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Authors: Chongmei Dong
Group: High molecular weight DNA extraction from all kingdoms
Summary: Current long read sequencing, e. g. PacBio and Nanopore, requires high molecular weight (HMW) and highly pure DNA. Many fungal and plant species have high content of polysaccharides and other contaminants that are co-precipitated with DNA during ethanol precipitation. This protocol aims to purify HMW DNA from the DNA solution obtained using Benjamin Schwessinger's protocol (dx.doi.org/10.17504/protocols.io.ewtbfen).The protocol was tested fuccessfully in wheat leaf rust, barley leaf rust, wheat stem rust and myrtle rust.Critcal step to seperate HMW DNA from low molecular weight DNA and other contaminants during ethanol precipitation is to allow cotton-fiber-like HMW DNA to sediment by gravity, while other small molecule DNA and contaminants remain in the supernatant.

Proper citation: Chongmei Dong 2017. Purification of HMW DNA from Fungi for long read sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.hbvb2n6 Copy   

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Authors: Baptiste Mayjonade
Group: High molecular weight DNA extraction from all kingdoms
Summary: Third generation sequencing aims to sequence long DNA molecules. High-quality and high-molecular-weight DNA is needed to fully benefit from the potential of third-generation sequencers. The cost of sequencing continues to decrease but DNA extraction kits remain expensive. Here is a fast and cheap protocol to extract genomic DNA from Gram-negative bacteria. The original protocol has been fine-tuned for Xanthomonas ssp., which is a tricky bacteria as it secrets a lot of extracellular polysaccharides. We found that bacterial culture can impact the purity of the final DNA solution. In addition, the lysis temperature and mixing by pipetting are critical to get an efficient homogenization as the sample becomes very viscous at some steps (see 'Assessment of various parameters.xlsx' attached below).High yield Nanopore runs can be achieved with these gDNA samples (see 'Nanopore sequencing report.pdf' attached below).Acknowledgements:I would like to thank Harihar Jaishree Subrahmaniam, Rekha Gopalan Nair, and BenjaminSchwessinger for their feedbacks and their corrections on this protocol.

Proper citation: Baptiste Mayjonade 2018. High Molecular Weight genomic DNA extraction from Gram-negative bacteria for long reads sequencing (Xanthomonas ssp.). protocols.io https://dx.doi.org/10.17504/protocols.io.r9id94e Copy   

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Authors: Erin Vaughn
Group: High molecular weight DNA extraction from all kingdoms
Summary: Long-read sequencing (e.g. Nanopore's MinION or PacBio) has the potential to dramatically improve genome assembly but the quality of the reads is critically dependent upon the quality of the input gDNA. Here I describe the extraction of high molecular weight DNA from the calcareous sponge, Sycon capricorn. I choose to use Qiagen RLT buffer in the initial lysis step as this solution results in higher molecular weight DNA compared to alternatives. I suspect that RLT is deactivating endogenous DNases that would otherwise digest the gDNA.

Proper citation: Erin Vaughn 2018. High quality DNA extraction from Sycon capricorn for MinION long read sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.i3mcgk6 Copy   

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Authors: Ashley Jones, Justin Borevitz
Group: High molecular weight DNA extraction from all kingdoms
Summary: Evolution has driven genetic diversity of life on Earth, but also created highly complex genomes that are difficult to sequence. Current draft genomes can have thousands to hundreds of thousands of contigs rather than chromosomes, containing incorrect assemblies, gaps and errors. With rapid advances in long-read technologies, it is becoming possible to resolve complex genomes, including repetitive, polyploid plant genomes. Despite the technology being available, a challenge persists: the extraction of pure high molecular weight DNA suitable for long-read sequencing. This is particularly true of recalcitrant native Australian trees such as Eucalypts and Acacias. To resolve this, firstly we optimised a density gradient and detergent based nuclei extraction to limit reads from high copy count plastid genomes. Secondly, we optimised a gentle high molecular weight DNA extraction free of columns and high centrifugation, to limit DNA fragmentation. Finally, the DNA was purified and size selected by gel electrophoresis. For sequencing, we adopted the portable MinION sequencer from Oxford Nanopore Technologies. Using these approaches, we have been obtaining >9 gigabases of sequencing from a single MinION flowcell, including reads over 100 kb in length. Such ultra-long reads assist the assembly of high quality genomes, from telomere to telomere.

