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Volume 8, Issue 2

DNA isolation methods for Nanopore sequencing of the genome Open Access

Abstract

is a Gram-positive bacterium, member of the oral commensal microbiota, which can occasionally be the etiologic agent of diseases such as infective endocarditis, bacteraemia and septicaemia. The highly recombinogenic and repetitive nature of the genome impairs the assembly of a complete genome relying only on short sequencing reads. Oxford Nanopore sequencing can overcome this limitation by generating long reads, enabling the resolution of genomic repeated regions and the assembly of a complete genome sequence. Since the output of a Nanopore sequencing run is strongly influenced by genomic DNA quality and molecular weight, the DNA isolation is the crucial step for an optimal sequencing run. In the present work, we have set up and compared three DNA isolation methods on two strains, evaluating their capability of preserving genomic DNA integrity and purity. Sequencing of DNA isolated with a mechanical lysis-based method, despite being cheaper and quicker, did not generate ultra-long reads (maximum read length of 59516 bases) and did not allow the assembly of a circular complete genome. Two methods based on enzymatic lysis of the bacterial cell wall, followed by either (i) a modified CTAB DNA isolation procedure, or (ii) a DNA purification after osmotic lysis of the protoplasts allowed the sequencing of ultra-long reads up to 107294 and 181199 bases in length, respectively. The reconstruction of a circular complete genome was possible sequencing DNAs isolated using the enzymatic lysis-based methods.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): genomic DNA extraction , Gram positive , high molecular weight DNA , Oxford Nanopore sequencing and Streptococcus mitis
Funding
This study was supported by the:
  • Seventh Framework Programme (Award 241446)
    • Principle Award Recipient: GianniPozzi
  • Ministero dell’Istruzione, dell’Università e della Ricerca (Award 20177J5Y3P)
    • Principle Award Recipient: GianniPozzi
© 2022 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution NonCommercial License.
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2022-02-16
2024-04-29
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DNA isolation methods for Nanopore sequencing of the Streptococcus mitis genome
M Gen 8, 000764 (2022); https://doi.org/10.1099/mgen.0.000764
/content/journal/mgen/10.1099/mgen.0.000764
/content/journal/mgen/10.1099/mgen.0.000764
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