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HMW gDNA purification and ONT ultra-long-read data generation Resource Report Resource Website |
Glennis Logsdon | 10.17504/protocols.io.bchhit36 | High molecular weight DNA extraction from all kingdoms | This protocol describes the purification of high-molecular-weight genomic DNA from mammalian cells and the generation of ultra-long (N50 >100 kbp) Oxford Nanopore data. It is based on the Sambrook and Russell protocol and Josh Quick's protocol with additional modifications. This protocol improves upon previous protocols developed for ultra-long read sequencing, as it gives longer reads with greater yield. In our hands, we obtain about 1-2 Gb of ultra-long-read ONT data with an N50 ~150 kbp on one MinION flow cell. | University of Washington | 1 | 2020 | Glennis Logsdon 2020. HMW gDNA purification and ONT ultra-long-read data generation. protocols.io dx.doi.org/10.17504/protocols.io.bchhit36 | 2021-03-29 03:10:52 | |||
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DNA size selection (>3-4kb) and purification of DNA using an improved homemade SPRI beads solution. Resource Report Resource Website |
Ramawatar, Benjamin Schwessinger | 10.17504/protocols.io.n7hdhj6 | High molecular weight DNA extraction from all kingdoms | Most noncommercial tradition DNA extraction protocols result in a crude DNA preparation. If the DNA is intended to be used for a high-end application like Nanopore sequencing, it requires a thorough clean-up and size selection before it could be used for sequencing. Solid Phase Reversible Immobilisation (SPRI) magnetic beads is a quick and convenient way of purifying and size selecting intact double-stranded DNA from crude DNA. Most commercially available SPRI beads based DNA purification mix is quite expensive so our lab endeavored to develop an inexpensive beads mix which is as good as the commercially available ones. In this effort, our lab has optimized a beads mix for purifying and size selecting crude DNA extracted from eucalyptus and posted on protocol.io, https://www.protocols.io/edit/high-purity-high-molecular-weight-dna-extraction-f-n5ydg7w?step=16.However, this solution was not very effective in purifying crude DNA extracted from fungal material. DNA extracted from fungal material is highly viscous which is indicative of high levels of impurities in the DNA preparation. I tried to improve the beads mix for purifying rust DNA by adding 0.25 % (v/v) Tween-20 into the beads mix.I tested beads mix with Tween-20 to see if adding tween into the solution makes any difference in the recovery, purity and size selection. Turns out that bead solutions with Tween-20 make big difference in the size selection and recovery of the DNA compared to the bead solutions without. I calibrated/tested the bead solution with and without Tween-20 on the 1 kb DNA ladder to establish which DNA solution to beads volume ratio gives optimal recovery and size selection. We found that beads mix with 0.25 % Tween-20 works much better in size selection and recovery than beads mix without Tween-20. The best DNA to beads volume ratios were 1.0: 0.9 and 1.0: 1.0. | Schalamun M, Nagar R, Kainer D, Beavan E, Eccles D, Rathjen JP, Lanfear R, Schwessinger B, Harnessing the MinION: An example of how to establish long‐read sequencing in a laboratory using challenging plant tissue from . Molecular Ecology Resources 19(1). doi: 10.1111/1755-0998.12938 | Australian National University, Australian National University | https://doi.org/10.1111/1755-0998.12938 | 1 | 2018 | Ramawatar, Benjamin Schwessinger 2018. DNA size selection (>3-4kb) and purification of DNA using an improved homemade SPRI beads solution.. protocols.io dx.doi.org/10.17504/protocols.io.n7hdhj6 | 2021-03-29 03:09:58 | |
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High molecular weight DNA extraction for long read sequencing Resource Report Resource Website |
Karen Cristine Gonçalves Dos Santos, Megan Mcdonald | 10.17504/protocols.io.uppevmn | High molecular weight DNA extraction from all kingdoms | Adaptation of the protocol dx.doi.org/10.17504/protocols.io.k6qczdwOptimized for DNA extration from Bipolaris sorokiniana spores. | Université du Québec à Trois-Rivières, Australian National University | 1 | 2018 | Karen Cristine Gonçalves Dos Santos, Megan Mcdonald 2018. High molecular weight DNA extraction for long read sequencing. protocols.io dx.doi.org/10.17504/protocols.io.uppevmn | 2021-03-29 03:10:06 | |||
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Amplicon sequencing using MinION optimized from 1D native barcoding genomic DNA Resource Report Resource Website |
