Nanopore sequencing and de novo assembly of a misidentified Camelpox vaccine reveals putative epigenetic modifications and alternate protein signal peptides

AbstractDNA viruses can exploit host cellular epigenetic processes to their advantage; however, the epigenome status of most DNA viruses remains undetermined. Third generation sequencing technologies allow for the identification of modified nucleotides from sequencing experiments without specialized sample preparation, permitting the detection of non-canonical epigenetic modifications that may distinguish viral nucleic acid from that of their host, thus identifying attractive targets for advanced therapeutics and diagnostics. We present a novel nanopore de novo assembly pipeline used to assemble a misidentified Camelpox vaccine. Two confirmed deletions of this vaccine strain in comparison to the closely related Vaccinia virus strain modified vaccinia Ankara make it one of the smallest non-vector derived orthopoxvirus genomes to be reported. Annotation of the assembly revealed a previously unreported signal peptide at the start of protein A38 and several predicted signal peptides that were found to differ from those previously described. Putative epigenetic modifications around various motifs have been identified and the assembly confirmed previous work showing the vaccine genome to most closely resemble that of Vaccinia virus strain Modified Vaccinia Ankara. The pipeline may be used for other DNA viruses, increasing the understanding of DNA virus evolution, virulence, host preference, and epigenomics.

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Nanopore sequencing and de novo assembly of a misidentified Camelpox vaccine reveals putative epigenetic modifications and alternate protein signal peptides

10.21203/rs.3.rs-89149/v1 ◽

2020 ◽

Author(s):

Zack Saud ◽

Matthew D. Hitchings ◽

Tariq M. Butt

Keyword(s):

Vaccinia Virus ◽

Virus Strain ◽

De Novo ◽

Epigenetic Modifications ◽

Signal Peptides ◽

Dna Viruses ◽

Sequencing Technologies ◽

Third Generation Sequencing ◽

Assembly Pipeline ◽

Vaccinia Virus Strain

Abstract DNA viruses can exploit host cellular epigenetic processes to their advantage; however, the epigenome status of most DNA viruses remains undetermined. Third generation sequencing technologies allow for the identification of modified nucleotides from sequencing experiments without specialized sample preparation, permitting the detection of non-canonical epigenetic modifications that may distinguish viral nucleic acid from that of their host, thus identifying attractive targets for advanced therapeutics and diagnostics. We present a novel nanopore de novo assembly pipeline used to assemble a misidentified Camelpox vaccine. Two confirmed deletions of this vaccine strain in comparison to the closely related Vaccinia virus strain modified vaccinia Ankara make it one of the smallest non-vector derived orthopoxvirus genomes to be reported. Annotation of the assembly revealed a previously unreported signal peptide at the start of protein A38 and several signal peptides that were found to differ from those previously described. Putative epigenetic modifications around various motifs have been identified and the assembly confirmed previous work showing the vaccine genome to most closely resemble that of Vaccinia virus strain Modified Vaccinia Ankara. The pipeline may be used for other DNA viruses, increasing the understanding of DNA virus evolution, virulence, host preference, and epigenomics.

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Telomere length de novo assembly of all 7 chromosomes and mitogenome sequencing of the model entomopathogenic fungus, Metarhizium brunneum, by means of a novel assembly pipeline

BMC Genomics ◽

10.1186/s12864-021-07390-y ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Zack Saud ◽

Alexandra M. Kortsinoglou ◽

Vassili N. Kouvelis ◽

Tariq M. Butt

Keyword(s):

De Novo Assembly ◽

De Novo ◽

Gene Prediction ◽

Fungal Species ◽

Orthologous Protein ◽

Metarhizium Brunneum ◽

Sequencing Technologies ◽

Protein Clusters ◽

Assembly Pipeline ◽

Generation Sequencing

Abstract Background More accurate and complete reference genomes have improved understanding of gene function, biology, and evolutionary mechanisms. Hybrid genome assembly approaches leverage benefits of both long, relatively error-prone reads from third-generation sequencing technologies and short, accurate reads from second-generation sequencing technologies, to produce more accurate and contiguous de novo genome assemblies in comparison to using either technology independently. In this study, we present a novel hybrid assembly pipeline that allowed for both mitogenome de novo assembly and telomere length de novo assembly of all 7 chromosomes of the model entomopathogenic fungus, Metarhizium brunneum. Results The improved assembly allowed for better ab initio gene prediction and a more BUSCO complete proteome set has been generated in comparison to the eight current NCBI reference Metarhizium spp. genomes. Remarkably, we note that including the mitogenome in ab initio gene prediction training improved overall gene prediction. The assembly was further validated by comparing contig assembly agreement across various assemblers, assessing the assembly performance of each tool. Genomic synteny and orthologous protein clusters were compared between Metarhizium brunneum and three other Hypocreales species with complete genomes, identifying core proteins, and listing orthologous protein clusters shared uniquely between the two entomopathogenic fungal species, so as to further facilitate the understanding of molecular mechanisms underpinning fungal-insect pathogenesis. Conclusions The novel assembly pipeline may be used for other haploid fungal species, facilitating the need to produce high-quality reference fungal genomes, leading to better understanding of fungal genomic evolution, chromosome structuring and gene regulation.

