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This protocols is part of the ANU Biosecurity mini-research project #2 "An SARS-COV2 incursion scenario: Genomics, phylogenetics, and incursions." This mini-research project is modeled on the yearly Quality Assurance Program of The Royal College of Pathologists of Australia (RCPAQAP), we take part in together with ACT Pathology. This research project is split into two major parts, identical to how the official RCPAQAP is run every year. Part #1 is focusing on the 'wet- lab' by sequencing SARS-COV2 from real world RNA samples provided by ACT Pathology especially for our ANU biosecurity course (Thank YOU!). Here you will amplify and sequence five (5) RNA samples per research group. You will assess the SARS-COV2 genome sequences for their lineage assignments using online programs, put sequences into a global context, estimate the collection date based on genetic information, and describe mutations in the spike protein. Part #2 is focusing on the 'dry-lab' by investigating a hypothetical incursion scenario in the so-called city Fantastica. You will combine genomic surveillance of SARS-COV2 with case interview data to trace the spread into of SARS-COV2 in the community and into high risk settings. We will provide you with real publicly available SARS-COV2 genome and fantasized case interviews. You will put these two together to trace the spread and suggest potential improvements in containment strategies with a focus on high risk settings. This protocol describes the analysis component of Part #1. The metrics you are suppose to report for each of your samples are mostly borrowed from the official SARS-CoV-2 QAP. Don't worry if not all of these mean something to you at the moment as we will explain them again during the prac. In case all/most of your samples have < 50% genome coverage please also include the analysis of MakeUp for points 1 to 7 and TimeMakeUp for point 8. You can access the MakeUp data here (ANU only). Make sure to read the README file so you understand what each item relates to. The metrics you have to report for each of our samples (or MakeUps) include the following. Consensus genome coverage. Average read depth; You might want to include detailed read depth plots here as well. Pangolin Lineage. NextClade Lineage. Base pair differences relative to the original SARS-CoV-2 genome. Amino acid replacements and deletions in the S (spike) protein sequence. Evaluation if your and/or the MakeUp samples would make the QC cut-off 90% genome coverage and other metrics that you deem important for QC. Would you 'flag' any of your samples as standing out e.g. being negative control? Approximate sampling date of your sequences for month and year. You must report the versions of all tools used in your report and the day the analysis was performed. This is extremely important for reporting as lineage naming and such change VERY frequently during the pandemic and any outbreak. This protocol is applicable for week 9. The following links might be useful for your report: The original publication that describes the sequencing protocol is here: https://academic.oup.com/biomethods/article/5/1/bpaa014/5873518?login=true Original sources describing the consensus reconstruction from raw reads are here: https://artic.network/ncov-2019, https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.html, https://labs.epi2me.io/, https://www.nature.com/articles/s41467-020-20075-6 Other websites and resources used in the protocol: https://igv.org/, https://clades.nextstrain.org/, https://pangolin.cog-uk.io/, https://genome.ucsc.edu/cgi-bin/hgPhyloPlace

This protocols is part of the ANU Biosecurity mini-research project #1 "Plant Pathogen Diagnostics: Visuals, subcultures, and genomics". You will be provided four pots of 3-4 week old wheat plants that have been infected with different wheat pathogens. Each pot has been infected with one major pathogen. You will not know which pot has been infected with which pathogen. However, you will be provided a compendium of 10-15 wheat pathogens that will guide you to identify the infective agent for each treatment group. The fifth treatment group will be uninfected wheat plants which will be clearly identified. You can use treatment group #5 as negative control for your experiments. In total, each group will obtain five pots each: This specific protocol is a step by step guide to assess DNA quality via agarose gel electrophoresis. Agarose gel electrophoresis lets you assess the size of DNA molecules. In addition, you can estimate DNA concentrations and impurities of RNA. During agarose gel electrophoresis DNA gets linearised. Larger molecules take longer to migrate through the gel when migration is driven by a electric potential at the specific pH of the buffer. When running a standard DNA ladder with bands of known molecular size, one can compare the DNA size of ones DNA or PCR sample with the known standards. The final goal is to achieve the following: To assess DNA length of molecules in DNA stock samples and for all PCR reactions. To approximately estimate relative DNA concentrations. To test if negative PCR controls did not amplify anything as appropriate. To test if whole DNA extracts contain RNA in addition to DNA. This protocol is applicable for week 5. Protocols progress overview: Week 5 Run a 1% Agarose Gel electrophoresis for all samples of each research group. Some useful explainers and resources: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846332/ https://en.wikipedia.org/wiki/Agarose_gel_electrophoresis https://www.youtube.com/watch?v=ZDZUAleWX78

