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The Illumina Sequencing Protocol and the NovaSeq 6000 System

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Bacterial Pangenomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2242))


The NovaSeq 6000 is a sequencing platform from Illumina that enables the sequencing of short reads with an output up to 6 Tb. The NovaSeq 6000 uses the typical Illumina sequencing workflow based on library preparation, cluster generation by in situ amplification, and sequencing by synthesis. Flexibility is one of the major features of the NovaSeq 6000. Several types of sequencing kits coupled with dual flow cell mode enable high scalability of sequencing outputs to match a wide range of applications from complete genome sequencing to metagenomics analysis. In this chapter, after explaining how to assemble a normalized pool of libraries for sequencing, we will describe the experimental steps required to run the pools on the NovaSeq 6000 platform.

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  1. 1.

    At the time of writing, the authors are not aware of alternative suppliers offering sequencing reagents and materials compatible with Illumina NovaSeq6000 System.


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The authors acknowledge Chiara Natali for the help in revising protocols and for taking pictures. The Department of Biology at University of Firenze is supported by the Italian Ministry of Education, University and Research (project “Dipartimenti di Eccellenza 2018-2022”).

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Correspondence to Martina Lari .

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Modi, A., Vai, S., Caramelli, D., Lari, M. (2021). The Illumina Sequencing Protocol and the NovaSeq 6000 System. In: Mengoni, A., Bacci, G., Fondi, M. (eds) Bacterial Pangenomics. Methods in Molecular Biology, vol 2242. Humana, New York, NY.

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1098-5

  • Online ISBN: 978-1-0716-1099-2

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