scholarly journals MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution

2021 ◽  
Author(s):  
CM Gallardo ◽  
S Wang ◽  
DJ Montiel-Garcia ◽  
SJ Little ◽  
DM Smith ◽  
...  
Keyword(s):  
Long Range ◽  
Single Molecule ◽  
Vaccine Development ◽  
Complex Mixture ◽  
Therapeutic Benefit ◽  
Read Length ◽  
Clinical Samples ◽  
Viral Genomes ◽  
Immune Pressure ◽  
Sequencing Platforms

AbstractTechnical challenges remain in the sequencing of RNA viruses due to their high intra-host diversity. This bottleneck is particularly pronounced when interrogating long-range co-evolution given the read-length limitations of next-generation sequencing platforms. This has hampered the direct observation of long-range genetic interactions that code for protein-protein interfaces with relevance in both drug and vaccine development. Here we overcome these technical limitations by developing a nanopore-based long-range viral sequencing pipeline that yields accurate single molecule sequences of circulating virions from clinical samples. We demonstrate its utility in observing the evolution of individual HIV Gag-Pol genomes in response to antiviral pressure. Our pipeline, called Multi-read Hairpin Mediated Error-correction Reaction (MrHAMER), yields >1000s viral genomes per sample at 99.9% accuracy, maintains the original proportion of sequenced virions present in a complex mixture, and allows the detection of rare viral genomes with their associated mutations present at <1% frequency. This method facilitates scalable investigation of genetic correlates of resistance to both antiviral therapy and immune pressure, and enable the identification of novel host-viral and viral-viral interfaces that can be modulated for therapeutic benefit.

scholarly journals MINERVA: A facile strategy for SARS-CoV-2 whole genome deep sequencing of clinical samples

2020 ◽  
Author(s):  
Chen Chen ◽  
Jizhou Li ◽  
Lin Di ◽  
Qiuyu Jing ◽  
Pengcheng Du ◽  
...  
Keyword(s):  
High Performance ◽  
Vaccine Development ◽  
Viral Evolution ◽  
High Sensitivity ◽  
High Yield ◽  
Clinical Samples ◽  
High Coverage ◽  
Preparation Methods ◽  
Viral Genomes ◽  
Viral Spread

AbstractThe novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from clinical samples is crucial for the understanding of viral spread and viral evolution, as well as for vaccine development. Existing sample preparation methods for viral genome sequencing are demanding on user technique and time, and thus not ideal for time-sensitive clinical samples; these methods are also not optimized for high performance on viral genomes. We have developed MetagenomIc RNA EnRichment VirAl sequencing (MINERVA), a facile, practical, and robust approach for metagenomic and deep viral sequencing from clinical samples. This approach uses direct tagmentation of RNA/DNA hybrids using Tn5 transposase to greatly simplify the sequencing library construction process, while subsequent targeted enrichment can generate viral genomes with high sensitivity, coverage, and depth. We demonstrate the utility of MINERVA on pharyngeal, sputum and stool samples collected from COVID-19 patients, successfully obtaining both whole metatranscriptomes and complete high-depth high-coverage SARS-CoV-2 genomes from these clinical samples, with high yield and robustness. MINERVA is compatible with clinical nucleic extracts containing carrier RNA. With a shortened hands-on time from sample to virus-enriched sequencing-ready library, this rapid, versatile, and clinic-friendly approach will facilitate monitoring of viral genetic variations during outbreaks, both current and future.

scholarly journals Genome-wide epigenetic profiling of 5-hydroxymethylcytosine by long-read optical mapping

2018 ◽  
Author(s):  
Tslil Gabrieli ◽  
Hila Sharim ◽  
Gil Nifker ◽  
Jonathan Jeffet ◽  
Tamar Shahal ◽  
...  
Keyword(s):  
Long Range ◽  
Single Molecule ◽  
Human Peripheral Blood ◽  
Read Length ◽  
Epigenetic Mark ◽  
Sequencing Data ◽  
Chromosomal Dna ◽  
Genome Wide ◽  
Long Read ◽  
Genomic Regions

AbstractThe epigenetic mark 5-hydroxymethylcytosine (5-hmC) is a distinct product of active enzymatic demethylation that is linked to gene regulation, development and disease. Genome-wide 5-hmC profiles generated by short-read next-generation sequencing are limited in providing long-range epigenetic information relevant to highly variable genomic regions, such as the 3.7 Mbp disease-related Human Leukocyte Antigen (HLA) region. We present a long-read, single-molecule mapping technology that generates hybrid genetic/epigenetic profiles of native chromosomal DNA. The genome-wide distribution of 5- hmC in human peripheral blood cells correlates well with 5-hmC DNA immunoprecipitation (hMeDIP) sequencing. However, the long read length of 100 kbp-1Mbp produces 5-hmC profiles across variable genomic regions that failed to showup in the sequencing data. In addition, optical 5-hmC mapping shows strong correlation between the 5-hmC density in gene bodies and the corresponding level of gene expression. The single molecule concept provides information on the distribution and coexistence of 5-hmC signals at multiple genomic loci on the same genomic DNA molecule, revealing long-range correlations and cell-to-cell epigenetic variation.