Proper citation: Ashley Jones, Justin Borevitz 2018. Nuclear DNA purification from recalcitrant plant species for long-read sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.ssveee6 Copy   

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Authors: Jihoon Jo, Jooseong Oh, Hyun Gwan Lee, Hyun Hee Hong, Sung Gwon Lee, Seongmin Cheon, Elizabeth MA Kern, Soyeong Jin, Sung Jin Cho, Joong Ki Park, Chungoo Park
Group: GigaScience Press, High molecular weight DNA extraction from all kingdoms
Summary: This is a DNA extraction protocol from sea cucumber (Apostichopus japonicus) body-wall tissue. It accompanies the following GigaScience publication:Jihoon Jo, et al.  (2016): Draft genome of the sea cucumber Apostichopus japonicus and genetic polymorphism among color variants. GigaScience...

Proper citation: Jihoon Jo, Jooseong Oh, Hyun Gwan Lee, Hyun Hee Hong, Sung Gwon Lee, Seongmin Cheon, Elizabeth MA Kern, Soyeong Jin, Sung Jin Cho, Joong Ki Park, Chungoo Park 2016. Genomic DNA extraction from sea cucumber body-wall tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.gk8buzw Copy   

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Authors: Patrick Driguez, Karen Carty, Alexander Putra, Luca Ermini
Group: High molecular weight DNA extraction from all kingdoms
Summary: We have developed a method based on the Qiagen Genomics-Tip kit for the extraction of HMW DNA from a wide range of plants suitable for third generation sequencing platforms such as PacBio and Oxford Nanopore. This HMW plant DNA was used to make sequencing libraries with high insert N50 (30-40 Kb) for GridION and PromethION Oxford Nanopore machines and Sequel I and Sequel II PacBio machines. This HMW DNA is also suitable for PacBio HiFi libraries. The resulting sequencing data is ideally suited for de novo assembly of plant genomes.

Proper citation: Patrick Driguez, Karen Carty, Alexander Putra, Luca Ermini 2021. Workflow for generating HMW plant DNA for third generation sequencing with high N50 and high accuracy. protocols.io https://dx.doi.org/10.17504/protocols.io.bafmibk6 Copy   

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Authors: Sukhwinder Kaur, Quyen Anh Pham, and Lynn Epstein
Group: High molecular weight DNA extraction from all kingdoms, Plantae
Summary: This protocol is for isolation of high quality, high molecular weight DNA (20 kb and larger) that is suitable for PacBio library preparation. This protocol has been tested on lyophilized conidia from multiple isolates of Fusarium oxysporum, including f. sp. apii, f. sp. ciceris, and f. sp. lycopersici.

Proper citation: Sukhwinder Kaur, Quyen Anh Pham, and Lynn Epstein 2017. High quality DNA from Fusarium oxysporum conidia suitable for library preparation and long read sequencing with PacBio. protocols.io https://dx.doi.org/10.17504/protocols.io.i8ichue Copy   

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Authors: Yaowu Yuan
Group: High molecular weight DNA extraction from all kingdoms, Mimulus

Proper citation: Yaowu Yuan 2019. CTAB DNA Extraction for high quality/molecular weight DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.3rsgm6e Copy   

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Authors: Benjamin Schwessinger and Megan McDonald
Group: High molecular weight DNA extraction from all kingdoms
Summary: Extraction of high quality DNA for long read sequencing e.g. PacBioOptimized for DNA extraction from wheat stripe rust spores and also tested on barley leaf rust.Buffers are best when fresh and not older than 3-6 months. Buffered Phenol:Chloroform:Isoamylalcohol (25:24:1) should not be older than 3 months.Critical steps to obtain high quality DNA:Do NOT heat samples during DNA extractions! Perform all steps at RT or 4oC as indicated.Do NOT incubate samples with KAc for prolonged time periodsPerform two steps of buffered Phenol:Chloroform:Isoamylalcohol purification to reduce co-purifying metabolites.DNA fragments were well above the 40kb mark based on Pippin Pulse Gels. The sequencing center performed a second AMPure purification step before library construction. Summary statistics of sequencing runs to follow.

