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High quality DNA from Fungi for long read sequencing e.g. PacBio Resource Report Resource Website |
Benjamin Schwessinger | DOI:10.17504/protocols.io.ewtbfen | High molecular weight DNA extraction from all kingdoms, RUSTIS | 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. | Langner T, Harant A, Gomez-Luciano LB, Shrestha RK, Malmgren A, Latorre SM, Burbano HA, Win J, Kamoun S, Genomic rearrangements generate hypervariable mini-chromosomes in host-specific isolates of the blast fungus. PLoS Genetics 17(2). doi: 10.1371/journal.pgen.1009386 | ANU | https://doi.org/10.1371/journal.pone.0238611 | 10 | 2016 | 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 | 2021-04-15 09:14:27 | |
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Megabase DNA Extraction from Animal Blood Resource Report Resource Website |
Huang Zhihai, Xu Jiang, Xiao Shuiming, Liao Baosheng, Gao Yuan, Zhai Chaochao, Qiu Xiaohui, Xu Wen, Chen Shilin | DOI:10.17504/protocols.io.gagbsbw | GigaScience Press, High molecular weight DNA extraction from all kingdoms | 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. | GigaScience, GigaScience, GigaScience, GigaScience, GigaScience, GigaScience, GigaScience, GigaScience, GigaScience | https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giw001/2756882/Comparative-optical-genome-analysis-of-two | 1 | 2016 | 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 | 2021-04-15 09:14:33 | ||
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Plant DNA extraction and preparation for ONT sequencing Resource Report Resource Website |
Boas Pucker | DOI:10.17504/protocols.io.bcvyiw7w | High molecular weight DNA extraction from all kingdoms | 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). | Department of Plant Sciences, University of Cambridge (UK) | https://www.cebitec.uni-bielefeld.de/~bpucker | 1 | 2020 | Boas Pucker 2020. Plant DNA extraction and preparation for ONT sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bcvyiw7w | 2021-04-15 09:14:37 | ||
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High quality DNA extraction from Fungi_small scale Resource Report Resource Website |
Yiheng Hu | DOI:10.17504/protocols.io.exmbfk6 | High molecular weight DNA extraction from all kingdoms, Team Schwessinger | 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 | 1 | 2016 | Yiheng Hu 2016. High quality DNA extraction from Fungi_small scale. protocols.io https://dx.doi.org/10.17504/protocols.io.exmbfk6 | 2021-04-15 09:14:40 | ||||
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Algal DNA extraction for HMW Nanopore sequencing Resource Report Resource Website |
Robert Auber, Jennifer Wisecaver | DOI:10.17504/protocols.io.45dgy26 | High molecular weight DNA extraction from all kingdoms, Wisecaver Lab | 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. | Purdue University, Purdue University | 1 | 2019 | Robert Auber, Jennifer Wisecaver 2019. Algal DNA extraction for HMW Nanopore sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.45dgy26 | 2021-04-15 09:14:34 | |||
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DNA extraction from gram negative bacteria for ONT MinION sequencing Resource Report Resource Website |
Louise Judd | DOI:10.17504/protocols.io.p5mdq46 | High molecular weight DNA extraction from all kingdoms | 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 ( | University of Melbourne | 1 | 2018 | Louise Judd 2018. DNA extraction from gram negative bacteria for ONT MinION sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.p5mdq46 | 2021-04-15 09:14:57 | |||
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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 | DOI:10.17504/protocols.io.i6vche6 | High molecular weight DNA extraction from all kingdoms | 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. | 7 | 2017 | 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 | 2021-04-15 09:14:38 | ||||
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Modified Samberg Phenol:Chloforom HMW DNA prep for (some) plants Resource Report Resource Website |
Alex Harkess | DOI:10.17504/protocols.io.kpwcvpe | High molecular weight DNA extraction from all kingdoms | Danforth Center | 1 | 2017 | Alex Harkess 2017. Modified Samberg Phenol:Chloforom HMW DNA prep for (some) plants. protocols.io https://dx.doi.org/10.17504/protocols.io.kpwcvpe | 2021-04-15 09:14:52 | ||||
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Protocole for Mycobacterium ulcerans DNA detection in environmental samples using real time PCR Resource Report Resource Website |
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 | DOI:10.17504/protocols.io.pb7dirn | High molecular weight DNA extraction from all kingdoms | 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. | Maman I, Tchacondo T, Kere AB, Beissner M, Badziklou K, Tedihou E, Nyaku E, Amekuse K, Wiedemann FX, Karou DS, Bretzel G (2018) Molecular detection of Mycobacterium ulcerans in the environment and its relationship with Buruli ulcer occurrence in Zio and Yoto districts of maritime region in Togo. PLoS Negl Trop Dis 12(5): e0006455. doi: 10.1371/journal.pntd.0006455 | National reference laboratory for Buruli ulcer disease in Togo, Institut national d’hygiène (INH), Lomé, Togo, Laboratoire des sciences biomédicales et substances bioactives (LSBSB), Ecole supérieure des techniques biologiques et alimentaires (ESTBA), Université de Lomé, Togo, National reference laboratory for Buruli ulcer disease in Togo, Institut national d’hygiène (INH), Lomé, Togo, Department for infectious diseases and tropical medicine (DITM), Medical center of the University of Munich (LMU), Munich, Germany, National reference laboratory for Buruli ulcer disease in Togo, Institut national d’hygiène (INH), Lomé, Togo, Laboratoire de défense des cultures -Laboratoire national de biosécurité, Institut togolais de recherche agronomique, ITRA, Lomé, Togo, Laboratoire de défense des cultures -Laboratoire national de biosécurité, Institut togolais de recherche agronomique, ITRA, Lomé, Togo, German leprosy and tuberculosis relief association (DAHW-T), Togo office, Lomé, Togo, German leprosy and tuberculosis relief association (DAHW-T), Togo office, Lomé, Togo, Laboratoire des sciences biomédicales et substances bioactives (LSBSB), Ecole supérieure des techniques biologiques et alimentaires (ESTBA), Université de Lomé, Togo, Department for infectious diseases and tropical medicine (DITM), Medical center of the University of Munich (LMU), Munich, Germany | https://doi.org/10.1371/journal.pntd.0006455 | 1 | 2018 | 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 | 2021-04-15 09:15:28 | |
