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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 https://dx.doi.org/10.17504/protocols.io.ewtbfen Copy   

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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 https://dx.doi.org/10.17504/protocols.io.gagbsbw Copy   

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Authors: Boas Pucker
Group: High molecular weight DNA extraction from all kingdoms
Summary: Plant DNA extraction and preparation for ONT sequencingThis CTAB-based protocol is suitable for extraction of genomic DNA from a wide range of plant species. The DNA quality is sufficient for ONT sequencing after SRE enrichment of long fragments and removal of short fragments. Quality control steps are described as part of this protocol.Successful application for the genome sequencing of the following plant species:Arabidopsis species, Beta vulgaris (sugar beet), Brassica napus (rapeseed/canola), Dioscorea dumetorum (yams), Helichrysum umbraculigerum, mosses, Vitis vinifera (grapevine)References:Siadjeu C.*, Pucker B.*, Viehoever P., Albach D. and Weisshaar B. (2020). High contiguity de novo genome sequence assembly of Trifoliate yam (Dioscorea dumetorum) using long read sequencing. Genes. doi:10.3390/genes11030274.Pucker B, Rückert C, Stracke R, Viehöver P, Kalinowski J, Weisshaar B. Twenty-Five Years of Propagation in Suspension Cell Culture Results in Substantial Alterations of the Arabidopsis Thaliana Genome. Genes. 2019. doi:10.3390/genes10090671.Photo provided by Hanna Schilbert (@HSchilbert).

Proper citation: Boas Pucker 2020. Plant DNA extraction and preparation for ONT sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bcvyiw7w Copy   

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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 https://dx.doi.org/10.17504/protocols.io.exmbfk6 Copy   

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Authors: Robert Auber, Jennifer Wisecaver
Group: High molecular weight DNA extraction from all kingdoms, Wisecaver Lab
Summary: This is a protocol adapted for a haptophte algae. It is a single cellular organism lacking any type of silica or calcium carbonate armor. If your organism doesn't lyse by a simple snap freeze, some type of grinding step is recommended. Confirmation of properly extracting the nuclei can be done with propidium iodide staining and an epiflourescent microscope. The actual DNA extraction gives ultra long reads (greater than 200kb) by using a circulomics NanoBind kit.

Proper citation: Robert Auber, Jennifer Wisecaver 2019. Algal DNA extraction for HMW Nanopore sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.45dgy26 Copy   

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Authors: Louise Judd
Group: High molecular weight DNA extraction from all kingdoms
Summary: This is the protocol I have developed to genterate High'ish' Molecular Weight gDNA from gram negative bacterial speices to use for ONT MinION sequencing.This protocol was intially developed for the high throughput extraction of bacterial gDNA for Illumina short read sequencing. To perform our high throughput work we utislise Beckman Coulter Biomek liquid handling robots and the Beckman Coutler gDNA extraction kit Genfind v2. This kit utilises magnetic pariticles and is relatively gentle. We have found that gDNA extracted using this method has a high'ish' molecular weight. On a fragment analyser the GNDA is typically a clean single peak >60 kb (the limits of the fragment analyser). ONT MinION libraries generated wiht this gDNA typically have N50s in excess of 20 kb and the data is suitable for the assembly automated of almost bacterial genomes we have tested to date. This protocol will not give you the ultra long 'whale' reads that others have reported but it is a good compromise between HMW DNA, speed and ease of use and capacity to scale up to high throughput. NOTEWe have found that gDNA extracted using this method often have underepresentation of smaller plamids (

Proper citation: Louise Judd 2018. DNA extraction from gram negative bacteria for ONT MinION sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.p5mdq46 Copy   

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Authors: Miriam Schalamun
Group: High molecular weight DNA extraction from all kingdoms
Summary: 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. 

