A Simplified and Efficient Method for Himar-1 Transposon Sequencing in Bacteria, Demonstrated by Creation and Analysis of a Saturated Transposon-Mutant Library in <named-content content-type="genus-species">Mycobacterium abscessus</named-content>

ABSTRACT We present a technically simple, easy-to-perform method for generating the genomic libraries for Himar-1 transposon site sequencing (Tn-seq). In addition to being simpler than present methods in the technical aspect, it also allows more robust and straightforward identification of the inser...

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Autores principales:	Mark Foreman, Moran Gershoni, Daniel Barkan
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	transposon genomics bacterial genetics Mycobacterium abscessus bioinformatics mycobacteria Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/b4be4e940148445aac5ec82e79ab3f5b
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spelling	oai:doaj.org-article:b4be4e940148445aac5ec82e79ab3f5b2021-12-02T18:15:46ZA Simplified and Efficient Method for Himar-1 Transposon Sequencing in Bacteria, Demonstrated by Creation and Analysis of a Saturated Transposon-Mutant Library in <named-content content-type="genus-species">Mycobacterium abscessus</named-content>10.1128/mSystems.00976-202379-5077https://doaj.org/article/b4be4e940148445aac5ec82e79ab3f5b2020-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00976-20https://doaj.org/toc/2379-5077ABSTRACT We present a technically simple, easy-to-perform method for generating the genomic libraries for Himar-1 transposon site sequencing (Tn-seq). In addition to being simpler than present methods in the technical aspect, it also allows more robust and straightforward identification of the insertion site, by generating a longer sequence surrounding the insertion TA in the genome. The method makes Tn-seq more user-friendly and accessible to laboratories with more-limited bioinformatic resources. Finally, we created a saturated transposon-mutant library in Mycobacterium abscessus and demonstrated the usefulness of the method in analysis of genes involved in colony morphology, as well as in analysis of the whole Tn-mutant library, with identification of over 8,000 unique mutants. IMPORTANCE Transposon insertion sequencing is a powerful tool, but many researchers are discouraged by the apparent technical complexity of preparing the genomic library for deep sequencing and by the complicated computational analysis needed for insertion site identification. Our proposed method makes the preparation of the library easy and straightforward, relying on well-known molecular biology techniques. In addition, the results obtained from the deep sequencing are easily analyzed in terms of transposon insertion site identification, placing library preparation and analysis within the reach of more researchers in the microbiology community, including those with less computational and bioinformatic resources and experience. This is demonstrated by analysis of the most saturated Tn-mutant library created to date in the emerging pathogen Mycobacterium abscessus.Mark ForemanMoran GershoniDaniel BarkanAmerican Society for Microbiologyarticletransposongenomicsbacterial geneticsMycobacterium abscessusbioinformaticsmycobacteriaMicrobiologyQR1-502ENmSystems, Vol 5, Iss 5 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	transposon genomics bacterial genetics Mycobacterium abscessus bioinformatics mycobacteria Microbiology QR1-502
spellingShingle	transposon genomics bacterial genetics Mycobacterium abscessus bioinformatics mycobacteria Microbiology QR1-502 Mark Foreman Moran Gershoni Daniel Barkan A Simplified and Efficient Method for Himar-1 Transposon Sequencing in Bacteria, Demonstrated by Creation and Analysis of a Saturated Transposon-Mutant Library in <named-content content-type="genus-species">Mycobacterium abscessus</named-content>
description	ABSTRACT We present a technically simple, easy-to-perform method for generating the genomic libraries for Himar-1 transposon site sequencing (Tn-seq). In addition to being simpler than present methods in the technical aspect, it also allows more robust and straightforward identification of the insertion site, by generating a longer sequence surrounding the insertion TA in the genome. The method makes Tn-seq more user-friendly and accessible to laboratories with more-limited bioinformatic resources. Finally, we created a saturated transposon-mutant library in Mycobacterium abscessus and demonstrated the usefulness of the method in analysis of genes involved in colony morphology, as well as in analysis of the whole Tn-mutant library, with identification of over 8,000 unique mutants. IMPORTANCE Transposon insertion sequencing is a powerful tool, but many researchers are discouraged by the apparent technical complexity of preparing the genomic library for deep sequencing and by the complicated computational analysis needed for insertion site identification. Our proposed method makes the preparation of the library easy and straightforward, relying on well-known molecular biology techniques. In addition, the results obtained from the deep sequencing are easily analyzed in terms of transposon insertion site identification, placing library preparation and analysis within the reach of more researchers in the microbiology community, including those with less computational and bioinformatic resources and experience. This is demonstrated by analysis of the most saturated Tn-mutant library created to date in the emerging pathogen Mycobacterium abscessus.
format	article
author	Mark Foreman Moran Gershoni Daniel Barkan
author_facet	Mark Foreman Moran Gershoni Daniel Barkan
author_sort	Mark Foreman
title	A Simplified and Efficient Method for Himar-1 Transposon Sequencing in Bacteria, Demonstrated by Creation and Analysis of a Saturated Transposon-Mutant Library in <named-content content-type="genus-species">Mycobacterium abscessus</named-content>
title_short	A Simplified and Efficient Method for Himar-1 Transposon Sequencing in Bacteria, Demonstrated by Creation and Analysis of a Saturated Transposon-Mutant Library in <named-content content-type="genus-species">Mycobacterium abscessus</named-content>
title_full	A Simplified and Efficient Method for Himar-1 Transposon Sequencing in Bacteria, Demonstrated by Creation and Analysis of a Saturated Transposon-Mutant Library in <named-content content-type="genus-species">Mycobacterium abscessus</named-content>
title_fullStr	A Simplified and Efficient Method for Himar-1 Transposon Sequencing in Bacteria, Demonstrated by Creation and Analysis of a Saturated Transposon-Mutant Library in <named-content content-type="genus-species">Mycobacterium abscessus</named-content>
title_full_unstemmed	A Simplified and Efficient Method for Himar-1 Transposon Sequencing in Bacteria, Demonstrated by Creation and Analysis of a Saturated Transposon-Mutant Library in <named-content content-type="genus-species">Mycobacterium abscessus</named-content>
title_sort	simplified and efficient method for himar-1 transposon sequencing in bacteria, demonstrated by creation and analysis of a saturated transposon-mutant library in <named-content content-type="genus-species">mycobacterium abscessus</named-content>
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/b4be4e940148445aac5ec82e79ab3f5b
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A Simplified and Efficient Method for Himar-1 Transposon Sequencing in Bacteria, Demonstrated by Creation and Analysis of a Saturated Transposon-Mutant Library in <named-content content-type="genus-species">Mycobacterium abscessus</named-content>

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