scholarly journals SARS-CoV-2 genomes recovered by long amplicon tiling multiplex approach using nanopore sequencing and applicable to other sequencing platforms

2020 ◽  
Author(s):  
Paola Cristina Resende ◽  
Fernando Couto Motta ◽  
Sunando Roy ◽  
Luciana Appolinario ◽  
Allison Fabri ◽  
...  
Keyword(s):  
Clinical Samples ◽  
Nanopore Sequencing ◽  
Total Rna ◽  
Viral Genomes ◽  
Long Reads ◽  
Sequencing Platforms

SummaryGenomic surveillance has become a useful tool for better understanding virus pathogenicity, origin and spread. Obtaining accurately assembled, complete viral genomes directly from clinical samples is still a challenging. Here, we describe three protocols using a unique primer set designed to recover long reads of SARS-CoV-2 directly from total RNA extracted from clinical samples. This protocol is useful, accessible and adaptable to laboratories with varying resources and access to distinct sequencing methods: Nanopore, Illumina and/or Sanger.

scholarly journals Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 779
Author(s):  
Man Teng ◽  
Yongxiu Yao ◽  
Venugopal Nair ◽  
Jun Luo
Keyword(s):  
Biological Sciences ◽  
Gene Therapy ◽  
Genetic Engineering ◽  
Gene Function ◽  
Viral Genome ◽  
Gene Editing ◽  
Vaccine Development ◽  
Future Prospects ◽  
Dna Viruses ◽  
Viral Genomes

In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.

scholarly journals Phylogenomics reveals viral sources, transmission, and potential superinfection in early-stage COVID-19 patients in Ontario, Canada

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Calvin P. Sjaarda ◽  
Nazneen Rustom ◽  
Gerald A. Evans ◽  
David Huang ◽  
Santiago Perez-Patrigeon ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Disease Surveillance ◽  
Early Stage ◽  
Emerging Diseases ◽  
Contact Tracing ◽  
Travel History ◽  
Infectious Disease Surveillance ◽  
Viral Genomes ◽  
Heterozygous Variant ◽  
Sequencing Platforms

AbstractThe emergence and rapid global spread of SARS-CoV-2 demonstrates the importance of infectious disease surveillance, particularly during the early stages. Viral genomes can provide key insights into transmission chains and pathogenicity. Nasopharyngeal swabs were obtained from thirty-two of the first SARS-CoV-2 positive cases (March 18–30) in Kingston Ontario, Canada. Viral genomes were sequenced using Ion Torrent (n = 24) and MinION (n = 27) sequencing platforms. SARS-CoV-2 genomes carried forty-six polymorphic sites including two missense and three synonymous variants in the spike protein gene. The D614G point mutation was the predominate viral strain in our cohort (92.6%). A heterozygous variant (C9994A) was detected by both sequencing platforms but filtered by the ARTIC network bioinformatic pipeline suggesting that heterozygous variants may be underreported in the SARS-CoV-2 literature. Phylogenetic analysis with 87,738 genomes in the GISAID database identified global origins and transmission events including multiple, international introductions as well as community spread. Reported travel history validated viral introduction and transmission inferred by phylogenetic analysis. Molecular epidemiology and evolutionary phylogenetics may complement contact tracing and help reconstruct transmission chains of emerging diseases. Earlier detection and screening in this way could improve the effectiveness of regional public health interventions to limit future pandemics.

scholarly journals Genome-wide integration site detection using Cas9 enriched amplification-free long-range sequencing

2020 ◽  
Author(s):  
Joost van Haasteren ◽  
Altar M Munis ◽  
Deborah R Gill ◽  
Stephen C Hyde
Keyword(s):  
Long Range ◽  
Insertional Mutagenesis ◽  
Lentiviral Vector ◽  
Integration Site ◽  
Low Cost ◽  
Safety Evaluation ◽  
Read Length ◽  
Chromosomal Dna ◽  
Wide Range

Abstract The gene and cell therapy fields are advancing rapidly, with a potential to treat and cure a wide range of diseases, and lentivirus-based gene transfer agents are the vector of choice for many investigators. Early cases of insertional mutagenesis caused by gammaretroviral vectors highlighted that integration site (IS) analysis was a major safety and quality control checkpoint for lentiviral applications. The methods established to detect lentiviral integrations using next-generation sequencing (NGS) are limited by short read length, inadvertent PCR bias, low yield, or lengthy protocols. Here, we describe a new method to sequence IS using Amplification-free Integration Site sequencing (AFIS-Seq). AFIS-Seq is based on amplification-free, Cas9-mediated enrichment of high-molecular-weight chromosomal DNA suitable for long-range Nanopore MinION sequencing. This accessible and low-cost approach generates long reads enabling IS mapping with high certainty within a single day. We demonstrate proof-of-concept by mapping IS of lentiviral vectors in a variety of cell models and report up to 1600-fold enrichment of the signal. This method can be further extended to sequencing of Cas9-mediated integration of genes and to in vivo analysis of IS. AFIS-Seq uses long-read sequencing to facilitate safety evaluation of preclinical lentiviral vector gene therapies by providing IS analysis with improved confidence.

