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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 dx.doi.org/10.17504/protocols.io.bcvyiw7w 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. Transcript Coverage Analysis from Long Reads. protocols.io dx.doi.org/10.17504/protocols.io.re2d3ge Copy
Authors: Chongmei Dong
Group: High molecular weight DNA extraction from all kingdoms
Summary: Current long read sequencing, e. g. PacBio and Nanopore, requires high molecular weight (HMW) and highly pure DNA. Many fungal and plant species have high content of polysaccharides and other contaminants that are co-precipitated with DNA during ethanol precipitation. This protocol aims to purify HMW DNA from the DNA solution obtained using Benjamin Schwessinger's protocol (dx.doi.org/10.17504/protocols.io.ewtbfen).The protocol was tested fuccessfully in wheat leaf rust, barley leaf rust, wheat stem rust and myrtle rust.Critcal step to seperate HMW DNA from low molecular weight DNA and other contaminants during ethanol precipitation is to allow cotton-fiber-like HMW DNA to sediment by gravity, while other small molecule DNA and contaminants remain in the supernatant.
Proper citation: Chongmei Dong 2017. Purification of HMW DNA from Fungi for long read sequencing. protocols.io dx.doi.org/10.17504/protocols.io.hbvb2n6 Copy
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 dx.doi.org/10.17504/protocols.io.45dgy26 Copy
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 dx.doi.org/10.17504/protocols.io.i6vche6 Copy
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 dx.doi.org/10.17504/protocols.io.kpwcvpe Copy
Authors: Sukhwinder Kaur, Quyen Anh Pham, and Lynn Epstein
Group: High molecular weight DNA extraction from all kingdoms, Plantae
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.i8ichue Copy
Authors: Yaowu Yuan
Group: High molecular weight DNA extraction from all kingdoms, Mimulus
Proper citation: Yaowu Yuan 2019. CTAB DNA Extraction for high quality/molecular weight DNA. protocols.io dx.doi.org/10.17504/protocols.io.3rsgm6e 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.jxxcppn Copy
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 dx.doi.org/10.17504/protocols.io.5isg4ee Copy
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 dx.doi.org/10.17504/protocols.io.bhftj3nn Copy
Authors: Robert Auber, Jennifer Wisecaver
Group: High molecular weight DNA extraction from all kingdoms, Wisecaver Lab
Summary: This protocol was developed for extraction of high molecular weight (HMW) DNA from Prymnesium parvum, a unicellular haptophyte alga, for the purpose of whole genome sequencing using Oxford Nanopore Technology (ONT) long reads. P. parvum is known to produce several specialized metabolic compounds that may compromise isolated DNA, leading to decreased sequencing yield. We found that separating intact nuclei from cellular debris prior to DNA isolation, improved read length and throughput. Isolated nuclei were processed using a Circulomics NanoBind kit to extract HMW DNA.
Proper citation: Robert Auber, Jennifer Wisecaver 2019. Algal nuclei isolation for Nanopore sequencing of HMW DNA. protocols.io dx.doi.org/10.17504/protocols.io.7b7hirn Copy
Authors: Miriam Schalamun, Benjamin Schwessinger
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, Benjamin Schwessinger 2017. High molecular weight gDNA extraction after Mayjonade et al. optimised for eucalyptus for nanopore sequencing. protocols.io dx.doi.org/10.17504/protocols.io.ka2csge Copy
Authors: Miriam Schalamun, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms
Summary: This protocol describes a clean up and size selection method for nucleic acids (tested on DNA) to deplete and remove fragments below 1 - 2 kb.
