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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. Finally, a Short Read Eliminator (SRE) kit by Circulomics is utilised for size selection, which also appears to clean the DNA. This has been 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 are expected across many organisms, but have not been tested.

Proper citation: Ashley Jones, Neeraj Purushotham, Jamila Nasim, Benjamin Schwessinger 2019. DNA clean-up and size selection for long-read sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.6kahcse Copy   

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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 https://dx.doi.org/10.17504/protocols.io.6bthann Copy   

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Authors: David Eccles
Group: High molecular weight DNA extraction from all kingdoms
Summary: This protocol is for a semi-manual method for read demultiplexing, as used after my presentation Sequencing DNA with Linux Cores and Nanopores to work out the number of reads captured by different barcodes.Input: reads as a FASTQ file, barcode sequences as a FASTA fileOutput: reads split into single FASTQ files per target [barcode]Note: barcode / adapter sequences are not trimmed by this protocol

Proper citation: David Eccles 2019. Demultiplexing Nanopore reads with LAST. protocols.io https://dx.doi.org/10.17504/protocols.io.xj3fkqn Copy   

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Authors: Miriam Schalamun, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms, Plantae
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 https://dx.doi.org/10.17504/protocols.io.khkct4w Copy   

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

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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 https://dx.doi.org/10.17504/protocols.io.8vehw3e Copy   

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Authors: Yiheng Hu, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms, Team Schwessinger
Summary: This is a protocol optimized from Oxford Nanopore Technology 1D native barcoding genomic DNA (with EXP-NBD103 and SQK-LSK108) for sequencing amplicons of fungal marker genes (ITS, EF1a) using PCR.Thanks to Dr. Benjamin Schwesinger for his suggestions about the protocol.Thanks to Dr. Leon Smith (ANU, Linde lab), Prof. Wieland Meyer (USydney) and Dr. Laszlo Irinyi for their contribution of fungal samples.

Proper citation: Yiheng Hu, Benjamin Schwessinger 2018. Amplicon sequencing using MinION optimized from 1D native barcoding genomic DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.mhkc34w Copy   

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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 https://dx.doi.org/10.17504/protocols.io.i8jchun Copy   

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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. Finally, a Short Read Eliminator (SRE) kit by Circulomics is utilised for size selection, which also appears to clean the DNA. This has been 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 are expected across many organisms, but have not been tested.

Proper citation: Ashley Jones, Neeraj Purushotham, Jamila Nasim, Benjamin Schwessinger 2019. DNA clean-up and size selection for long-read sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.87ehzje Copy   

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

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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 https://dx.doi.org/10.17504/protocols.io.mrxc57n Copy   

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Authors: Josh Quick
Group: High molecular weight DNA extraction from all kingdoms
Summary: This is our ‘one-pot ligation’ protocol for Oxford Nanopore ligation libraries. It benefits from increased recovery of library and faster preparation time by using the Ultra II ligation module in conjunction with the Ultra II end repair/dA-tailing module therefore removing a clean-up step. It can be used with or without the optional FFPE DNA repair step which shoudl help to improve read-lengths in nicked DNA. The incubation times given here are sufficient to generate high quality libraries in under one hour. In the past we have cut these times in half without noticably impacting performance but we will leave this up to you.

Proper citation: Josh Quick 2018. One-pot ligation protocol for Oxford Nanopore libraries. protocols.io https://dx.doi.org/10.17504/protocols.io.k9acz2e Copy   

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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 https://dx.doi.org/10.17504/protocols.io.gvxbw7n Copy   

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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 https://dx.doi.org/10.17504/protocols.io.28bghsn Copy   

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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 https://dx.doi.org/10.17504/protocols.io.ka2csge Copy   

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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 https://dx.doi.org/10.17504/protocols.io.idmca46 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: 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 https://dx.doi.org/10.17504/protocols.io.bnjhmcj6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.7b7hirn Copy   

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Authors: David Hayward
Group: High molecular weight DNA extraction from all kingdoms
Summary: This protocol is based on that described by Blin and Stafford (1976).Blin, N., Stafford, D., 1976. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Research. 3, 2303.It uses RNAse and ProteinaseK follwed by phenol/Chlorofrom extraction.  For the highest molecular weight, the DNA is purified by dialysis, avoiding alcohol precipitation.

Proper citation: David Hayward 2018. High Molecular Weight genomic DNA from coral sperm. protocols.io https://dx.doi.org/10.17504/protocols.io.nwhdfb6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.4vbgw2n Copy   

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