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Authors: Tom Harrop
Group: High molecular weight DNA extraction from all kingdoms
Summary: Adapted from Qiagen's genomic DNA handbook and the user protocol for mosquito DNA extraction.
Proper citation: Tom Harrop 2018. HMW DNA extraction for insects. protocols.io dx.doi.org/10.17504/protocols.io.pnwdmfe Copy
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 dx.doi.org/10.17504/protocols.io.2yfgftn Copy
Authors: John Tyson
Group: High molecular weight DNA extraction from all kingdoms, Long Read Club
Summary: We have been playing quite a bit with native genomic DNA sequencing for a project we have running on Epigenetic modifications of Rat models of neurological disease. While we are still rapidly iterating on tweaks etc. to some new protocols around increasing our production efficiency of ultra-long reads (100kb+) using LSK109 ligation kit components and PEG/NaCl mediated DNA precipitation, we are settling into a workflow now that I think is worth sharing and has been generating very good results for us. That said remember this is a little “wild west” and perhaps for the more adventurous at this stage, use at your own risk etc. etc. and perhaps help with some tweaks of your own ;o). I’ll say now that I have been optimizing this on a very easy Rat cell line grown in culture and realise some people don’t have this luxury, however I think this is a very good jumping off point and allowed us to iterate on a consistent starting material and will myself be venturing off into tissue extractions now.There are currently three main areas to highlight and detail changes within a sample to sequence workflow that we think has allowed us to push the efficiency and yields of sequence data out. Firstly DNA extractions using Phenol/Chloroform and spooling out of HWM DNA to generate the starting DNA sample. Secondly input material shearing and modifications to the LSK109 ligation protocol that have yielded high efficiency libraries, and production of greater ultra-long reads numbers. Lastly the use of flowcell refueling and DNAseI clearing of the flowcell surface to remove “blocking” DNA and allow fresh library addition after a surface “reset”.I’m detailing all we have here is an attempt to allow people to fully understand what is going on as much as possible and so we can all use this information to make intelligent changes to the protocols etc. if you are so inclined. Commercial “black box kits” and solutions while great for defined on mass purpose and reproducibility make this process more opaque and often overly complex and expensive. A good example of this is the addition of just NaCl after the ligation reaction to precipitate the adapted DNA showing how knowing what you have provides simplification and economic sense for the end user in some situations, but more on that later……Final thoughts….One of my interests around nanopore sequencing and moving sequencing back to small labs and beyond with the MinION device is also mitigating the sample preparation costs while not sacrificing performance. Using cheap needles, Polyethylene Glycol, salt and old school molecular biology techniques we are seeing uncompromising performance for both yield and read lengths that I think even the “big” guys will use :o)). I will reproduce this post and methods at www.longreadclub.org soon so they will be easier to find and develop going forward, and we will try and provide protocols on www.protocols.io as well.Things on the to do list include:Further optimisation of PEG/salt parameters for size selection / short read elimination at different stages of library preparation.Refinement of shearing to provide increased 100kb+ reads from HMW DNALook at replacing Phenol/Chloroform with salting out in initial HMW Genomic DNA preparation.Who knew that PEG and salt would be a route to cheap ultra-long reads, we all just need to keep tweaking away in an open fashion and who knows where we can get. Anyway that’s enough from me for now, happy Nanopore adventuring!
Proper citation: John Tyson 2020. Rocky Mountain adventures in Genomic DNA sample preparation, ligation protocol optimisation / simplification and Ultra long read generation. protocols.io dx.doi.org/10.17504/protocols.io.7euhjew Copy
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 dx.doi.org/10.17504/protocols.io.beixjcfn Copy
Authors: Miriam Schalamun
Group: High molecular weight DNA extraction from all kingdoms
Summary: Extraction of high molecular weight DNA after Bolger et al. Carried out for eucalyptus grandis. DNA fragments were ranging up to 270 kb on a Pulsfield Electrophoresis Gel
Proper citation: Miriam Schalamun 2017. High molecular weight DNA extraction after Bolger et al.. protocols.io dx.doi.org/10.17504/protocols.io.hhqb35w Copy
Authors: Ashley Jones, Neeraj Purushotham, Jamila Nasim, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms
Summary: 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.
Proper citation: Ashley Jones, Neeraj Purushotham, Jamila Nasim, Benjamin Schwessinger 2020. DNA clean-up and size selection for long-read sequencing. protocols.io dx.doi.org/10.17504/protocols.io.betdjei6 Copy
Authors: Sukhwinder Kaur, Quyen Anh Pham, and Lynn Epstein
Group: High molecular weight DNA extraction from all kingdoms
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 dx.doi.org/10.17504/protocols.io.inycdfw Copy
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 dx.doi.org/10.17504/protocols.io.vyne7ve Copy
Authors: Josh Quick
Group: High molecular weight DNA extraction from all kingdoms, Long Read Club
Summary: The intention of this protocol is to isolate high molecular weight DNA. This means you should avoid any pipetting without using a wide-bore or cut off pipette tip, vortexing, mixer shakers or anything else which generate a velocity gradient which may shear the DNA. In addition you should be very careful not to introduce nucleases by making up buffers with nuclease-free water. Avoid unnecessary heating and do not freeze, isolated DNA should be stored in the fridge, a good extraction will be stable for months. Currently tested on E. coli and human cell lines, however it is likely to work with many gram-negative bacteria and mammalian cells.