Yiheng Hu, Benjamin Schwessinger | 10.17504/protocols.io.mhkc34w | High molecular weight DNA extraction from all kingdoms, Team Schwessinger | This is a protocol optimized from Oxford Nanopore Technology 1D native barcoding genomic DNA (with EXP-NBD103 and SQK-LSK108) for sequencing amplicons of fungal marker genes (ITS, EF1a) using PCR.Thanks to Dr. Benjamin Schwesinger for his suggestions about the protocol.Thanks to Dr. Leon Smith (ANU, Linde lab), Prof. Wieland Meyer (USydney) and Dr. Laszlo Irinyi for their contribution of fungal samples. | Australian National University, Australian National University | 1 | 2018 | Yiheng Hu, Benjamin Schwessinger 2018. Amplicon sequencing using MinION optimized from 1D native barcoding genomic DNA. protocols.io dx.doi.org/10.17504/protocols.io.mhkc34w | 2021-03-29 03:10:27 | |||
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The 'Three Peaks' faecal DNA extraction method for long-read sequencing Resource Report Resource Website |
Josh Quick | 10.17504/protocols.io.584g9yw | High molecular weight DNA extraction from all kingdoms, Long Read Club | Sam Nicholls [University of Birmingham], Nicholas Loman [University of Birmingham] | https://www.slideshare.net/scalene/the-three-peak-challenge-for-longread-ultradeep-stool-metagenomics-on-the-promethion | 1 | 2019 | Josh Quick 2019. The 'Three Peaks' faecal DNA extraction method for long-read sequencing. protocols.io dx.doi.org/10.17504/protocols.io.584g9yw | 2021-03-29 03:08:46 | |||
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Stranded Transcript Count Table Generation from Long Reads Resource Report Resource Website |
David Eccles | 10.17504/protocols.io.smuec6w | High molecular weight DNA extraction from all kingdoms | 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 | Malaghan Institute of Medical Research (NZ) | 1 | 2018 | David Eccles 2018. Stranded Transcript Count Table Generation from Long Reads. protocols.io dx.doi.org/10.17504/protocols.io.smuec6w | 2021-03-29 03:08:47 | |||
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Demultiplexing Nanopore reads with LAST Resource Report Resource Website |
David Eccles | 10.17504/protocols.io.xj3fkqn | High molecular weight DNA extraction from all kingdoms | This protocol is for a semi-manual method for read demultiplexing, as used after my presentation Sequencing DNA with Linux Cores and Nanopores to work out the number of reads captured by different barcodes.Input: reads as a FASTQ file, barcode sequences as a FASTA fileOutput: reads split into single FASTQ files per target [barcode]Note: barcode / adapter sequences are not trimmed by this protocol | Malaghan Institute of Medical Research (NZ) | https://doi.org/10.5281/zenodo.2535894 | 1 | 2019 | David Eccles 2019. Demultiplexing Nanopore reads with LAST. protocols.io dx.doi.org/10.17504/protocols.io.xj3fkqn | 2021-03-29 03:08:58 | ||
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High quality DNA from Fungi for long read sequencing e.g. PacBio, Nanopore MinION Resource Report Resource Website |
Benjamin Schwessinger and Megan McDonald | 10.17504/protocols.io.k6qczdw | Solomon Lab - Australian National University, High molecular weight DNA extraction from all kingdoms, Zymoseptoria community protocols (STBnet) | Extraction of high quality DNA for long read sequencing e.g. PacBio and MinIonOptimized for DNA extraction from Bipolaris sorokiniana. Also tested on Parastaognospora nodorum, Zymoseptoria tritici, wheat stripe rust, barley stripe rust and Pyrenophora tritici-repentisBuffers 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. | Australian National University | 4 | 2017 | Benjamin Schwessinger and Megan McDonald 2017. High quality DNA from Fungi for long read sequencing e.g. PacBio, Nanopore MinION. protocols.io dx.doi.org/10.17504/protocols.io.k6qczdw | 2021-03-29 03:08:56 | |||
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CTAB DNA Extraction Protocol of P. pruinosa Resource Report Resource Website |
Wenlu Yang | 10.17504/protocols.io.icgcatw | High molecular weight DNA extraction from all kingdoms | 2 | 2017 | Wenlu Yang 2017. CTAB DNA Extraction Protocol of P. pruinosa. protocols.io dx.doi.org/10.17504/protocols.io.icgcatw | 2021-03-29 03:08:59 | |||||
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Multi-step high purity high molecular weight DNA extraction protocol from challenging fungal tissues Resource Report Resource Website |
Ramawatar Nagar, Benjamin Schwessinger | 10.17504/protocols.io.rzkd74w | High molecular weight DNA extraction from all kingdoms | 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. | Australian National University, Australian National University | 1 | 2018 | Ramawatar Nagar, Benjamin Schwessinger 2018. Multi-step high purity high molecular weight DNA extraction protocol from challenging fungal tissues. protocols.io dx.doi.org/10.17504/protocols.io.rzkd74w | 2021-03-29 03:08:31 | |||
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High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing Resource Report Resource Website |