Proper citation: Benjamin Schwessinger and Megan McDonald 2017. High quality DNA from Fungi for long read sequencing e.g. PacBio, Nanopore MinION. protocols.io https://dx.doi.org/10.17504/protocols.io.jxxcppn Copy   

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Authors: Ashley Jones, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms
Summary: The extraction of pure DNA can be challenging due to the presence of sugars, oils and other endogenous chemicals present within biological samples. This is particularly true for many plants and fungi due to the presence of secondary metabolites such as polyphenols and polysaccharides. Polyphenols within the cytosol can become irreversibly DNA-bound after cell lysis and polysaccharides can co-precipitate with DNA during the extraction. Sorbitol is an osmotically active sugar alcohol and washing homogenate with sorbitol before cell lysis has been shown to significantly improve the purity of DNA extractions. Sorbitol does not pass cell membranes and likely acts by drawing the cytosol out of the cell. Therefore polyphenols and polyscacharides would be removed.

Proper citation: Ashley Jones, Benjamin Schwessinger 2020. Sorbitol washing complex homogenate for improved DNA extractions. protocols.io https://dx.doi.org/10.17504/protocols.io.beuvjew6 Copy   

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Authors: Benjamin Schwessinger, David Stanley, Ming-Dao Chia
Group: High molecular weight DNA extraction from all kingdoms
Summary: Pulsefield gel electrophoresis is an easy and affordable method of quality control for high molecular weight DNA extractions, particularly for use in long-read Oxford Nanopore sequencing. This method is currently used in the Borevitz Lab (ANU) and is adapted from the method used by Benjamin Schwessinger (ANU).For more details on the PFGE hardware, refer to the manual at http://www.bio-rad.com/webroot/web/pdf/lsr/literature/M1703690.pdfYou may need to use different settings for different DNA size ranges - see pg. 25 of the manual ("5.2 Pulsed Field Conditions by DNA Size")

Proper citation: Benjamin Schwessinger, David Stanley, Ming-Dao Chia 2020. Pulse Field Gel Electrophoresis for Long Read Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.83jhykn Copy   

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Authors: Robert Auber, Jennifer Wisecaver
Group: High molecular weight DNA extraction from all kingdoms, Wisecaver Lab
Summary: This protocol was developed for extraction of high molecular weight (HMW) DNA from Prymnesium parvum, a unicellular haptophyte alga, for the purpose of whole genome sequencing using Oxford Nanopore Technology (ONT) long reads. P. parvum is known to produce several specialized metabolic compounds that may compromise isolated DNA, leading to decreased sequencing yield. We found that separating intact nuclei from cellular debris prior to DNA isolation, improved read length and throughput. Isolated nuclei were processed using a Circulomics NanoBind kit to extract HMW DNA.

Proper citation: Robert Auber, Jennifer Wisecaver 2019. Algal nuclei isolation for Nanopore sequencing of HMW DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.5i2g4ge Copy   

