Reconstitution and Mutagenesis of Avian Infectious Laryngotracheitis Virus from Cosmid and Yeast Centromeric Plasmid Clones

Spatz, Stephen J.; Garcı́a, Maricarmen; Fuchs, Walter; Loncoman, Carlos; Volkening, Jeremy D.; Ross, Teresa A.; Riblet, Sylva M.; Kim, Taejoong; Likens, Nathan; Mettenleiter, Thomas C.

doi:10.1128/jvi.01406-22

“…This would tremendously lower the ‘’hit to discovery timeframe’’. The proposed method could be expanded to plasmids, cosmids or more challenging vectors such as BACs and be applied to projects outside functional screenings where sequencing of these molecules is needed (32, 33).…”

Section: Discussionmentioning

confidence: 99%

High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies

Chuzel,

Sinha,

Cunningham

et al. 2024

Preprint

0

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Fosmids and cosmids are vectors frequently used in functional metagenomic studies. With a large insert capacity (around 30 kb) they can encode dozens of cloned genes or in some cases, entire biochemical pathways. Fosmids with cloned inserts can be transferred to heterologous hosts and propagated to enable screening for new enzymes and metabolites. After screening, fosmids from clones with an activity of interest must be de novo sequenced, a critical step towards identification of the gene(s) of interest. In this work, we present a new approach for rapid and high-throughput fosmid sequencing directly from Escherichia coli colonies without liquid culturing or fosmid purification. Our sample preparation involves fosmid amplification with phi29 polymerase and then direct nanopore sequencing using the Oxford Nanopore Technologies system. We also present a bioinformatics pipeline termed "phiXXer" that facilitates both de novo read assembly and vector trimming to generate a linear sequence of the fosmid insert. Finally, we demonstrate accurate sequencing of 96 fosmids in a single run and validate the method using two fosmid libraries that contain cloned large insert (~30-40 kb) genomic or metagenomic DNA.

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“…This would tremendously lower the “hit to discovery timeframe’’. The proposed method could be expanded to plasmids, cosmids, or more challenging vectors such as BACs and be applied to projects outside functional screenings where sequencing of these molecules is needed ( 34 , 35 ).…”

Section: Discussionmentioning

confidence: 99%

High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies

Chuzel,

Sinha,

Cunningham

et al. 2024

Appl Environ Microbiol

0

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Fosmids and cosmids are vectors frequently used in functional metagenomic studies. With a large insert capacity (around 30 kb) they can encode dozens of cloned genes or in some cases, entire biochemical pathways. Fosmids with cloned inserts can be transferred to heterologous hosts and propagated to enable screening for new enzymes and metabolites. After screening, fosmids from clones with an activity of interest must be de novo sequenced, a critical step toward the identification of the gene(s) of interest. In this work, we present a new approach for rapid and high-throughput fosmid sequencing directly from Escherichia coli colonies without liquid culturing or fosmid purification. Our sample preparation involves fosmid amplification with phi29 polymerase and then direct nanopore sequencing using the Oxford Nanopore Technologies system. We also present a bioinformatics pipeline termed “phiXXer” that facilitates both de novo read assembly and vector trimming to generate a linear sequence of the fosmid insert. Finally, we demonstrate the accurate sequencing of 96 fosmids in a single run and validate the method using two fosmid libraries that contain cloned large insert (~30–40 kb) genomic or metagenomic DNA. IMPORTANCE Large-insert clone (fosmids or cosmids) sequencing is challenging and arguably the most limiting step of functional metagenomic screening workflows. Our study establishes a new method for high-throughput nanopore sequencing of fosmid clones directly from lysed Escherichia coli cells. It also describes a companion bioinformatic pipeline that enables de novo assembly of fosmid DNA insert sequences. The devised method widens the potential of functional metagenomic screening by providing a simple, high-throughput approach to fosmid clone sequencing that dramatically speeds the pace of discovery.

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“…Another impressive example of TAR-based assembly of the entire viral genome is reconstruction of infectious laryngotracheitis virus (ILTV), known as Gallid alphaherpesvirus-1 [ 121 ]. In this case, the authors generated overlapping cosmid clones, that encompassed 90% of the 151 kb ILTV genome.…”

Section: Applications Of Tar Cloningmentioning

confidence: 99%

“…In this case, the authors generated overlapping cosmid clones, that encompassed 90% of the 151 kb ILTV genome. Homologous recombination between the clones in yeast allowed to develop the full-length genome of the ILTV virus [ 121 ]. TAR-based approach allowed to manipulate the constructs by modifying the genes encoding virulence factors that facilitated the development of the improved virus vaccines and establishing ILTV-based viral vectors for expressing immunogens of other avian pathogens.…”

Section: Applications Of Tar Cloningmentioning

confidence: 99%

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Transformation-associated recombination (TAR) cloning and its applications for gene function; genome architecture and evolution; biotechnology and biomedicine

Kouprina,

Larionov

2023

Oncotarget

1

0

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Transformation-associated recombination (TAR) cloning represents a unique tool to selectively and efficiently recover a given chromosomal segment up to several hundred kb in length from complex genomes (such as animals and plants) and simple genomes (such as bacteria and viruses). The technique exploits a high level of homologous recombination in the yeast Sacharomyces cerevisiae . In this review, we summarize multiple applications of the pioneering TAR cloning technique, developed previously for complex genomes, for functional, evolutionary, and structural studies, and extended the modified TAR versions to isolate biosynthetic gene clusters (BGCs) from microbes, which are the major source of pharmacological agents and industrial compounds, and to engineer synthetic viruses with novel properties to design a new generation of vaccines. TAR cloning was adapted as a reliable method for the assembly of synthetic microbe genomes for fundamental research. In this review, we also discuss how the TAR cloning in combination with HAC (human artificial chromosome)- and CRISPR-based technologies may contribute to the future.

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Reconstitution and Mutagenesis of Avian Infectious Laryngotracheitis Virus from Cosmid and Yeast Centromeric Plasmid Clones

Abstract: Infectious laryngotracheitis virus (ILTV) is an important pathogen of chicken with morbidity of 100% and mortality rates as high as 70%. Factoring in decreased production, mortality, vaccination, and medication, a single outbreak can cost producers over a million dollars.

Cited by 3 publications

References 60 publications

High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies

High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies

High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies

Transformation-associated recombination (TAR) cloning and its applications for gene function; genome architecture and evolution; biotechnology and biomedicine

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