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Authors: Baptiste Mayjonade
Group: High molecular weight DNA extraction from all kingdoms
Summary: Third generation sequencing aims to sequence long DNA molecules. High-quality and high-molecular-weight DNA is needed to fully benefit from the potential of third-generation sequencers. The cost of sequencing continues to decrease but DNA extraction kits remain expensive. Here is a fast and cheap protocol to extract genomic DNA from Gram-negative bacteria. The original protocol has been fine-tuned for Xanthomonas ssp., which is a tricky bacteria as it secrets a lot of extracellular polysaccharides. We found that bacterial culture can impact the purity of the final DNA solution. In addition, the lysis temperature and mixing by pipetting are critical to get an efficient homogenization as the sample becomes very viscous at some steps (see 'Assessment of various parameters.xlsx' attached below).High yield Nanopore runs can be achieved with these gDNA samples (see 'Nanopore sequencing report.pdf' attached below).Acknowledgements:I would like to thank Harihar Jaishree Subrahmaniam, Rekha Gopalan Nair, and BenjaminSchwessinger for their feedbacks and their corrections on this protocol.
Proper citation: Baptiste Mayjonade 2018. High Molecular Weight genomic DNA extraction from Gram-negative bacteria for long reads sequencing (Xanthomonas ssp.). protocols.io dx.doi.org/10.17504/protocols.io.r9id94e Copy
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 dx.doi.org/10.17504/protocols.io.p5mdq46 Copy
Authors: Jihoon Jo, Jooseong Oh, Hyun Gwan Lee, Hyun Hee Hong, Sung Gwon Lee, Seongmin Cheon, Elizabeth MA Kern, Soyeong Jin, Sung Jin Cho, Joong Ki Park, Chungoo Park
Group: GigaScience Press, High molecular weight DNA extraction from all kingdoms
Summary: This is a DNA extraction protocol from sea cucumber (Apostichopus japonicus) body-wall tissue. It accompanies the following GigaScience publication:Jihoon Jo, et al. (2016): Draft genome of the sea cucumber Apostichopus japonicus and genetic polymorphism among color variants. GigaScience...
Proper citation: Jihoon Jo, Jooseong Oh, Hyun Gwan Lee, Hyun Hee Hong, Sung Gwon Lee, Seongmin Cheon, Elizabeth MA Kern, Soyeong Jin, Sung Jin Cho, Joong Ki Park, Chungoo Park 2016. Genomic DNA extraction from sea cucumber body-wall tissue. protocols.io dx.doi.org/10.17504/protocols.io.gk8buzw Copy
Authors: Patrick Driguez, Karen Carty, Alexander Putra, Luca Ermini
Group: High molecular weight DNA extraction from all kingdoms
Summary: We have developed a method based on the Qiagen Genomics-Tip kit for the extraction of HMW DNA from a wide range of plants suitable for third generation sequencing platforms such as PacBio and Oxford Nanopore. This HMW plant DNA was used to make sequencing libraries with high insert N50 (30-40 Kb) for GridION and PromethION Oxford Nanopore machines and Sequel I and Sequel II PacBio machines. This HMW DNA is also suitable for PacBio HiFi libraries. The resulting sequencing data is ideally suited for de novo assembly of plant genomes.
Proper citation: Patrick Driguez, Karen Carty, Alexander Putra, Luca Ermini 2021. Workflow for generating HMW plant DNA for third generation sequencing with high N50 and high accuracy. protocols.io dx.doi.org/10.17504/protocols.io.bafmibk6 Copy
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 dx.doi.org/10.17504/protocols.io.pb7dirn Copy
Authors: Ashley Jones, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms
Summary: 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.
Proper citation: Ashley Jones, Benjamin Schwessinger 2020. Sorbitol washing complex homogenate for improved DNA extractions. protocols.io dx.doi.org/10.17504/protocols.io.beuvjew6 Copy
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 dx.doi.org/10.17504/protocols.io.jytcpwn Copy
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 dx.doi.org/10.17504/protocols.io.hadb2a6 Copy
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 dx.doi.org/10.17504/protocols.io.bhx8j7rw Copy
Authors: 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: Megan Mcdonald 2017. High quality DNA from Fungi for long read sequencing e.g. PacBio, Nanopore MinION. protocols.io dx.doi.org/10.17504/protocols.io.g8cbzsw Copy
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