PLoS ONE plos plosone PLOS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0253830 PONE-D-21-06732 Lab Protocol Biology and life sciencesMolecular biologyMolecular biology techniquesSequencing techniquesDNA sequencing Research and analysis methodsMolecular biology techniquesSequencing techniquesDNA sequencing Biology and life sciencesPhysiologyPhysiological parametersBody weight Research and analysis methodsExtraction techniquesDNA extraction Biology and life sciencesGeneticsFungal geneticsFungal genomics Biology and life sciencesMycologyFungal geneticsFungal genomics Biology and life sciencesGeneticsGenomicsFungal genomics Biology and life sciencesOrganismsEukaryotaFungi Biology and life sciencesGeneticsPlant genetics Biology and life sciencesPlant sciencePlant genetics Biology and life sciencesMolecular biologyMolecular biology techniquesSequencing techniquesGenome sequencing Research and analysis methodsMolecular biology techniquesSequencing techniquesGenome sequencing Biology and life sciencesBiochemistryGlycobiologyPolysaccharides High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing https://orcid.org/0000-0002-7368-1666 Jones Ashley Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Supervision Validation Visualization Writing – original draft Writing – review & editing 1 * Torkel Cynthia Data curation Methodology Project administration Validation 1 Stanley David Data curation Methodology Project administration Validation 1 2 Nasim Jamila Data curation Methodology Project administration Validation 1 3 Borevitz Justin Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Supervision Validation Visualization 1 Schwessinger Benjamin Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Supervision Validation Visualization Writing – review & editing 1 Research School of Biology, Australian National University, Canberra, Australian Capital Territory, Australia Diversity Arrays Technology, Bruce, Australian Capital Territory, Australia Soil Carbon Co., Orange, New South Wales, Australia Eppinger Mark Editor University of Texas at San Antonio, UNITED STATES

“For two plant species, Wahlenbergia ceracea and Phebalium stellatum, sequencing funds have been generously provided by Bioplatforms Australia, under the Genomics for Australian Plants initiative. The funder had no influence on the interpretation of the data.” This does not alter our adherence to PLOS ONE policies on sharing data and materials. Many datasets are publicly available on NCBI, others will become available in due time with publication of the genome assembly. There are no competing interests relating to employment, consultancy, patents, products in development, or marketed products. We present a full and objective manuscript that has no interference to its objectivity.

 * E-mail: ashley.jones@anu.edu.au 15 7 2021 2021 16 7 e0253830 1 3 2021 9 6 2021 2021 Jones et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Rapid advancements in long-read sequencing technologies have transformed read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection; high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale in low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Oxford Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30–50 kb, with reads exceeding 200 kb and outputs ranging from 15–30 Gbp. This has been routinely achieved with various plant, fungi, animal and bacteria samples.

 Bioplatforms Australia, Genomics for Australian Plants https://orcid.org/0000-0002-7368-1666 Jones Ashley Bioplatforms Australia, Genomics for Australian Plants Schwessinger Benjamin http://dx.doi.org/10.13039/501100006067 Centre of Excellence in Plant Energy Biology, Australian Research Council CE140100008 Borevitz Justin Australian Research Council Discovery Project DP150103591 Borevitz Justin Australian Research Council Future Fellowship FT180100024 Schwessinger Benjamin A.J. and B.S. received sequencing funds from Bioplatforms Australia, as part of the Genomics for Australian Pants initiative www.genomicsforaustralianplants.com J.B. received funds from an Australian Research Council Centre of Excellence (Plant Energy Biology) (CE140100008) and Discovery Project (DP150103591) www.arc.gov.au B.S. received funds from an Australian Research Council Future Fellowship (FT180100024) www.arc.gov.au The funders had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability Sequencing data and reference genomes generated with this protocol are being made publicly available on the Sequence Read Archive (SRA, NCBI) under BioProjects PRJNA509734 and PRJNA510265. https://www.ncbi.nlm.nih.gov/bioproject/509734 https://www.ncbi.nlm.nih.gov/sra?linkname=bioproject_sra_all&from_uid=510265. Introduction

