Comparative evaluation of commercial DNA isolation approaches for nanopore-only bacterial genome assembly and plasmid recovery.

Publisher: Nature Publishing Group Country of Publication: England NLM ID: 101563288 Publication Model: Electronic Cited Medium: Internet ISSN: 2045-2322 (Electronic) Linking ISSN: 20452322 NLM ISO Abbreviation: Sci Rep Subsets: MEDLINE

Original Publication: London : Nature Publishing Group, copyright 2011-

Genome, Bacterial* ; Plasmids*/genetics ; DNA, Bacterial*/genetics ; DNA, Bacterial*/isolation & purification ; Nanopores*; Nanopore Sequencing/methods ; High-Throughput Nucleotide Sequencing/methods ; Bacteria/genetics ; Bacteria/isolation & purification ; Sequence Analysis, DNA/methods

The advent of Oxford Nanopore Technologies has undergone significant improvements in terms of sequencing costs, accuracy, and sequencing read lengths, making it a cost-effective, and readily accessible approach for analyzing microbial genomes. A major challenge for bacterial whole genome sequencing by Nanopore technology is the requirement for a higher quality and quantity of high molecular weight DNA compared to short-read sequencing platforms. In this study, using eight pathogenic bacteria, we evaluated the quality, quantity, and fragmented size distribution of extracted DNA obtained from three different commercial DNA extraction kits, and one automated robotic platform. Our results demonstrated significant variation in DNA yield and purity among the extraction kits. The ZymoBIOMICS DNA Miniprep Kit (ZM) provided a higher purity of DNA compared to other kit-based extractions. All kit-based DNA extractions were successfully performed on all twenty-four samples using a single MinION flow cell, with the Nanobind CBB Big DNA kit (NB) yielding the longest raw reads. The Fire Monkey HMW-DNA Extraction Kit (FM) and the automated Roche MagNaPure 96 platform (RO) outperformed in genome assembly, particularly in gram-negative bacteria. Based on our finding, we recommend a minimum read coverage and raw read N50, obtained from the appropriate DNA extraction kit for each bacterial species, to optimize genome assembly and plasmid recovery. This approach will assist end-users in selecting the most effective kit-based extraction method for bacterial whole-genome assembly using only long-read nanopore sequences.
Competing Interests: Declarations Competing interests The authors declare that they have no competing interests. Use of trade names is for research only and does not imply endorsement by all authors and the Division of Medical Bioinformatics, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
(© 2024. The Author(s).)

Front Cell Infect Microbiol. 2021 Oct 01;11:678522. (PMID: 34660333)
Bioinformatics. 2022 Nov 30;38(23):5315-5316. (PMID: 36218463)
PLoS One. 2021 Oct 1;16(10):e0257521. (PMID: 34597327)
Microbiol Resour Announc. 2020 Nov 12;9(46):. (PMID: 33184156)
Nat Biotechnol. 2019 May;37(5):540-546. (PMID: 30936562)
Science. 2002 Jan 18;295(5554):517-8. (PMID: 11799250)
Genome Res. 2015 Jul;25(7):1043-55. (PMID: 25977477)
J Microbiol Methods. 2010 Feb;80(2):209-11. (PMID: 19995580)
Int J Mol Sci. 2022 Aug 02;23(15):. (PMID: 35955702)
Access Microbiol. 2023 Feb 21;5(2):. (PMID: 36910509)
Bioinformatics. 2015 Oct 15;31(20):3350-2. (PMID: 26099265)
Front Microbiol. 2020 Dec 22;11:621041. (PMID: 33424822)
Microb Genom. 2021 Mar;7(3):. (PMID: 33683192)
Microb Genom. 2017 Jun 9;3(8):e000118. (PMID: 29026658)
Sci Rep. 2019 Nov 8;9(1):16350. (PMID: 31704961)
Microb Genom. 2023 Feb;9(2):. (PMID: 36790430)
PLoS One. 2019 Dec 31;14(12):e0226818. (PMID: 31891602)
Genomics. 2012 Sep;100(3):167-75. (PMID: 22750556)
Vet Res. 2023 Apr 5;54(1):33. (PMID: 37020296)
Antimicrob Agents Chemother. 2023 Jan 24;67(1):e0107222. (PMID: 36533931)
Microb Genom. 2023 May;9(5):. (PMID: 37224062)
PLoS Comput Biol. 2017 Jun 8;13(6):e1005595. (PMID: 28594827)
Syst Appl Microbiol. 2018 Mar;41(2):151-157. (PMID: 29305057)
BMC Microbiol. 2020 Jan 24;20(1):20. (PMID: 31980014)
Bioinformatics. 2018 Aug 1;34(15):2666-2669. (PMID: 29547981)
Microb Genom. 2019 Sep;5(9):. (PMID: 31483244)
Appl Plant Sci. 2023 Jun 06;11(3):e11524. (PMID: 37342170)
Evol Lett. 2022 Nov 19;6(6):426-437. (PMID: 36579163)
PLoS One. 2022 Jul 13;17(7):e0270751. (PMID: 35830426)
Microb Genom. 2018 Aug;4(8):. (PMID: 30052170)
Bioinformatics. 2013 Apr 15;29(8):1072-5. (PMID: 23422339)
FEMS Microbiol Rev. 2009 May;33(3):539-71. (PMID: 19396957)
Ecol Evol. 2017 Jul 07;7(16):6382-6389. (PMID: 28861241)
Imeta. 2023 May 08;2(2):e107. (PMID: 38868435)
Microb Genom. 2021 Nov;7(11):. (PMID: 34739368)
Bioinformatics. 2018 Sep 15;34(18):3094-3100. (PMID: 29750242)
Sci Rep. 2016 Jun 17;6:28063. (PMID: 27312200)
Nucleic Acids Res. 2019 Jan 8;47(D1):D195-D202. (PMID: 30380090)

HSRI 65-118 Health Systems Research Institute of Thailand under the Genomics Thailand Initiative; HSRI 65-118 Health Systems Research Institute of Thailand under the Genomics Thailand Initiative; HSRI 65-118 Health Systems Research Institute of Thailand under the Genomics Thailand Initiative; HSRI 65-118 Health Systems Research Institute of Thailand under the Genomics Thailand Initiative; N42A660897 National Research Council of Thailand (NRCT); N42A660897 National Research Council of Thailand (NRCT); B13F660073 NSRF via the Program Management Unit for Human Resources & Institutional Development, Research Innovation; B13F660073 NSRF via the Program Management Unit for Human Resources & Institutional Development, Research Innovation

Keywords: Bead-beating; DNA extraction; Enzymatic lysis; GridION; Long-read sequencing; Pathogen

0 (DNA, Bacterial)

Date Created: 20241112 Date Completed: 20241112 Latest Revision: 20241116

20241116

PMC11557978

10.1038/s41598-024-78066-2

39532954