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Transcript Coverage Analysis from Long Reads Resource Report Resource Website |
David Eccles | DOI:10.17504/protocols.io.re2d3ge | 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) | 2 | 2018 | David Eccles 2018. Transcript Coverage Analysis from Long Reads. protocols.io https://dx.doi.org/10.17504/protocols.io.re2d3ge | 2021-04-15 09:15:07 | |||
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Genomic DNA extraction from the pennate diatom Asterionella formosa optimised for next generation sequencing. Resource Report Resource Website |
Carine Puppo, Tomas Voisin, Brigitte Gontero | DOI:10.17504/protocols.io.jytcpwn | High molecular weight DNA extraction from all kingdoms | 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. | Aix Marseille Univ CNRS, BIP, Imperial College of London, Aix Marseille Univ CNRS, BIP | 1 | 2017 | 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 | 2021-04-15 09:15:06 | |||
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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 | DOI:10.17504/protocols.io.hadb2a6 | High molecular weight DNA extraction from all kingdoms | 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. | Australian National University | 2 | 2017 | 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 | 2021-04-15 09:15:35 | |||
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Fungal CTAB DNA Extraction Resource Report Resource Website |
Derreck Carter-House, Jason Stajich, Sarah Unruh, Tania Kurbessoian | DOI:10.17504/protocols.io.bhx8j7rw | High molecular weight DNA extraction from all kingdoms, Chytrid Fungi - Molecular and Genomic Techniques, stajichlab | This protocol is a CTAB DNA extraction method for filamentous fungi. Its purpose is to extract high molecular weight genomic DNA for genome sequencing. | UC Riverside, UC Riverside, UC Riverside, UC Riverside | 1 | 2020 | 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 | 2021-04-15 09:15:39 | |||
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Extraction of high molecular weight DNA from aphids and other sap-feeding insects for long-read sequencing. Resource Report Resource Website |
Roland Wouters, Sam Mugford, Roberto Biello, Darren Heavens, Saskia Hogenhout | DOI:10.17504/protocols.io.bhftj3nn | High molecular weight DNA extraction from all kingdoms | 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 | John Innes Centre, Norwich, UK, John Innes Centre, Norwich, UK, John Innes Centre, Norwich, UK, Earlham Institute (EI), Norwich, UK, John Innes Centre, Norwich, UK | 1 | 2020 | 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 | 2021-04-15 09:15:44 | |||
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High-molecular weight DNA extraction from challenging fungi using CTAB and gel purification Resource Report Resource Website |
Ashley Jones, Ramawatar Nagar, Anna Sharp, Benjamin Schwessinger | DOI:10.17504/protocols.io.5isg4ee | High molecular weight DNA extraction from all kingdoms | 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. | Australian National University, Australian National University, Australian National University, Australian National University | 2 | 2019 | 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 | 2021-04-15 09:15:43 | |||
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High molecular weight DNA extraction after Bolger et al. Resource Report Resource Website |
Miriam Schalamun | DOI:10.17504/protocols.io.hhqb35w | High molecular weight DNA extraction from all kingdoms | 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 | 1 | 2017 | Miriam Schalamun 2017. High molecular weight DNA extraction after Bolger et al.. protocols.io https://dx.doi.org/10.17504/protocols.io.hhqb35w | 2021-04-15 09:15:26 | ||||
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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 | DOI: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 https://dx.doi.org/10.17504/protocols.io.bss7nehn | 2021-04-15 09:15:50 | |||
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Rocky Mountain adventures in Genomic DNA sample preparation, ligation protocol optimisation / simplification and Ultra long read generation Resource Report Resource Website |
John Tyson | DOI:10.17504/protocols.io.7euhjew | High molecular weight DNA extraction from all kingdoms, Long Read Club | 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! | Snutch Lab, UBC, Vancouver, BC, Canada | https://www.longreadclub.org/mountain-protocol/ | 1 | 2020 | 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 | 2021-04-15 09:15:52 | ||
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The 'Three Peaks' faecal DNA extraction method for long-read sequencing Resource Report Resource Website |
Josh Quick | DOI: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 https://dx.doi.org/10.17504/protocols.io.584g9yw | 2021-04-15 09:15:59 | |||
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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 | DOI: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 https://dx.doi.org/10.17504/protocols.io.k6qczdw | 2021-04-15 09:16:03 |
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