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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 https://dx.doi.org/10.17504/protocols.io.rved63e Copy   

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Authors: Ramawatar, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms
Summary: Most noncommercial tradition DNA extraction protocols result in a crude DNA preparation. If the DNA is intended to be used for a high-end application like Nanopore sequencing, it requires a thorough clean-up and size selection before it could be used for sequencing. Solid Phase Reversible Immobilisation (SPRI) magnetic beads is a quick and convenient way of purifying and size selecting intact double-stranded DNA from crude DNA. Most commercially available SPRI beads based DNA purification mix is quite expensive so our lab endeavored to develop an inexpensive beads mix which is as good as the commercially available ones. In this effort, our lab has optimized a beads mix for purifying and size selecting crude DNA extracted from eucalyptus and posted on protocol.io, https://www.protocols.io/edit/high-purity-high-molecular-weight-dna-extraction-f-n5ydg7w?step=16.However, this solution was not very effective in purifying crude DNA extracted from fungal material. DNA extracted from fungal material is highly viscous which is indicative of high levels of impurities in the DNA preparation. I tried to improve the beads mix for purifying rust DNA by adding 0.25 % (v/v) Tween-20 into the beads mix.I tested beads mix with Tween-20 to see if adding tween into the solution makes any difference in the recovery, purity and size selection.  Turns out that bead solutions with Tween-20 make big difference in the size selection and recovery of the DNA compared to the bead solutions without.  I calibrated/tested the bead solution with and without Tween-20 on the 1 kb DNA ladder to establish which DNA solution to beads volume ratio gives optimal recovery and size selection. We found that beads mix with 0.25 % Tween-20 works much better in size selection and recovery than beads mix without Tween-20. The best DNA to beads volume ratios were  1.0: 0.9 and 1.0: 1.0.

Proper citation: Ramawatar, Benjamin Schwessinger 2018. DNA size selection (>3-4kb) and purification of DNA using an improved homemade SPRI beads solution.. protocols.io https://dx.doi.org/10.17504/protocols.io.n7hdhj6 Copy   

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Authors: Karen Cristine Gonçalves Dos Santos, Megan Mcdonald
Group: High molecular weight DNA extraction from all kingdoms
Summary: Adaptation of the protocol dx.doi.org/10.17504/protocols.io.k6qczdwOptimized for DNA extration from Bipolaris sorokiniana spores.

Proper citation: Karen Cristine Gonçalves Dos Santos, Megan Mcdonald 2018. High molecular weight DNA extraction for long read sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.uppevmn 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 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 https://dx.doi.org/10.17504/protocols.io.hcgb2tw Copy   

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Authors: Glennis Logsdon
Group: High molecular weight DNA extraction from all kingdoms
Summary: This protocol describes the purification of high-molecular-weight genomic DNA from mammalian cells and the generation of ultra-long (N50 >100 kbp) Oxford Nanopore data. It is based on the Sambrook and Russell protocol and Josh Quick's protocol with additional modifications. This protocol improves upon previous protocols developed for ultra-long read sequencing, as it gives longer reads with greater yield. In our hands, we obtain about 1-2 Gb of ultra-long-read ONT data with an N50 ~150 kbp on one MinION flow cell.

Proper citation: Glennis Logsdon 2020. HMW gDNA purification and ONT ultra-long-read data generation. protocols.io https://dx.doi.org/10.17504/protocols.io.bchhit36 Copy   

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Authors: Elena Hilario
Group: High molecular weight DNA extraction from all kingdoms, Plantae, Long Read Club
Summary: Optical mapping technologies assist the assembly of large and complex genomes. Although protocols for preparing this kind of samples are now conveniently available as kits, some plants will not respond to standardized conditions or the researcher might not have access to kits.This protocol describes how to isolate plant nuclei from fresh very young leaves. The nuclei are purified over two Percoll® gradients and embedded in agarose. The nuclear DNA is released by solubizing the nuclear membrane with detergents and removing the proteins by digestion with Proteinse K. The nuclear DNA is analyzed by pulse field gel electrophoresis. If most of the DNA is > 1 Mbp, the sample could be further evaluated for optical mapping analyzis by the service provider.

Proper citation: Elena Hilario 2019. Optical mapping preps for Petunia spp.. protocols.io https://dx.doi.org/10.17504/protocols.io.baq3idyn 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. An optional needle shearing is described which can help create a more uniform DNA length to maximise sequencing output. Finally, a Short-Read Eliminator (SRE) kit by Circulomics is utilised for size selection, which also appears to clean the DNA. This was 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 have been obtained with other fungi, plants, reptiles and insects.

Proper citation: Ashley Jones, Neeraj Purushotham, Jamila Nasim, Benjamin Schwessinger 2020. DNA clean-up and size selection for long-read sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.betdjei6 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. Stranded Transcript Count Table Generation from Long Reads. protocols.io https://dx.doi.org/10.17504/protocols.io.vyne7ve Copy   

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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 https://dx.doi.org/10.17504/protocols.io.g8cbzsw Copy   

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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 https://dx.doi.org/10.17504/protocols.io.rncd5aw Copy   

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