DNA sequencing technologies have transformed genomics due to rapid advances in read length, throughput and application, combined with an ever competitive price. Short-read sequencing platforms provide billions of reads 100–250 bp in length at unrivalled accuracy, while long-read platforms a can provide millions of reads 1 kbp to 1 Mbp, at the cost of accuracy [1]. Long-read platforms have been at the forefront of recent advancements, as they offer unprecedented opportunities for de novo assembly of full length chromosomes and phasing of haplotypes [2,3]. With long-read sequencing platforms, advancements have shifted read length being limited by technology to being limited by quality and length of the DNA input. This has given rise to a new challenge; the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly troublesome in plants and fungi. This is often caused by the presence of secondary metabolites such as polyphenols and polysaccharides. Polyphenols within the cytosol will be exposed to DNA after cell lysis and can have irreversible interactions [4]. Polysaccharides can co-precipitate with DNA in the presence of alcohol and can have downstream inhibitory effects in many molecular biology techniques [5]. Isolation of nuclei can help resolve these issues and obtain high-molecular weight DNA [6]. Indeed, nuclei preps have been further developed for long-read sequencing but remain laborious and low throughput [7]. One approach that is becoming widely utilized for long-read sequencing is the use of carboxylated magnetic beads, which DNA can bind to under the presence of polyethylene glycol and sodium chloride [8]. This method does not isolate nuclei but still circumvents the use of binding columns and high centrifugation, which are techniques that can fragment DNA. Here we present a modified protocol of Mayjonade et al. [8] that has been used across a wide variety genera of samples, including recalcitrant plants. For plants containing excessive phenols and polysaccharides, an optional washing of homogenate with sorbitol is included which help remove these contaminants [9]. Lastly, DNA clean-up and size selection options are presented which can greatly enhance the success of long-read sequencing platforms. This protocol is part of a bigger repository hosted on Protocols.io, as part of the public workspace ‘High-molecular weight DNA extraction from all kingdoms’ (https://www.protocols.io/workspaces/high-molecular-weight-dna-extraction-from-all-kingdoms).

 Methods

The protocol described in this article is published on protocols.io, https://dx.doi.org/10.17504/protocols.io.bss7nehn.

Expected results

Using the protocol described, we have been obtaining large yields of high-molecular weight DNA (Table 1, Fig 1). DNA fragment size ranges from 20–200 kb in length, which is ideal for long-read sequencing (Fig 1). To remove the small DNA fragments and clean plant DNA preps which can be somewhat crude, PippinHT (Sage Science) to select fragments 20 kb and above has been very efficient (Table 1). Other DNA clean-up options are presented in the protocol and achieve similar results, however are more labour intensive. During sequencing, we can reproducibly obtain over 15–30 Gbp of reads from a single Oxford Nanopore MinION flow cell, with read length N50s 30–50 kb (Table 2, Fig 2). This includes quality reads over 200 kb in length (> Q7, Phred scale). It is likely smaller fragments are favoured during sequencing (higher molarity) and the library prep is likely to cause some DNA shearing. Sequencing with PacBio Sequel II (circular consensus sequencing mode for HiFi reads), yields over 20 Gbp can be achieved at very high accuracy (> Q30), but at a smaller length, as this technology is optimised for 10–20 kb fragments. High performing sequencing results have been achieved with various plant, fungi, animal and bacteria samples (Table 2). The sequencing data is being used for de novo genome assemblies and in some instances haplotype phasing. Sequencing data and the subsequent reference genomes being generated in this project are being made publicly available Sequence Read Archive (SRA, NCBI). Multiple Eucalyptus genomic datasets are available under BioProject PRJNA509734 and Acacia datasets are available under BioProject PRJNA510265. Supporting publications and other genera are soon to follow.

10.1371/journal.pone.0253830.g001 DNA size distribution of <italic>Eucalyptus caleyi</italic> based on a pulsed-field capillary electrophoresis system, a Femto Pulse (Agilent).

Peak at 200 kb represents all fragments > 200 kb, as they cannot be resolved with the technology. Sample was crude DNA prior to and size selection or further DNA clean-up.

 10.1371/journal.pone.0253830.g002 Read length by average read quality for <italic>Eucalyptus caleyi</italic> long-read sequencing with an Oxford Nanopore MinION flow cell.

Image generated with NanoPlot 1.28.2 [10].

 10.1371/journal.pone.0253830.t001 Fluorometer and Spectrophotometer results of a DNA extraction for <italic>Eucalyptus caleyi</italic>.

Firstly, crude DNA was extracted, which was then size selected for 20 kb and above with a PippinHT (Sage Science).

