PCI Genomics

335

Title *

T7 DNA polymerase treatment improves quantitative sequencing of both double-stranded and single-stranded DNA virusesuse asterix (*) to get italics

Authors *

Maud Billaud, Ilias Theodorou, Quentin Lamy-Besnier, Shiraz Shah, François Lecointe, Luisa De Sordi, Marianne De Paepe, Marie-Agnès PetitPlease use the format "First name initials family name" as in "Marie S. Curie, Niels H. D. Bohr, Albert Einstein, John R. R. Tolkien, Donna T. Strickland"

Year *

2024

Picture *

Abstract *

Background: Bulk microbiome, as well as virome-enriched shotgun sequencing only reveals the double-stranded DNA (dsDNA) content of a given sample, unless specific treatments are applied. However, genomes of viruses often consist of a circular single-stranded DNA (ssDNA) molecule. Pre-treatment and amplification of DNA using the multiple displacement amplification (MDA) method enables conversion of ssDNA to dsDNA, but this process can lead to over-representation of these circular ssDNA genomes. A more recent alternative permits to bypass the amplification step, as library adapters are ligated to sheared and denatured DNA, after an end-modification step (xGen kit). However, the sonication step might shear ssDNA more efficiently than dsDNA, therefore introducing another bias in virome sequencing. These limitations prompted us to explore an alternative method of DNA preparation for sequencing mixed ssDNA and dsDNA viromes.  Results: Using a synthetic mix of viral particles, we made use of the T7 DNA polymerase (T7pol) to convert viral circular ssDNA molecules to dsDNA, while preventing over-replication of such molecules, as is the case with the Phi29 DNA polymerase. Our findings indicate that using  T7pol  and a mix of degenerated primers to convert ssDNA to dsDNA prior library preparation is a good alternative to the currently used methods. It better represents the original synthetic mixtures compared to MDA or direct application of the xGen kit. Furthermore, when applied to two complex virome samples, the T7pol treatment improved both the richness and abundance in the Microviridae fraction. Conclusion: We conclude that T7pol pretreatment is preferable to MDA for the shotgun sequencing of viromes, which is easy to implement and inexpensive.

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https://doi.org/10.57745/C0BAPRYou should fill this box only if you chose 'All or part of the results presented in this preprint are based on data'. URL must start with http:// or https://

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Keywords (optional)

T7 DNA polymerase, shotgun sequencing, single-stranded DNA

Methods that require specific expertise (optional)

NonePlease indicate the methods that may require specialised expertise during the peer review process (use a comma to separate various required expertises).

Thematic fields *

Viruses and transposable elements

Suggested reviewers - Suggest up to 10 reviewers (provide names and Email addresses). (Optional)

Castro-Mejia JL (jcame@food.ku.dk), Shkoporov Andrey (andrey.shkoporov@ucc.ie), Kim Minsoo (minsookim@cnu.ac.kr), Blanco Luis (lblanco@cbm.csic.es), Thierry Candresse [thierry.candresse@inrae.fr] suggested: Bonjour Sebastien, Thierry Candresse [thierry.candresse@inrae.fr] suggested: Ca m'aurait bien interessé d'analyser ce manuscript. Mais j'ai pris beaucoup de retard dans mes engagements de reviewing du fait que j'ai ete arreté une semaine a la suite d'une grosse grippe..., Thierry Candresse [thierry.candresse@inrae.fr] suggested: Se suis maintenant debordé et donc je me vois contrait de décliner. Toutes mes excuses !, Thierry Candresse [thierry.candresse@inrae.fr] suggested: Merci d'avance de ta compréhension, Thierry Candresse [thierry.candresse@inrae.fr] suggested: Bien amicalement, Thierry Candresse [thierry.candresse@inrae.fr] suggested: Thierry

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Submission date

2023-12-20 16:50:00

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Sebastien Massart

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