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Telomere length de novo assembly of all 7 chromosomes and mitogenome sequencing of the model entomopathogenic fungus, Metarhizium brunneum, by means of a novel assembly pipeline

10.21203/rs.3.rs-60098/v3 ◽

2020 ◽

Author(s):

Zack Saud ◽

Alexandra M. Kortsinoglou ◽

Vassili N. Kouvelis ◽

Tariq M. Butt

Keyword(s):

De Novo Assembly ◽

De Novo ◽

Gene Prediction ◽

Fungal Species ◽

Orthologous Protein ◽

Metarhizium Brunneum ◽

Sequencing Technologies ◽

Protein Clusters ◽

Assembly Pipeline ◽

Generation Sequencing

Abstract Background More accurate and complete reference genomes have improved understanding of gene function, biology, and evolutionary mechanisms. Hybrid genome assembly approaches leverage benefits of both long, relatively error-prone reads from third-generation sequencing technologies and short, accurate reads from second-generation sequencing technologies, to produce more accurate and contiguous de novo genome assemblies in comparison to using either technology independently. In this study, we present a novel hybrid assembly pipeline that allowed for both mitogenome de novo assembly and telomere length de novo assembly of all 7 chromosomes of the model entomopathogenic fungus, Metarhizium brunneum . Results The improved assembly allowed for better ab initio gene prediction and a more BUSCO complete proteome set has been generated in comparison to the eight current NCBI reference Metarhizium spp. genomes. Remarkably, we note that including the mitogenome in ab initio gene prediction training improved overall gene prediction. The assembly was further validated by comparing contig assembly agreement across various assemblers, assessing the assembly performance of each tool. Genomic synteny and orthologous protein clusters were compared between Metarhizium brunneum and three other Hypocreales species with complete genomes, identifying core proteins, and listing orthologous protein clusters shared uniquely between the two entomopathogenic fungal species, so as to further facilitate the understanding of molecular mechanisms underpinning fungal-insect pathogenesis. Conclusions The novel assembly pipeline may be used for other haploid fungal species, facilitating the need to produce high-quality reference fungal genomes, leading to better understanding of fungal genomic evolution, chromosome structuring and gene regulation.

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Telomere length de novo assembly of all 7 chromosomes and mitogenome sequencing of the model entomopathogenic fungus, Metarhizium brunneum, by means of a novel assembly pipeline

10.21203/rs.3.rs-60098/v4 ◽

2021 ◽

Author(s):

Zack Saud ◽

Alexandra M. Kortsinoglou ◽

Vassili N. Kouvelis ◽

Tariq M. Butt

Keyword(s):

De Novo Assembly ◽

De Novo ◽

Gene Prediction ◽

Fungal Species ◽

Orthologous Protein ◽

Metarhizium Brunneum ◽

Sequencing Technologies ◽

Protein Clusters ◽

Assembly Pipeline ◽

Generation Sequencing

Abstract Background More accurate and complete reference genomes have improved understanding of gene function, biology, and evolutionary mechanisms. Hybrid genome assembly approaches leverage benefits of both long, relatively error-prone reads from third-generation sequencing technologies and short, accurate reads from second-generation sequencing technologies, to produce more accurate and contiguous de novo genome assemblies in comparison to using either technology independently. In this study, we present a novel hybrid assembly pipeline that allowed for both mitogenome de novo assembly and telomere length de novo assembly of all 7 chromosomes of the model entomopathogenic fungus, Metarhizium brunneum . Results The improved assembly allowed for better ab initio gene prediction and a more BUSCO complete proteome set has been generated in comparison to the eight current NCBI reference Metarhizium spp. genomes. Remarkably, we note that including the mitogenome in ab initio gene prediction training improved overall gene prediction. The assembly was further validated by comparing contig assembly agreement across various assemblers, assessing the assembly performance of each tool. Genomic synteny and orthologous protein clusters were compared between Metarhizium brunneum and three other Hypocreales species with complete genomes, identifying core proteins, and listing orthologous protein clusters shared uniquely between the two entomopathogenic fungal species, so as to further facilitate the understanding of molecular mechanisms underpinning fungal-insect pathogenesis. Conclusions The novel assembly pipeline may be used for other haploid fungal species, facilitating the need to produce high-quality reference fungal genomes, leading to better understanding of fungal genomic evolution, chromosome structuring and gene regulation.