Proper citation: Miriam Schalamun 2017. High molecular weight gDNA extraction after Mayjonade et al. optimised for eucalyptus for nanopore sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.i6vche6 Copy   

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Authors: Alex Harkess
Group: High molecular weight DNA extraction from all kingdoms

Proper citation: Alex Harkess 2017. Modified Samberg Phenol:Chloforom HMW DNA prep for (some) plants. protocols.io https://dx.doi.org/10.17504/protocols.io.kpwcvpe Copy   

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Authors: Issaka Maman1, Tchadjobo Tchacondo2, Abiba Banla Kere1, Marcus Beissner3, Kossi badziklou1, Ekanao Tedihou4, Edith Nyaku4, Komi Amekuse5, Franz Xaver Wiedemann5, Damintoti Simplice Karou2, Gisela Bretzel3
Group: High molecular weight DNA extraction from all kingdoms
Summary: This protocole provide guidance to detect Mycobacterium ulcerans in environmental samples. The protocole share the DNA extraction process using the FastDNA kit for soil samples and the amplification of gene based on a validated protocole published by Fyfe which is the most protocole use to date for such analyis.

Proper citation: Issaka Maman1, Tchadjobo Tchacondo2, Abiba Banla Kere1, Marcus Beissner3, Kossi badziklou1, Ekanao Tedihou4, Edith Nyaku4, Komi Amekuse5, Franz Xaver Wiedemann5, Damintoti Simplice Karou2, Gisela Bretzel3 2018. Protocole for Mycobacterium ulcerans DNA detection in environmental samples using real time PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.pb7dirn 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. Transcript Coverage Analysis from Long Reads. protocols.io https://dx.doi.org/10.17504/protocols.io.re2d3ge Copy   

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Authors: Carine Puppo, Tomas Voisin, Brigitte Gontero
Group: High molecular weight DNA extraction from all kingdoms
Summary: Asterionella formosa is a freshwater pennate diatom that forms star-shaped colonies, whose mitochondrial DNA was recently sequenced [1]. This diatom is of interest for genetic studies [2,3] and for the study of photosynthesis [4,5]. However, extraction of genomic DNA (gDNA) from A. formosa is difficult because of its silica cell-wall and the presence of photosynthetic pigments that contaminate the DNA. Here, we present an optimised protocol for gDNA extraction from A. formosa that overcomes those two problems. We also assessed its efficacity in yielding high-purity gDNA compared to a standard protocol using hexadecyltrimethylammonium bromide (CTAB).References[1] Villain A. et al. (2017) Mitochondr. DNA. 2 (1), 97-98.[2] De Bruin A. et al. (2004) J. Phycol. 40, 823-830.[3] van Den Wyngaert S. et al. (2015) Mol. Ecol. 24, 2955-2972.[4] Boggetto N. et al. (2007) J. Phycol. 43, 1227-1235.[5] Erales J. et al. (2008) J. Phycol. 44, 1455-1464.

Proper citation: Carine Puppo, Tomas Voisin, Brigitte Gontero 2017. Genomic DNA extraction from the pennate diatom Asterionella formosa optimised for next generation sequencing.. protocols.io https://dx.doi.org/10.17504/protocols.io.jytcpwn 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.hadb2a6 Copy   

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Authors: Derreck Carter-House, Jason Stajich, Sarah Unruh, Tania Kurbessoian
Group: High molecular weight DNA extraction from all kingdoms, Chytrid Fungi - Molecular and Genomic Techniques, stajichlab
Summary: This protocol is a CTAB DNA extraction method for filamentous fungi. Its purpose is to extract high molecular weight genomic DNA for genome sequencing.

Proper citation: Derreck Carter-House, Jason Stajich, Sarah Unruh, Tania Kurbessoian 2020. Fungal CTAB DNA Extraction . protocols.io https://dx.doi.org/10.17504/protocols.io.bhx8j7rw Copy   