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Authors: Ramawatar Nagar, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms
Summary: This protocol is based on the fact that we struggled for a long time to obtain high purity DNA from the fungal material, especially from several rust species. We finally thought we made it when we obtained DNA with perfect QC measures using a CTAB based DNA precipitation. Yet we still got low yields caused by high amount of 'active feedback' during the sequencing run. We and many others had similar observations that awesome looking DNA doesn't sequence well (https://www.protocols.io/groups/awesome-DNA-from-all-kingdoms-of-life/discussions/awesome-dna-purity-measures-but-quickly-dying-pores). In this protocol, we combined several ideas we accumulated over the last 1.5 years since we started working on HMW DNA extractions for Nanopore sequencing. -> Different precipitants have different affinities for different contaminants.-> Some contaminants may have a higher affinity and lower solubility in NaCl/PEG/SPRI 'clean' up steps.-> Adding enzyme cocktails during the extraction may help to get rid of some contaminants. We combined all these three steps in the current protocol as combinatorial testing is currently cost prohibitive. It may well be that one of these steps is already enough.These ideas are laid out in more detail below and in publication soon to come. Our general recommendation is to test different buffer conditions and precipitants and if necessary combine them in a sequential manner. We hypothesize that different precipitants, e.g. NaCl/PEG, isopropanol, ethanol, or CTAB, display varying affinities for precipitating different contaminants. By applying them in a sequential manner it may be possible to obtain clean DNA via preferential precipitation of DNA over contaminants. In addition, in this newly developed protocol, we add enzyme mixes to the extraction buffer containing pectinases and cellulases reducing the amount of co-purifying contaminants from the fungal tissue. In case of other tissue types, different enzymes may have to be tested. It is important to add these enzymes during the extraction and not apply them to the final DNA suspension as most are not completely pure enzyme preparations and contain traces of DNAase activity that degrades the DNA when applied in simple solutions like TE buffer. We (see above) and many others have reported that NaCl/PEG-SPRI bead solutions are not always ideally suited to clean up DNA as contaminants simply co-precipitate. Following a similar logic of preferential precipitation, We hypothesize that is possible to first precipitate contaminants onto SPRI beads at low NaCl/PEG concentrations when HMW DNA stays in solution. In a subsequent step, DNA can be precipitated out of the remaining supernatant by increasing NaCl/PEG concentration adding more of the initial NaCl/PEG-SPRI beads solution. Contaminants with higher affinity to SPRI beads and lower solubility than DNA can thereby be removed from the solution.It is important to mention that we have had DNA preparations that fulfilled all our recommended quality control criteria but did not sequence well on the MinION. This was likely caused by ‘invisible’ contaminants. However, applying a combination of the above-suggested approaches enabled us to overcome this problem with our latest protocol.

Proper citation: Ramawatar Nagar, Benjamin Schwessinger 2018. Multi-step high purity high molecular weight DNA extraction protocol from challenging fungal tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.rzkd74w Copy   

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Authors: Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms, Team Schwessinger
Summary: Extraction of high quality DNA for long read sequencing e.g. PacBioOptimized for DNA extraction from wheat stripe rust spores and also tested on barley leaf rust.Buffers are best when fresh and not older than 3-6 months. Buffered Phenol:Chloroform:Isoamylalcohol (25:24:1) should not be older than 3 months.Critical steps to obtain high quality DNA:Do NOT heat samples during DNA extractions! Perform all steps at RT or 4oC as indicated.Do NOT incubate samples with KAc for prolonged time periodsPerform two steps of buffered Phenol:Chloroform:Isoamylalcohol purification to reduce co-purifying metabolites.DNA fragments were well above the 40kb mark based on Pippin Pulse Gels. The sequencing center performed a second AMPure purification step before library construction. Summary statistics of sequencing runs to follow.

Proper citation: Benjamin Schwessinger 2019. High quality DNA from Fungi for long read sequencing e.g. PacBio. protocols.io https://dx.doi.org/10.17504/protocols.io.2yfgftn Copy   