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Vaccinia virus strain differences in cell attachment and entry

Virology ◽

10.1016/j.virol.2009.04.012 ◽

2009 ◽

Vol 389 (1-2) ◽

pp. 132-140 ◽

Cited By ~ 66

Author(s):

Zain Bengali ◽

Alan C. Townsley ◽

Bernard Moss

Keyword(s):

Vaccinia Virus ◽

Virus Strain ◽

Cell Attachment ◽

Strain Differences ◽

Vaccinia Virus Strain

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Notes on Transient Host Range Selection for Engineering Vaccinia Virus Strain MVA

BioTechniques ◽

10.2144/02331dd01 ◽

2002 ◽

Vol 33 (1) ◽

pp. 186-188 ◽

Cited By ~ 2

Author(s):

David C. Tscharke ◽

Geoffrey L. Smith

Keyword(s):

Vaccinia Virus ◽

Host Range ◽

Virus Strain ◽

Selection For ◽

Vaccinia Virus Strain ◽

Range Selection

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Optimizing de novo genome assembly from PCR-amplified metagenomes

10.7287/peerj.preprints.27453 ◽

2018 ◽

Author(s):

Simon Roux ◽

Gareth Trubl ◽

Danielle Goudeau ◽

Nandita Nath ◽

Estelle Couradeau ◽

...

Keyword(s):

Genome Assembly ◽

De Novo Assembly ◽

De Novo ◽

Pcr Amplification ◽

Error Rates ◽

De Novo Genome Assembly ◽

Low Input ◽

Assembly Algorithm ◽

Coverage Bias ◽

Assembly Pipeline

Background. Metagenomics has transformed our understanding of microbial diversity across ecosystems, with recent advances enabling de novo assembly of genomes from metagenomes. These metagenome-assembled genomes are critical to provide ecological, evolutionary, and metabolic context for all the microbes and viruses yet to be cultivated. Metagenomes can now be generated from nanogram to subnanogram amounts of DNA. However, these libraries require several rounds of PCR amplification before sequencing, and recent data suggest these typically yield smaller and more fragmented assemblies than regular metagenomes. Methods. Here we evaluate de novo assembly methods of 169 PCR-amplified metagenomes, including 25 for which an unamplified counterpart is available, to optimize specific assembly approaches for PCR-amplified libraries. We first evaluated coverage bias by mapping reads from PCR-amplified metagenomes onto reference contigs obtained from unamplified metagenomes of the same samples. Then, we compared different assembly pipelines in terms of assembly size (number of bp in contigs ≥ 10kb) and error rates to evaluate which are the best suited for PCR-amplified metagenomes. Results. Read mapping analyses revealed that the depth of coverage within individual genomes is significantly more uneven in PCR-amplified datasets versus unamplified metagenomes, with regions of high depth of coverage enriched in short inserts. This enrichment scales with the number of PCR cycles performed, and is presumably due to preferential amplification of short inserts. Standard assembly pipelines are confounded by this type of coverage unevenness, so we evaluated other assembly options to mitigate these issues. We found that a pipeline combining read deduplication and an assembly algorithm originally designed to recover genomes from libraries generated after whole genome amplification (single-cell SPAdes) frequently improved assembly of contigs ≥ 10kb by 10 to 100-fold for low input metagenomes. Conclusions. PCR-amplified metagenomes have enabled scientists to explore communities traditionally challenging to describe, including some with extremely low biomass or from which DNA is particularly difficult to extract. Here we show that a modified assembly pipeline can lead to an improved de novo genome assembly from PCR-amplified datasets, and enables a better genome recovery from low input metagenomes.

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Complete Genome Sequence of Vaccinia Virus Strain L-IVP

Genome Announcements ◽

10.1128/genomea.00372-16 ◽

2016 ◽

Vol 4 (3) ◽

Cited By ~ 2

Author(s):

Alexander N. Shvalov ◽

Galina F. Sivolobova ◽

Elena V. Kuligina ◽

Galina V. Kochneva

Keyword(s):

Vaccinia Virus ◽

Genome Sequence ◽

Virus Strain ◽

Complete Genome Sequence ◽

Complete Genome ◽

Sequence Similarity ◽

Consensus Sequence ◽

Live Vaccine ◽

Eradication Programme ◽

Vaccinia Virus Strain

Most of the live vaccine doses of vaccinia virus donated to the Intensified Smallpox Eradication Programme after 1971 were prepared using the L-IVP strain. A mixture of three clones of the L-IVP strain was sequenced using MySEQ. Consensus sequence similarity with the vaccinia virus Lister strain is 99.5%.

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