The success of this depends on the cleanliness of your sample (it doesn't have to be super clean but a whole lot of contaminants make working with the beads more difficult, diluting the sample out before usage can help with that). The concentrations of PEG and NaCl and the volume of the beads solution are crutial for recovery and proper removal of small fragments. As a basic guidline it can be said: more PEG and NaCl - higher recovery but hence less removal of small fragments and the other way round. I found for my samples (eucalyptus) that with 1 volume of the beads solution respectively to DNA sample I'm on the safe side recovery wise, but if I want to make sure to get rid of more smaller fragments I use 0.8 volumes.So in numbers that means: Final reaction concentration of PEG8000: 1 V: 5.5%
0.8 V: 4.8%Final reaction concentration of NaCl: 1V: 0.8 M0.8V: 0.7 M
Proper citation: Miriam Schalamun, Benjamin Schwessinger 2017. DNA size selection (>1kb) and clean up using an optimized SPRI beads mixture. protocols.io dx.doi.org/10.17504/protocols.io.idmca46 Copy
Authors: Elena Hilario
Group: High molecular weight DNA extraction from all kingdoms
Summary: The isolation of nuclear genomic DNA of high molecular weight is becoming a crucial step for obtaining long read sequencing data produced by the PacBio and Oxford Nanopore platforms. Although it involves a few more steps than a standard total genomic DNA preparations, it is worth optimizing the protocol to avoid wasting valuable sequence data on organellar DNA. The current protocol has been successfully applied to kiwifruit, apple, mānuka, Nicotiana benthamiana, some solanaceas, and two insects (pupae of the light brown apple moth (Epiphyas postvittana) and hind leg of a weta (Deinacrida spp)). Three alternative methods to extract DNA are also presented.
Proper citation: Elena Hilario 2018. Plant nuclear genomic DNA preps. protocols.io dx.doi.org/10.17504/protocols.io.rncd5aw Copy
Authors: Elena Hilario
Group: High molecular weight DNA extraction from all kingdoms, Plantae, Long Read Club
Summary: Chromatin capture-based protocols to produce Hi-C libraries start with a crude nuclei extract or a total cell extract. Either way, plants can be challenging due to the high content of contaminants that could interfere at the chromatin capture step.This protocol will help clean up the starting material needed for preparing Hi-C libraries. The nuclei yield varies, but should be enough for at least two preps. This protocol has been tested on blueberry, bilberry, pepino, Gillenia and rewarewa.
Proper citation: Elena Hilario 2019. Plant nuclei enrichment for chromatin capture-based Hi-C library protocols. protocols.io dx.doi.org/10.17504/protocols.io.8vehw3e Copy
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 NanoporeOptimized for DNA extraction from eucalyptus grandis and also tested on wheat stripe rust and brachypodium distachyon.
Proper citation: Miriam Schalamun 2017. High molecular weight gDNA extraction after Mayjonade et al. optimised for Eucalyptus. protocols.io dx.doi.org/10.17504/protocols.io.hcgb2tw Copy
Authors: Benjamin Istace, Anne Friedrich, Léo d’Agata, Sébastien Faye, Emilie Payen, Odette Beluche, Claudia Caradec, Sabrina Davidas, Corinne Cruaud, Gianni Liti, Arnaud Lemainque, Stefan Engelen, Patrick Wincker, Joseph Schacherer, Jean-Marc Aury
Group: GigaScience Press, High molecular weight DNA extraction from all kingdoms
Summary: These protocols accompany the following GigaScience publication:Benjamin Istace, et al. (2017) De novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer. GigaScience...
Proper citation: Benjamin Istace, Anne Friedrich, Léo d’Agata, Sébastien Faye, Emilie Payen, Odette Beluche, Claudia Caradec, Sabrina Davidas, Corinne Cruaud, Gianni Liti, Arnaud Lemainque, Stefan Engelen, Patrick Wincker, Joseph Schacherer, Jean-Marc Aury 2017. Protocols from "De novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer". protocols.io dx.doi.org/10.17504/protocols.io.gvxbw7n Copy
Authors: Ashley Jones, Justin Borevitz
Group: High molecular weight DNA extraction from all kingdoms
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 based nuclei extraction to remove cytoplasmic secondary metabolites, phenols and 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, 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 can approximately obtain over 10 gigabases of sequencing from a single MinION revC flow cell and over 15 gigabases with new revD flow cells, up to 23 gigabases. This includes quality reads over 200 kb in length, average N50 values over 20 kb and some N50 values exceeding 45 kb. Such ultra-long reads assist the assembly of high quality genomes, from telomere to telomere.
Proper citation: Ashley Jones, Justin Borevitz 2019. Nuclear DNA purification from recalcitrant plant species for long-read sequencing. protocols.io dx.doi.org/10.17504/protocols.io.28bghsn Copy
Authors: Douda Bensasson
Group: High molecular weight DNA extraction from all kingdoms
Summary: A very detailed four-day protocol for DNA extraction for yeast PacBio sequencing modified from Promega's protocol for yeast p13-14.
Proper citation: Douda Bensasson 2018. DNA extraction protocol for yeast PacBio sequencing. protocols.io dx.doi.org/10.17504/protocols.io.rved63e Copy
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