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Authors: Ramawatar Nagar, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms
Summary: Nanopore sequencing  (Oxford Nanopore Technologies MinION instrument) requires high quality, high molecular weight DNA (HMW) to produce long sequence reads. Extracting high purity, HMW DNA is a difficult challenge, especially for a biotrophic rust fungus. This is because the biomass for DNA extraction is restricted to spores which are physically tough and rich in complex polysaccharides and lipids. When DNA extracted from spores precipitated with ethanol or isopropanol, many other impurities like polysaccharides and lipids get coprecipitated along with DNA which renders DNA purity unsuitable for Nanopore sequencing. These impurities cannot be removed with any subsequent DNA purification with either paramagnetic beads (SPRI) or re-precipitation with ethanol and isopropanol. These impurities always co-precipitated with the DNA which absorbs light at 230 nm. In an attempt to improve purity, I tried precipitating DNA with cationic detergent CTAB (Hexadecyl-Trimethyl-Ammonium Bromide) which has been used to precipitate DNA in the past but not employed extensively. CTAB is a cationic surfactant which selectively complex with DNA and precipitates it out of the solution in the presence low salt concentration (0.4M NaCl) while leaving other impurities. Unlike isopropanol or ethanol precipitated DNA which looks highly viscous, the CTAB precipitated DNA lack similar viscosity and therefore indicative less or no impurities. Moreover, with CTAB precipitation I get ~ 20-25 ug crude DNA per 100 mg of spores which is almost double the amount I get with isopropanol and ethanol precipitation. The crude DNA once washed with 1 V homemade SPRI beads solution, gives a 260/280 ratio ~1.8-2 and 260/230 2.0-2.2.

Proper citation: Ramawatar Nagar, Benjamin Schwessinger 2018. High purity, high molecular weight DNA extraction from rust spores via CTAB based DNA precipitation for long read sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.n5ydg7w Copy   

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Authors: Marie-Ka Tilak, Rémi Allio, Frédéric Delsuc
Group: High molecular weight DNA extraction from all kingdoms
Summary: This protocol was developed to optimize DNA sequencing from mammalian roadkill tissue samples using Oxford Nanopore Technology (ONT). Roadkill tissues contain necrotized cells and impurities that result in DNA degradation. First, we observed that DNA preservation was generally better in RNAlater than in 95% EtOH preserved tissues. Second, we found that physically removing necrotized and epidermal cells before DNA extraction resulted in better DNA quality and purity. Finally, adjusting the ratio of AMPure beads to 0.4x at the DNA purification step permitted optimizing size selection for subsequent ONT library construction. These optimization steps allowed to significantly increase both read length and yield per flow cell for roadkill samples sequencing on the ONT MinION device.

Proper citation: Marie-Ka Tilak, Rémi Allio, Frédéric Delsuc 2020. An optimized protocol for sequencing mammalian roadkill tissues with Oxford Nanopore Technology (ONT). protocols.io https://dx.doi.org/10.17504/protocols.io.beixjcfn Copy   

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Authors: David Eccles
Group: High molecular weight DNA extraction from all kingdoms
Summary: This protocol is for comparing two different samples at the transcript level, using long reads that are mapped to transcripts.Input(s): stranded fastq files (see steps 1-8 of Stranded Mapping from Long Reads), transcript reference fasta file, annotation fileOutput(s): transcript table, sorted by differential coverage, annotated with gene name / description / location

Proper citation: David Eccles 2018. Stranded Transcript Count Table Generation from Long Reads. protocols.io https://dx.doi.org/10.17504/protocols.io.smuec6w Copy   