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Authors: Roland Wouters, Sam Mugford, Roberto Biello, Darren Heavens, Saskia Hogenhout
Group: High molecular weight DNA extraction from all kingdoms
Summary: Chromosome-scale genome assembly usually requires the use of long-read sequencing technologies such as PacBio or ONT MinION. However, the development of suitable DNA extraction methods that can yield high molecular weight DNA suitable these platforms can be problematic and time-consuming. We found that some traditional DNA extraction methods that are suitable for short-read sequencing platforms did not produce satisfactory results using long-read sequencing technologies with aphids or other sap-feeding hemipteran insects. We hypothesised that plant-derived components of the aphid’s diet might inhibit the sequencing chemistry. Here we present a method suitable for extraction of high-molecular DNA for long-read sequencing of aphids and other sap-feeding insects. We employed the Illustra Nucleon Phytopure DNA extraction kit designed to remove plant-specific contaminants. We were able to consistently recover very high molecular weight DNA that was compatible with library preparation and sequencing by PacBio or ONT MinION. The protocol presented here describes the use of this plant-DNA extraction kit optimised to obtain high molecular weight DNA from insect samples.We have used this method across a range of aphid species, leafhoppers and froghoppers (spittlebugs). It has enabled sequencing and - in most cases -chromosome-level assembly of genomes from the green peach aphid Myzus persicae (1), the pea aphid Acyrthosiphon pisum (1), the woolly apple aphid, Eriosoma lanigerum (2), and the common meadow spittlebug Philaenus spumarius (3). 1. Chromosome-scale genome assemblies of aphids reveal extensively rearranged autosomes and long-term conservation of the X chromosomeThomas C.Mathers, Roland H. M.Wouters, Sam T.Mugford, DavidSwarbreck, CockVan Oosterhout, Saskia A.HogenhoutbioRxiv 2020.03.24.006411; doi: https://doi.org/10.1101/2020.03.24.006411 2. A chromosome-level genome assembly of the woolly apple aphid, Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae)RobertoBiello, ArchanaSingh, Cindayniah J.Godfrey, FelicidadFernández Fernández, Sam T.Mugford, GlenPowell, Saskia A.Hogenhout, Thomas C.MathersbioRxiv 2020.05.29.121947; doi: https://doi.org/10.1101/2020.05.29.121947 3. Draft genome assembly version 1 of the meadow spittlebug Philaenus spumarius (Linnaeus, 1758) (Hemiptera, Aphrophoridae) (Version 1) [Data set]. Roberto Biello, Thomas C. Mathers, Sam T. Mugford, Qun Liu, Ana S. B. Rodrigues, Ana Carina Neto, Maria Teresa Rebelo; Octávio S. Paulo; Sofia G. Seabra; Saskia A. Hogenhout. (2020). Zenodo. http://doi.org/10.5281/zenodo.3368385

Proper citation: Roland Wouters, Sam Mugford, Roberto Biello, Darren Heavens, Saskia Hogenhout 2020. Extraction of high molecular weight DNA from aphids and other sap-feeding insects for long-read sequencing.. protocols.io https://dx.doi.org/10.17504/protocols.io.bhftj3nn Copy   

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Authors: Ashley Jones, Ramawatar Nagar, Anna Sharp, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms
Summary: 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.

Proper citation: Ashley Jones, Ramawatar Nagar, Anna Sharp, Benjamin Schwessinger 2019. High-molecular weight DNA extraction from challenging fungi using CTAB and gel purification. protocols.io https://dx.doi.org/10.17504/protocols.io.5isg4ee Copy   

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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 https://dx.doi.org/10.17504/protocols.io.hhqb35w Copy   

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Authors: Ashley Jones, Cynthia Torkel, David Stanley, Jamila Nasim, Justin Borevitz, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms
Summary: 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.

Proper citation: 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 https://dx.doi.org/10.17504/protocols.io.bss7nehn Copy   

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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 https://dx.doi.org/10.17504/protocols.io.7euhjew Copy   

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Authors: Josh Quick
Group: High molecular weight DNA extraction from all kingdoms, Long Read Club

Proper citation: Josh Quick 2019. The 'Three Peaks' faecal DNA extraction method for long-read sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.584g9yw Copy   

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Authors: Benjamin Schwessinger and Megan McDonald
Group: Solomon Lab - Australian National University, High molecular weight DNA extraction from all kingdoms, Zymoseptoria community protocols (STBnet)
Summary: 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. 

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.k6qczdw Copy   

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