Strictly linear trinuclear Dy–Ca/Mg–Dy single-molecule magnets: the impact of long-range f–f ferromagnetic interactions on suppressing quantum tunnelling of magnetization leading to slow magnetic relaxation

2017 ◽  
Vol 46 (25) ◽  
pp. 8259-8268 ◽  
Author(s):  
Wan-Ying Zhang ◽  
Yong-Mei Tian ◽  
Hong-Feng Li ◽  
Peng Chen ◽  
Yi-Quan Zhang ◽  
...  
Keyword(s):  
Long Range ◽  
Single Molecule ◽  
Magnetic Relaxation ◽  
Single Molecule Magnets ◽  
Quantum Tunnelling ◽  
Trinuclear Complexes ◽  
Slow Magnetic Relaxation ◽  
Ferromagnetic Interactions ◽  
The Impact

A series of linear trinuclear complexes Ln2M(OQ)8 [Ln(iii) = Dy and Er, M(ii) = Ca and Mg] were structurally and magnetically investigated.

scholarly journals Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2093
Author(s):  
Shen-Yuan Hsieh ◽  
Mohammad A. Tariq ◽  
Andrea Telatin ◽  
Rebecca Ansorge ◽  
Evelien M. Adriaenssens ◽  
...  
Keyword(s):  
Alpha Diversity ◽  
Diversity Indices ◽  
P Value ◽  
Library Preparation ◽  
High Genetic Diversity ◽  
Sequencing Platform ◽  
Viral Genomes ◽  
Healthy Donors ◽  
Faecal Samples ◽  
Sequencing Platforms

The human intestinal microbiota is abundant in viruses, comprising mainly bacteriophages, occasionally outnumbering bacteria 10:1 and is termed the virome. Due to their high genetic diversity and the lack of suitable tools and reference databases, the virome remains poorly characterised and is often referred to as “viral dark matter”. However, the choice of sequencing platforms, read lengths and library preparation make study design challenging with respect to the virome. Here we have compared the use of PCR and PCR-free methods for sequence-library construction on the Illumina sequencing platform for characterising the human faecal virome. Viral DNA was extracted from faecal samples of three healthy donors and sequenced. Our analysis shows that most variation was reflecting the individually specific faecal virome. However, we observed differences between PCR and PCR-free library preparation that affected the recovery of low-abundance viral genomes. Using three faecal samples in this study, the PCR library preparation samples led to a loss of lower-abundance vOTUs evident in their PCR-free pairs (vOTUs 128, 6202 and 8364) and decreased the alpha-diversity indices (Chao1 p-value = 0.045 and Simpson p-value = 0.044). Thus, differences between PCR and PCR-free methods are important to consider when investigating “rare” members of the gut virome, with these biases likely negligible when investigating moderately and highly abundant viruses.

scholarly journals Imaging and energetics of single SSB-ssDNA molecules reveal intramolecular condensation and insight into RecOR function

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Jason C Bell ◽  
Bian Liu ◽  
Stephen C Kowalczykowski
Keyword(s):  
Long Range ◽  
Single Molecule ◽  
Magnetic Tweezers ◽  
Intramolecular Interactions ◽  
Ssdna Binding ◽  
Ssdna Binding Protein ◽  
Intramolecular Condensation ◽  
Nucleoprotein Complexes ◽  
Ssdna Binding Proteins ◽  
Dna Maintenance

Escherichia coli single-stranded DNA (ssDNA) binding protein (SSB) is the defining bacterial member of ssDNA binding proteins essential for DNA maintenance. SSB binds ssDNA with a variable footprint of ∼30–70 nucleotides, reflecting partial or full wrapping of ssDNA around a tetramer of SSB. We directly imaged single molecules of SSB-coated ssDNA using total internal reflection fluorescence (TIRF) microscopy and observed intramolecular condensation of nucleoprotein complexes exceeding expectations based on simple wrapping transitions. We further examined this unexpected property by single-molecule force spectroscopy using magnetic tweezers. In conditions favoring complete wrapping, SSB engages in long-range reversible intramolecular interactions resulting in condensation of the SSB-ssDNA complex. RecO and RecOR, which interact with SSB, further condensed the complex. Our data support the idea that RecOR--and possibly other SSB-interacting proteins—function(s) in part to alter long-range, macroscopic interactions between or throughout nucleoprotein complexes by microscopically altering wrapping and bridging distant sites.

scholarly journals Genome-Wide Identification of 5-Methylcytosine Sites in Bacterial Genomes By High-Throughput Sequencing of MspJI Restriction Fragments

2021 ◽  
Author(s):  
Brian P. Anton ◽  
Alexey Fomenkov ◽  
Victoria Wu ◽  
Richard J. Roberts
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Restriction Enzymes ◽  
Specific Sequence ◽  
Genome Wide ◽  
Cost Effective Alternative ◽  
Simple Column ◽  
Sequencing Platforms

ABSTRACTSingle-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

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