Sample Input Yield μg Qubit ng/μL Nanodrop ng/μL 260/280 260/230
E. caleyi Leaf to crude DNA 10 g leaves (fresh) 59.60 298 564 1.48 0.72
E. caleyi Pippin Prep 20 kb 30 μg crude DNA 13.68 228 283 1.91 2.38

Readings taken with a Qubit 2.0 Qubit fluorometer (Thermo Fisher Scientific) and a Nanodrop 1000 (Thermo Fisher Scientific).

 10.1371/journal.pone.0253830.t002 Example long-read sequencing results for multiple samples using different sequencing platforms.
Sample Kingdom Size selection Platform Library prep Flow cell Output (Gbp) N50 Read length (kb) Mean read length (kb) Median read length (kb) Mean read quality (Phred) Median read quality (Phred) Longest read (kb) Longest read ≥ Q7 (kb)
Eucalyptus caleyi Plantae PippinHT (≥ 20 kb) ONT MinION SQK-LSK109 FLO-MIN106D R9.4.1 25.57 38.99 26.04 22.68 Q10.9 Q11.50 245,735 (Q11.1) 245,735 (Q11.1)
Acacia melanoxylon Plantae PippinHT (≥ 20 kb) ONT MinION SQK-LSK109 FLO-MIN106D R9.4.1 20.78 43.07 29.33 25.00 Q9.8 Q10.3 304,552 (Q7.4) 304,552 (Q7.4)
Oryza australiensis Plantae PippinHT (≥ 20 kb) ONT MinION SQK-LSK109 FLO-MIN106D R9.4.1 15.40 44.86 27.14 21.34 Q9.7 Q10.4 321,653 (Q8.6) 321,653 (Q8.6)
Triticum aestivum Plantae SRE XS (≥ 10 kb) ONT MinION SQK-LSK109 FLO-MIN106D R9.4.1 33.21* 12.42 8.19 6.12 Q10.8 Q11.3 12,977 (Q7.8) 12,977 (Q7.8)
Nannizziopsis barbatae Fungi PippinHT (≥ 20 kb) ONT MinION SQK-LSK109 FLO-MIN106D R9.4.1 3.46† 32.95 23.74 22.62 Q11.3 Q12.2 182,089 (Q9.1) 182,089 (Q9.1)
Puccinia graminis germinated spores Fungi SRE XS (≥ 10 kb) ONT MinION SQK-LSK109 FLO-MIN106D R9.4.1 7.82† 28.86 17.11 12.72 Q11.8 Q12.6 464,014 (Q4.0) 145,682 (Q9.6)
Gehyra lapistola Animalia SRE (≥ 25 kb) ONT MinION SQK-LSK109 FLO-MIN106D R9.4.1 9.22 16.16 8.58 5.45 Q10.5 Q11.2 196,384 (Q3.2) 125,703 (Q7.7)
Escherichia coli BACS (multiplexed) Bacteria SRE (≥ 25 kb) ONT MinION SQK-LSK109 FLO-MIN106D R9.4.1 21.41 20.54 13.00 9.99 Q12.2 Q13.1 162,741 (Q2.9) 147,076 (Q7.9)
Phebalium stellatum Plantae PippinHT (≥ 20 kb) ONT PromethION SQK-LSK110 FLO-PRO002 132.39 44.75 25.85 20.62 Q8.1 Q8.3 1,109,097 (Q4.6) 352,235 (Q8.5)
Wahlenbergia ceracea Plantae SageELF (~13 kb) PacBio Sequell II HiFi SMRTbell Express 2.0 SMRTcell 8M 23,91 12.20 12.19 12.13 Q33.5 Q33.6 34,652 (Q21.3) 34,652 (Q21.3)

*DNA sheared with 29 gauge needle before size selection to optimise sequencing output.

†Flow cell could be run longer, including flow cell washes, to achieve more output. For small genomes flow cell was stopped when sufficient coverage was achieved.

DNA was isolated using the protocol presented and a final size selection was performed, using either a PippinHT prep (Sage Science), a SageELF (Sage Science) or Short Read Eliminator (SRE) (Circulomics). Sequencing was performed with either Oxford Nanopore Technologies (ONT) MinION, ONT PromethION or PacBio Sequel II in circular consensus sequencing (CCS) mode for HiFi reads. Total sequencing output, length N50, median quality (Phred scale) and longest reads are shown. ONT fast5 data was basecalled post-sequencing with the manufacturer’s algorithms provided in Guppy version 4.4.1 (ONT).

 Supporting information Step-by-step protocol, also available on protocols.io.