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Authors: Reuben Brown, Taylor Wass, Nishanti Sudhakar
Group: High molecular weight DNA extraction from all kingdoms
Summary: Preparing genomic DNA extractions from microalgae specifically for long read sequencing on platforms such as Nanopore and Pac bio requires ultra-high quality DNA free from contaminants that may interfere and inhibit the sequencing chemistry. In microalgae there are many contaminants that can co precipitate with DNA and cause poor run quality on the nanopore platform. Genome sequencing of microalgae can be a difficult task to approach given the microbial consortiums alga are found in. Separating microalgae from bacteria and fungi can be a daunting task and a multifaceted approach is often the most effective. Combining different techniques to obtain pure axenic cultures is often required. The most effective techniques require long regrowth times from single cell colonies, increasing the time required to obtain pure cultures.Many raw read entries into the NCBI SRA are plagued with artefacts from bacteria and fungi. Although sequencing monoclonal axenic cultures is ideal it may not always be practical, by employing density gradient centrifugation, and antibiotic cocktails it is possible to obtain Sequence ready cultures in very short time periods with enough cell mass to produce >5ug of gDNA for nanopore sequencing. The nanopore platform is particularly susceptible to contamination induce failure mode, whatsmore several fungi species have been shown to contain silent contaminants (https://www.protocols.io/groups/awesome-DNA-from-all-kingdoms-of-life/discussions/awesome-dna-purity-measures-but-quickly-dying-pores?comment=3445) where gDNA preparation test as high quality on the nanodrop ONE platform (thermofisher).Extraction kits are marketed as a one size fits all solution and, in many circumstances, will provide sub standard results and the price of these kits greatly exceeds the face value of the reagents and packaging. Proprietary additives and devices supposedly increase efficiency of extractions and attract a premium, but often perform just as well as a Classic CTAB extraction (carlson). One of the most expensive components of DNA extraction and library preparation are SPRI magnetic beads, which can be easily synthesized in house at a fraction of the cost (BOMB.bio) with the same functionality that enables high performance extractions. To increase the quality of extraction delicate handling and mixing is required, this is best achieved with a rotisserie mixer these have classically had high price tags in order to save costs on this component we 3d printed a model from a recent publication (Karankumar C et. al ). For many situations the ability to customize extractions by varying buffer components and concentrations as well as steps involved can vastly improve the quality of extract and speed up the entire process.By chaining together, a series of extraction and precipitation chemistry into 3 stages, each stage helps remove contaminants that may co-precipitate with other steps. 1. This method exploits the extraction surfactant CTABs ability to only solubilize nucleic acids at high salt concentrations (>1.2M NaCl) where nucleic acids precipitate in a complex with CTAB at a low salt concentration (1kb size selection and PEG based precipitation with DIY-SPRI mix. Each successive step removes more contaminants and improves quality metrics of the extract.Its evident from the nanodrop data there is a hidden contaminent that is not being detected by uv absorbtion spectroscopy. For many first pass extractions (namley KB1 and MUR-279) values appear to be pefectly inrange but then resuspending, cleaning and precipertating with ipa (pass 2) degrades the quality, which is then restored on the third pass with PEG/NaCl precipertation.

Proper citation: Reuben Brown, Taylor Wass, Nishanti Sudhakar 2019. Efficient NGS ready gDNA from microalga. protocols.io https://dx.doi.org/10.17504/protocols.io.zvmf646 Copy   

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Authors: Wenlu Yang
Group: High molecular weight DNA extraction from all kingdoms

Proper citation: Wenlu Yang 2017. CTAB DNA Extraction Protocol of P. pruinosa. protocols.io https://dx.doi.org/10.17504/protocols.io.icgcatw Copy   

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Authors: Ashley Jones, Justin Borevitz
Group: High molecular weight DNA extraction from all kingdoms, Team Schwessinger
Summary: Evolution has driven genetic diversity of life on Earth, but also created highly complex genomes that are difficult to sequence. Current draft genomes can have thousands to hundreds of thousands of contigs rather than chromosomes, containing incorrect assemblies, gaps and errors. With rapid advances in long-read technologies, it is becoming possible to resolve complex genomes, including repetitive, polyploid plant genomes. Despite the technology being available, a challenge persists: the extraction of pure high molecular weight DNA suitable for long-read sequencing. This is particularly true of recalcitrant native Australian trees such as Eucalypts and Acacias. To resolve this, firstly we optimised a density gradient and detergent based nuclei extraction to limit reads from high copy count plastid genomes. Secondly, we optimised a gentle high molecular weight DNA extraction free of columns and high centrifugation, to limit DNA fragmentation. Finally, the DNA was purified and size selected by gel electrophoresis. For sequencing, we adopted the portable MinION sequencer from Oxford Nanopore Technologies. Using these approaches, we have been obtaining >9 gigabases of sequencing from a single MinION flow cell, including reads over 100 kb in length. Such ultra-long reads assist the assembly of high quality genomes, from telomere to telomere.

Proper citation: Ashley Jones, Justin Borevitz 2018. Nuclear DNA purification from recalcitrant plant species for long-read sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.vmee43e Copy   

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