This protocols is part of the ANU Biosecurity mini-research project #1 "Plant Pathogen Diagnostics: Visuals, subcultures, and genomics". You will be provided four pots of 3-4 week old wheat plants that have been infected with different wheat pathogens. Each pot has been infected with one major pathogen. You will not know which pot has been infected with which pathogen. However, you will be provided a compendium of 10-15 wheat pathogens that will guide you to identify the infective agent for each treatment group. The fifth treatment group will be uninfected wheat plants which will be clearly identified. You can use treatment group #5 as negative control for your experiments. In total, each group will obtain five pots each: This specific protocol is a step by step guide to sample representative leaf material for each treatment group and to extract DNA from these samples with the Qiagen DNeasy Plant Mini Kit. We use a kit in the course to speed up the process and make it reliable. Though it is important to understand the rough principles of DNA extraction as this process might influence the outcome of pathogen detection and identification in a biosecurity setting. We provide a short description of each step and its function in the protocol. In addition, please consult the handbook of the DNeasy Plant Mini Kit provider for further details as required. The final goal is to achieve the following: sample representative leaves for each treatment group that contain enough infective agent for DNA based pathogen identification. extract pure and high quality DNA that allows for PCR amplification of marker gene regions for two kingdoms (bacteria and fungi). This protocol is applicable for week 2 and 3. Protocols progress overview: Week 2 Sampling of two representative leaves and subsections thereof. Wee 2-3 tissue rupture with metal beads when tissues is frozen (performed by demonstrator). Week 3 DNA extraction from ruptured leaf tissue. The whole process follows the following major steps. Sampling -> Tissue rupture -> Tissue lysis -> Clearance of material -> DNA precipitation -> DNA binding to silica matrix -> Washing of impurities -> DNA elution from column and collection.

Extracting pure high-molecular weight DNA from fungi is often difficult due to the presence of polysaccharides and potentially other compounds, which biochemically mimic DNA or interfere with the DNA extraction process. Such compounds can co-elute with DNA in many extraction methods, making them difficult to remove from solution. Some contaminants may even be undetected by spectrophotometers or fluorometric devices, however they still substantially interferes with long-read DNA sequencing. In attempt to resolve these challenges, a protocol is presented that utilses cetrimonium bromide (CTAB) for lysis and precipitation. CTAB in the lysis solution acts as a cationic detergent to dissolve cell membranes and remove polysaccharides by the formation of CTAB-polysaccharide complexes that co-precipitate during chloroform phase separation. Using CTAB for DNA precipitation under low salt, low ionic conditions, CTAB-DNA complexes form while leaving polysaccharides in solution, resulting in significantly less co-precipitation than alcohol methods. The presented protocol also includes some updates to current strategies and incorporates two different options for DNA clean-up and size selection. Using this protocol, we have been successfully sequencing various fungi with a MinION (Oxford Nanopore Technologies). For wheat stripe rust Puccinia striiformis and wheat leaf rust Puccinia triticina, we performed the presented method followed by size selection with an automated gel purification system, with sequencing yielding 5.88 Gbp with an N50 of 34.79 kb and 4.88 Gbp with an N50 of 27.61 kb, respectively for the two fungi. These fungi are notorious for being recalcitrant and these results have been positive improvements on many other methods. To increase sequencing output for these samples, more work is needed to identify and remove the elusive contaminants. Other fungi, such as sorghum rot fungus Macrophomina phaseolina, we chose an alternative size selection method, precipitation with a polymer and salt solution. This sequencing yielded 13.71 Gbp with an N50 of 21.75 kb, while another strain yielded 9.72 Gbp with an N50 of 43.50 kb.

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. An optional needle shearing is described which can help create a more uniform DNA length to maximise sequencing output. Finally, polymer and salt based solutions are used to selectively precipitate DNA based on size, which also appears to further clean the DNA. This was 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 have been obtained with other fungi, plants, reptiles and insects.

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.

Massively parallel, second-generation short-read DNA sequencing has become an integral tool in biology for genomic studies. Offering highly accurate base-pair resolution at the most competitive price, the technology has become widespread. However, high-throughput generation of multiplexed DNA libraries can be costly and cumbersome. Here, we present a cost-conscious protocol for generating multiplexed short-read DNA libraries using a bead-linked transposome from Illumina. By preparing libraries in high-throughput with small reaction volumes that use 1/50th the amount of transposome compared to Illumina DNA Prep tagmentation protocols, the cost per library can be substantially reduced, by approximately 1/20th. Furthermore, we optimised the protocol to minimise magnetic bead-based clean-ups between steps, further reducing cost, time and DNA input requirements. By developing our own dual index primers to multiplex nine 96-well microplates, up to 864 samples can be placed on a single flow cell. This enables efficient usage of large-scale sequencing platforms, such as the Illumina NovaSeq 6000, which offers up to three terabases of sequencing per S4 flow cell.

With rapid advances in long-read DNA sequencing 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 native plants, crops and fungi. To resolve this, we optimised a gentle magnetic bead based high-molecular weight DNA extraction free of columns and high-centrifugation, to limit DNA fragmentation. A protocol that is scalable based on tissue input is presented, that can be used on many species of plants, fungi, reptiles, insects and bacteria. An optional sorbitol wash is listed and is highly recommended for plant tissues. To remove any remaining contaminants such as phenols and polysaccharides, two optional DNA clean-up and size selection strategies are given. Sequencing with Oxford Nanopore Technologies MinION, we can approximately obtain over 15-30 Gbp of sequencing from a single MinION flow cell with N50 values 30-50 kb. 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.