(PDF)

 References Logsdon GA, Vollger MR, Eichler EE. Long-read human genome sequencing and its applications. Nature Reviews Genetics. 2020;21(10):597–614. doi: 10.1038/s41576-020-0236-x 32504078 Miga KH, Koren S, Rhie A, Vollger MR, Gershman A, Bzikadze A, et al. Telomere-to-telomere assembly of a complete human X chromosome. Nature. 2020;585(7823):79–84. doi: 10.1038/s41586-020-2547-7 32663838 Shafin K, Pesout T, Lorig-Roach R, Haukness M, Olsen HE, Bosworth C, et al. Nanopore sequencing and the Shasta toolkit enable efficient de novo assembly of eleven human genomes. Nature Biotechnology. 2020;38(9):1044–53. doi: 10.1038/s41587-020-0503-6 32686750 Leng M, Drocourt J-L, Helene C, Ramstein J. Interactions between phenol and nucleic acids. Biochimie. 1974;56(6):887–91. doi: 10.1016/s0300-9084(74)80511-0 4447802 Do N, Adams RP. A simple technique for removing plant polysaccharide contaminants from DNA. BioTechniques. 1991;10(2):162, 4, 6. 2059438 Bolger A, Scossa F, Bolger ME, Lanz C, Maumus F, Tohge T, et al. The genome of the stress-tolerant wild tomato species Solanum pennellii. Nature Genetics. 2014;46:1034. doi: 10.1038/ng.3046 25064008 Workman R, Fedak R, Kilburn D, Hao S, Liu K, Timp W. High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing. 2018. Mayjonade B, Gouzy J, Donnadieu C, Pouilly N, Marande W, Callot C, et al. Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules. BioTechniques. 2016;61(4):203–5. doi: 10.2144/000114460 27712583 Inglis PW, Pappas MdCR, Resende LV, Grattapaglia D. Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications. PLOS ONE. 2018;13(10):e0206085. doi: 10.1371/journal.pone.0206085 30335843 De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics. 2018;34(15):2666–9. doi: 10.1093/bioinformatics/bty149 29547981 10.1371/journal.pone.0253830.r001 Decision Letter 0 Eppinger Mark Academic Editor 2021 Mark Eppinger This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 0

27 Apr 2021

PONE-D-21-06732

High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing

PLOS ONE

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Reviewer #1: In the manuscript titled as “High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing” Jones et al present a lab protocol for the isolation of high molecular weight DNA from plant tissue. Since Oxford Nanopore Technology (ONT) sequencing is highly sensitive to the DNA quality, this protocol would be very useful to the plant community. Furthermore, the authors claim 15-30 Gbp of sequencing data from a single MinION flow-cell. I find it very impressive and would like to congratulate the authors on achieving that. Overall, the protocol is clearly written and well elaborated. However, I have some minor suggestions for the authors which could further improve the quality of the manuscript.

Line 26: the authors claim that the protocol works for both ONT and PacBio sequencing whereas they only present the data for ONT.

Line 30: “eucalypts” is wrongly spelled.

Line 30: The authors claim that this DNA isolation protocol has been used for several organisms such as eucalyptus, acacias, rice, themeda, wheat, various fungi, 31 geckos, skinks, ticks, ladybird beetles, caterpillars and E. coli but there is no literature reference pointing to the publications where this protocol was used for the above-mentioned species.

Line 84: “includes quality reads over”. Please mention the q-score for referring a read as high quality.

Table 1: The authors should mention if 10 gm was fresh wait or powdered tissue.

Table 2: I would also like to see the mean and the median read length together with N50 in this table. Also, the authors should state the Guppy (or Bonito) version used for base-calling.

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Reviewer #1: Yes: Harmeet Singh Chawla

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 10.1371/journal.pone.0253830.r002 Author response to Decision Letter 0 Submission Version 1

3 Jun 2021

We thank the editor and reviewers for the thoughtful feedback. We now have addressed all outstanding concerns. We also provide a point-by-point reply to all the reviewers comments.

In accordance with your specific recent review request, we have also included updated competing interests statements in the cover letter. Thank you

Submitted filename: response_to_reviewersBS.docx

 10.1371/journal.pone.0253830.r003 Decision Letter 1 Eppinger Mark Academic Editor 2021 Mark Eppinger This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

14 Jun 2021

High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing

PONE-D-21-06732R1

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5 Jul 2021

PONE-D-21-06732R1

High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing

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