Ashley Jones, Cynthia Torkel, David Stanley, Jamila Nasim, Justin Borevitz, Benjamin Schwessinger | 10.17504/protocols.io.bss7nehn | High molecular weight DNA extraction from all kingdoms | Rapid advancements in long-read sequencing technologies have transformed sequencing 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, insects and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale at 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 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 eucalypts, acacias, rice, themeda, wheat, wheat rusts, various other fungi, geckos, skinks, ticks, ladybird beetles, caterpillars and E. coli. | Research School of Biology, Australian National University, Canberra, ACT, Australia, Research School of Biology, Australian National University, Canberra, ACT, Australia, Research School of Biology, Australian National University, Canberra, ACT, Australia, Research School of Biology, Australian National University, Canberra, ACT, Australia, Research School of Biology, Australian National University, Canberra, ACT, Australia, Research School of Biology, Australian National University, Canberra, ACT, Australia | 1 | 2021 | Ashley Jones, Cynthia Torkel, David Stanley, Jamila Nasim, Justin Borevitz, Benjamin Schwessinger 2021. High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. protocols.io dx.doi.org/10.17504/protocols.io.bss7nehn | 2021-03-29 03:08:37 | |||
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DNA clean-up and size selection for long-read sequencing Resource Report Resource Website |
Ashley Jones, Neeraj Purushotham, Jamila Nasim, Benjamin Schwessinger | 10.17504/protocols.io.6kahcse | High molecular weight DNA extraction from all kingdoms | DNA extractions often contain impurities which limit the output of long-read sequencing technologies. Here a protocol is provided which removes impurities and size selects for longer fragments. To remove residual RNA and protein, an additional RNAse A and Proteinase K treatment is performed. A clean-up with chloroform: isoamyl alcohol (24:1) removes these proteins and other hydrophobic organics such as lipids. A low volume ethanol precipitation and wash is used to concentrate the DNA, hopefully also reducing polysaccharides. Finally, a Short Read Eliminator (SRE) kit by Circulomics is utilised for size selection, which also appears to clean the DNA. This has been trialled for the sorghum rot fungus Macrophomina phaseolina, providing highly promising results with an Oxford Nanopore MinION. One strain yielded 13.71 Gbases with an N50 of 21.75 kb, another strain yielded 9.72 Gbases with an N50 of 43.50 kb. Similar results are expected across many organisms, but have not been tested. | Australian National University, University of Southern Queensland, Australian National University, Australian National University | 1 | 2019 | Ashley Jones, Neeraj Purushotham, Jamila Nasim, Benjamin Schwessinger 2019. DNA clean-up and size selection for long-read sequencing. protocols.io dx.doi.org/10.17504/protocols.io.6kahcse | 2021-03-29 03:08:59 | |||
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High molecular weight gDNA extraction after Mayjonade et al. optimised for eucalyptus for nanopore sequencing Resource Report Resource Website |
Miriam Schalamun, Benjamin Schwessinger | 10.17504/protocols.io.khkct4w | High molecular weight DNA extraction from all kingdoms, Plantae | Extraction of high quality DNA for long read sequencing e.g. the Oxford Nanopore Optimized for DNA extraction from eucalyptus grandis and eucalyptus pauciflora. This protocol contains an optional Chloroform clean up step which is necessary for eucalyptus but might not be for other tissue. For long DNA fragments don't vortex the DNA sample. | Schalamun M, Nagar R, Kainer D, Beavan E, Eccles D, Rathjen JP, Lanfear R, Schwessinger B, Harnessing the MinION: An example of how to establish long‐read sequencing in a laboratory using challenging plant tissue from . Molecular Ecology Resources 19(1). doi: 10.1111/1755-0998.12938 | , Australian National University | https://doi.org/10.1111/1755-0998.12938 | 9 | 2017 | Miriam Schalamun, Benjamin Schwessinger 2017. High molecular weight gDNA extraction after Mayjonade et al. optimised for eucalyptus for nanopore sequencing. protocols.io dx.doi.org/10.17504/protocols.io.khkct4w | 2021-03-29 03:09:17 | |
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Algal nuclei isolation for Nanopore sequencing of HMW DNA Resource Report Resource Website |
Robert Auber, Jennifer Wisecaver | 10.17504/protocols.io.5i2g4ge | High molecular weight DNA extraction from all kingdoms, Wisecaver Lab | 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. | Purdue University, Purdue University | 2 | 2019 | Robert Auber, Jennifer Wisecaver 2019. Algal nuclei isolation for Nanopore sequencing of HMW DNA. protocols.io dx.doi.org/10.17504/protocols.io.5i2g4ge | 2021-03-29 03:08:14 | |||
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High purity, high molecular weight DNA extraction from rust spores via CTAB based DNA precipitation for long read sequencing Resource Report Resource Website |
Ramawatar Nagar, Benjamin Schwessinger | 10.17504/protocols.io.n5ydg7w | High molecular weight DNA extraction from all kingdoms | 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. | Australian National University, Australian National University | 1 | 2018 | 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 dx.doi.org/10.17504/protocols.io.n5ydg7w | 2021-03-29 03:08:29 | |||