Proper citation: Josh Quick 2018. Ultra-long read sequencing protocol for RAD004. protocols.io dx.doi.org/10.17504/protocols.io.mrxc57n Copy
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 dx.doi.org/10.17504/protocols.io.zvmf646 Copy
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 dx.doi.org/10.17504/protocols.io.vmee43e Copy
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 2019. An optimized protocol for sequencing mammalian roadkill tissues with Oxford Nanopore Technology (ONT). protocols.io dx.doi.org/10.17504/protocols.io.6bthann Copy
Authors: Todd GB McLay
Group: High molecular weight DNA extraction from all kingdoms, Plantae
Summary: This protocol was developed in order to remove secondary compounds which inhibited restriction enzyme digestion of Xanthorrhoea genomic DNA. It has also been used to successfully obtain high quality, high weight DNA from other recalcitrant plant tissues, including Eucalyptus, Eremophila, Spyridium, and various Malvaceae, Rutaceae, Orchidaceae, and Fabaceae species.It is based on a modified CTAB protocol from Doyle and Doyle (1990), and includes modifications from Shepherd and McLay (2011) and Tibbits et al (2006).
Proper citation: Todd GB McLay 2017. High quality DNA extraction protocol from recalcitrant plant tissues. protocols.io dx.doi.org/10.17504/protocols.io.i8jchun Copy
Authors: Elena Hilario
Group: High molecular weight DNA extraction from all kingdoms, Plantae, Long Read Club
Summary: Optical mapping technologies assist the assembly of large and complex genomes. Although protocols for preparing this kind of samples are now conveniently available as kits, some plants will not respond to standardized conditions or the researcher might not have access to kits.This protocol describes how to isolate plant nuclei from fresh very young leaves. The nuclei are purified over two Percoll® gradients and embedded in agarose. The nuclear DNA is released by solubizing the nuclear membrane with detergents and removing the proteins by digestion with Proteinse K. The nuclear DNA is analyzed by pulse field gel electrophoresis. If most of the DNA is > 1 Mbp, the sample could be further evaluated for optical mapping analyzis by the service provider.
Proper citation: Elena Hilario 2019. Optical mapping preps for Petunia spp.. protocols.io dx.doi.org/10.17504/protocols.io.baq3idyn Copy
Authors: Ashley Jones, Cynthia Torkel, David Stanley, Jamila Nasim, Justin Borevitz, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms
Summary: 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.
Proper citation: Ashley Jones, Cynthia Torkel, David Stanley, Jamila Nasim, Justin Borevitz, Benjamin Schwessinger 2020. Scalable high-molecular weight DNA extraction for long-read sequencing. protocols.io dx.doi.org/10.17504/protocols.io.bnjhmcj6 Copy
Authors: Huang Zhihai, Xu Jiang, Xiao Shuiming, Liao Baosheng, Gao Yuan, Zhai Chaochao, Qiu Xiaohui, Xu Wen, Chen Shilin
Group: GigaScience Press, High molecular weight DNA extraction from all kingdoms
Summary: This protocol provides an efficient and reliable technique for obtaining HMW DNA(>1Mb) from animal blood. It accompanies:Huang Zhihai, Xu Jiang, Xiao Shuiming, Liao Baosheng, Gao Yuan, Zhai Chaochao, Qiu Xiaohui, Xu Wen, Chen Shilin (2016): Supporting data for 'Comparative optical genome analysis of two Pangolin species Manis pentadactyla and Manis javanica'. GigaScience Database.
Proper citation: Huang Zhihai, Xu Jiang, Xiao Shuiming, Liao Baosheng, Gao Yuan, Zhai Chaochao, Qiu Xiaohui, Xu Wen, Chen Shilin 2016. Megabase DNA Extraction from Animal Blood. protocols.io dx.doi.org/10.17504/protocols.io.gagbsbw Copy
Authors: Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms, RUSTIS
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 2016. High quality DNA from Fungi for long read sequencing e.g. PacBio. protocols.io dx.doi.org/10.17504/protocols.io.ewtbfen Copy
Authors: Yiheng Hu
Group: High molecular weight DNA extraction from all kingdoms, Team Schwessinger
Summary: Modified from Benjamin Schwessinger: High quality DNA from Fungi for long read sequencing e.g. PacBio. protocols.ioOptimized for DNA extraction from wheat stripe rust spores.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
Proper citation: Yiheng Hu 2016. High quality DNA extraction from Fungi_small scale. protocols.io dx.doi.org/10.17504/protocols.io.exmbfk6 Copy
Authors: Rachael Workman, Renee Fedak , Duncan Kilburn, Stephanie Hao, Kelvin Liu, Winston Timp
Group: High molecular weight DNA extraction from all kingdoms, TimpLab
Summary: Single molecule sequencing requires optimized sample and library preparation protocols to obtain long-read lengths and high sequencing yields. Numerous protocols exist for the extraction of DNA from plant species, but the genomic DNA from these extractions is either too low yield, of insufficient purity for sensitive sequencing platforms, e.g. nanopore sequencing, too fragmented to achieve long reads, or otherwise unattainable from recalcitrant adult tissue. This renders many plant sequencing projects cost prohibitive or methodologically intractable. Existing protocols are also labor intensive, taking days to complete. Our protocol described here yields micrograms of high molecular weight gDNA from a single gram of adult or seedling leaf tissue in only a few hours, and produces high quality sequencing libraries for the Oxford Nanopore system, with typical yields ranging from 3-10 Gb per R9.4.1 flowcell and producing reads averaging 5-8 kb, with read length N50s ranging from 6-30 kb depending on the style of library preparation (details in sequencing outcomes section), and maximum lengths extending up to 200 kb+.
Proper citation: Rachael Workman, Renee Fedak , Duncan Kilburn, Stephanie Hao, Kelvin Liu, Winston Timp 2019. High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing. protocols.io dx.doi.org/10.17504/protocols.io.4vbgw2n Copy
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 dx.doi.org/10.17504/protocols.io.i3mcgk6 Copy
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