Extracting pure high-molecular weight DNA from some fungal species is difficult due to the presence of polysaccharides and potentially other compounds which biochemically mimic DNA or interfere with the DNA extraction process. Such compounds can co-elute with DNA in many extraction methods, being difficult to separate fom the DNA. Although the contaminant may not be detected by spectrophotometers or fluorometric devices, it substantially interferes with long-read DNA sequencing, such as Oxford Nanopore Technologies. To partially resolve this, a protocol is presented with some updates to current strategies and incorporates a small fragment removal step using Polyethylene Glycol. Using this protocol, we have been successfully sequencing the lentil pathogen Ascochyta lentis with a MinION (Oxford Nanopore Technologies). Sequencing yields have surpassed 13 gigabases with an N50 of approximately 15 kb. To increase sequencing output, more work is needed to identify and remove the elusive contaminants. DNA extraction modified from: https://www.protocols.io/view/high-molecular-weight-dna-extraction-from-challeng-5isg4ee PEG small fragment elimination after: https://www.protocols.io/view/size-selective-precipitation-of-dna-using-peg-amp-7erhjd6

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.

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. An optional needle shearing is described which can help create a more uniform DNA length to maximise sequencing output. Finally, a Short-Read Eliminator (SRE) kit by Circulomics is utilised for size selection, which also appears to clean the DNA. This was 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 have been obtained with other fungi, plants, reptiles and insects.

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.

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.pdf You 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")

This protocol was optimised from an existing protocol to achieve maximum pore occupancy in a MinION flow cell. This protocol was performed using Ligation sequencing kit (ONT) in combination with native barcoding kit (ONT). Using this protocol, I was able to achieve ~12 Gbp of data from one MinION run from samples with very low input concentration.

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.

This is an optimised protocol for 16S library preparation of V3-V4 region for sequencing through Illumina MiSeq platform (2 x 300 bp V3 chemistry). This protocol uses Platinum™ SuperFi™ PCR Master Mix instead of 2x KAPA HiFi HotStart ReadyMix given in Illumina 16S protocol. This polymerase master mix has lower error rate than KAPA, making it more suitable for sequencing.

Faecal tissues are difficult to lyse and contain lower amount of DNA compared to other animal tissues such as liver and duodenum. This protocol was optimised to extract maximum amount of DNA using 50 mg of faecal tissues following Qiagen DNeasy blood and tissue kit.

Extracting pure high-molecular weight DNA from some fungal species is difficult due to the presence of polysaccharides and potentially other compounds which biochemically mimic DNA or interfere with the DNA extraction process. Such compounds can co-elute with DNA in many extraction methods, being difficult to separate fom the DNA. Although the contaminant may not be detected by spectrophotometers or fluorometric devices, it substantially interferes with long-read DNA sequencing, such as Oxford Nanopore Technologies. To partially resolve this, a protocol is presented with some updates to current strategies and incorporates a gel purification with a Pippin Prep (Sage Science). Using this protocol, we have been successfully sequencing the wheat stripe rust Puccinia striiformis and leaf rust Puccinia triticina with a MinION (Oxford Nanopore Technologies). Sequencing yields have surpassed 4 gigabases with an N50 of approximately 30 kb. To increase sequencing output, more work is needed to identify and remove the elusive contaminants.

Extraction of high quality DNA for long read sequencing e.g. PacBio Optimized 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 periods Perform 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.

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.

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.

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.

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.

Extraction of high quality DNA for long read sequencing e.g. PacBio and MinIon Optimized for DNA extraction fromBipolaris sorokiniana. Also tested on Parastaognospora nodorum, Zymoseptoria tritici, wheat stripe rust, barley stripe rust and Pyrenophora tritici-repentis 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 periods Perform 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.

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.

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.

Extraction of high quality DNA for long read sequencing e.g. PacBio Optimized 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 periods Perform 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.

This protocol describesa clean up and size selection method for nucleic acids (tested on DNA) to deplete and remove fragments below 1 - 2 kb.The success of this depends on the cleanliness of your sample (it doesn't have to be super clean but a whole lot of contaminants make working with the beads more difficult, diluting the sample out before usage can help with that). The concentrations of PEG and NaCl and the volume of the beads solution are crutial for recovery and proper removal of small fragments. As a basic guidline it can be said: more PEG and NaCl - higher recovery but hence less removal of small fragments and the other way round. I found for my samples (eucalyptus) that with 1 volume of the beads solution respectively to DNA sample I'm on the safe side recovery wise, but if I want to make sure to get rid of more smaller fragments I use 0.8 volumes. So in numbers that means: Final reaction concentration of PEG8000: 1 V: 5.5% 0.8 V: 4.8% Final reaction concentration of NaCl: 1V: 0.8 M 0.8V: 0.7 M

Extraction of high quality DNA for long read sequencing e.g. PacBio Optimized 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 periods Perform 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.