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Pulse Field Gel Electrophoresis for Long Read Sequencing Resource Report Resource Website |
Benjamin Schwessinger, David Stanley, Ming-Dao Chia | 10.17504/protocols.io.83jhykn | High molecular weight DNA extraction from all kingdoms | 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") | Australian National University, Australian National University, Australian National University | 1 | 2020 | Benjamin Schwessinger, David Stanley, Ming-Dao Chia 2020. Pulse Field Gel Electrophoresis for Long Read Sequencing. protocols.io dx.doi.org/10.17504/protocols.io.83jhykn | 2021-03-29 03:07:29 | |||
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DNA clean-up and size selection for long-read sequencing Resource Report Resource Website |
Ashley Jones, Neeraj Purushotham, Jamila Nasim, Benjamin Schwessinger | 10.17504/protocols.io.87ehzje | High molecular weight DNA extraction from all kingdoms | DNA extractions often contain impurities which limit the output of long-read sequencing technologies. Here a protocol is provided which removes impurities and size selects for longer fragments. To remove residual RNA and protein, an additional RNAse A and Proteinase K treatment is performed. A clean-up with chloroform: isoamyl alcohol (24:1) removes these proteins and other hydrophobic organics such as lipids. A low volume ethanol precipitation and wash is used to concentrate the DNA, hopefully also reducing polysaccharides. Finally, a Short Read Eliminator (SRE) kit by Circulomics is utilised for size selection, which also appears to clean the DNA. This has been trialled for the sorghum rot fungus Macrophomina phaseolina, providing highly promising results with an Oxford Nanopore MinION. One strain yielded 13.71 Gbases with an N50 of 21.75 kb, another strain yielded 9.72 Gbases with an N50 of 43.50 kb. Similar results are expected across many organisms, but have not been tested. | Australian National University, University of Southern Queensland, Australian National University, Australian National University | 2 | 2019 | Ashley Jones, Neeraj Purushotham, Jamila Nasim, Benjamin Schwessinger 2019. DNA clean-up and size selection for long-read sequencing. protocols.io dx.doi.org/10.17504/protocols.io.87ehzje | 2021-03-29 03:11:23 | |||
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One-pot ligation protocol for Oxford Nanopore libraries Resource Report Resource Website |
Josh Quick | 10.17504/protocols.io.k9acz2e | High molecular weight DNA extraction from all kingdoms | This is our ‘one-pot ligation’ protocol for Oxford Nanopore ligation libraries. It benefits from increased recovery of library and faster preparation time by using the Ultra II ligation module in conjunction with the Ultra II end repair/dA-tailing module therefore removing a clean-up step. It can be used with or without the optional FFPE DNA repair step which shoudl help to improve read-lengths in nicked DNA. The incubation times given here are sufficient to generate high quality libraries in under one hour. In the past we have cut these times in half without noticably impacting performance but we will leave this up to you. | University of Birmingham | 1 | 2018 | Josh Quick 2018. One-pot ligation protocol for Oxford Nanopore libraries. protocols.io dx.doi.org/10.17504/protocols.io.k9acz2e | 2021-03-29 03:11:32 | |||
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Nuclear DNA purification from recalcitrant plant species for long-read sequencing Resource Report Resource Website |
Ashley Jones, Justin Borevitz | 10.17504/protocols.io.ssveee6 | High molecular weight DNA extraction from all kingdoms | 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. | Research School of Biology, Australian National University. Canberra, ACT Australia., Research School of Biology, Australian National University. Canberra, ACT Australia. | 1 | 2018 | Ashley Jones, Justin Borevitz 2018. Nuclear DNA purification from recalcitrant plant species for long-read sequencing. protocols.io dx.doi.org/10.17504/protocols.io.ssveee6 | 2021-03-29 03:13:26 | |||
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High Molecular Weight genomic DNA from coral sperm Resource Report Resource Website |
David Hayward | 10.17504/protocols.io.nwhdfb6 | High molecular weight DNA extraction from all kingdoms | This protocol is based on that described by Blin and Stafford (1976).Blin, N., Stafford, D., 1976. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Research. 3, 2303.It uses RNAse and ProteinaseK follwed by phenol/Chlorofrom extraction. For the highest molecular weight, the DNA is purified by dialysis, avoiding alcohol precipitation. | Australian National University | 1 | 2018 | David Hayward 2018. High Molecular Weight genomic DNA from coral sperm. protocols.io dx.doi.org/10.17504/protocols.io.nwhdfb6 | 2021-03-29 03:12:43 |
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