson ◽  
Mark Dupal ◽  
Masoud Toloue ◽  
Anubhav Tripathi
Keyword(s):  
Sample Preparation ◽  
Magnetic Bead ◽  
Library Preparation ◽  
Next Generation ◽  
Preparation Methods ◽  
Next Generation Sequencing Technology ◽  
Extraction And Purification ◽  
Spin Column ◽  
Sequencing Library ◽  
End Sequences

Next-generation sequencing technology requires amplified, short DNA fragments with known end sequences. Samples must undergo processing steps, including extraction and purification of genomic DNA (gDNA), fragmentation, end repair, adapter ligation, and amplification, to prepare a sequencing library. The process of sample preparation requires careful control of temperature and buffer conditions, as well as the stringent removal of contaminants. As a result, library preparation methods are often plagued by sample loss, long protocol times, numerous manual steps, and high cost. We attempt to understand and optimize each step of sample preparation on a microfluidic platform using magnetic bead motion through channels containing immiscible phases. Our platform integrates all steps associated with library preparation with no buffer exchanges and utilizes just 30–60 µL of reagents. Our chip shows a sixfold improvement in yield compared with an affinity spin column when capturing gDNA from samples of ~50 ± 4 MCF-7 cells. Finally, we show whole-genome shotgun sequencing results from 660 pg of human gDNA, in which >93 ± 1% of reads map to a reference genome at or above 99.9% confidence, matching a commercially available sample preparation kit optimized for low-cell-count samples.

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