High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing v1

Workman, Rachael E.; provided, Renee Fedak not; Kilburn, Duncan; Hao, Stephanie; Liu, Kelvin; Timp, Winston

doi:10.17504/protocols.io.4vbgw2n

Cited by 39 publications

(56 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nuclei were isolated from leaf tissue following a previously described protocol 27 with an input of 1 − 2 g of ground tissue; spin speeds were used based on estimated genome size, 2,700, 3,030, 3,030, and 2,900 g for O. basilicum , O. majorana , O. vulgare , and R. officinalis , respectively. DNA was extracted using the Nanobind Plant Nuclei Big DNA Kit (Circulomics, Baltimore, MD, Cat No.…”

Section: Methodsmentioning

confidence: 99%

Genome sequencing of four culinary herbs reveals terpenoid genes underlying chemodiversity in the Nepetoideae

et al. 2020

View full text Add to dashboard Cite

Species within the mint family, Lamiaceae, are widely used for their culinary, cultural, and medicinal properties due to production of a wide variety of specialized metabolites, especially terpenoids. To further our understanding of genome diversity in the Lamiaceae and to provide a resource for mining biochemical pathways, we generated high-quality genome assemblies of four economically important culinary herbs, namely, sweet basil (Ocimum basilicum L.), sweet marjoram (Origanum majorana L.), oregano (Origanum vulgare L.), and rosemary (Rosmarinus officinalis L.), and characterized their terpenoid diversity through metabolite profiling and genomic analyses. A total 25 monoterpenes and 11 sesquiterpenes were identified in leaf tissue from the four species. Genes encoding enzymes responsible for the biosynthesis of precursors for mono- and sesqui-terpene synthases were identified in all four species. Across all four species, a total of 235 terpene synthases were identified, ranging from 27 in O. majorana to 137 in the tetraploid O. basilicum. This study provides valuable resources for further investigation of the genetic basis of chemodiversity in these important culinary herbs.

show abstract

Section: Methodsmentioning

confidence: 99%

Genome sequencing of four culinary herbs reveals terpenoid genes underlying chemodiversity in the Nepetoideae

et al. 2020

View full text Add to dashboard Cite

show abstract

“…High molecular weight (HMW) DNA for Nanopore sequencing (Oxford Nanopore Technologies Inc., UK) was isolated through a nuclei extraction and lysis protocol. First, mature leaf tissue from the same tree used for the original J. regia genome (Martínez-García et al 2016) was homogenized with mortar and pestle in liquid nitrogen until well ground, then added to the Nuclei Isolation Buffer (Workman et al 2018), and stirred at 4°C for 10 minutes. The cellular homogenate was filtered through 5 layers of Miracloth (Millipore-Sigma) into a 50 mL Falcon tube, then centrifuged at 4°C for 20 minutes at 3000 x g. This speed of centrifugation was selected based on the estimated walnut genome size of 1 Gb (Zhang et al 2012).…”

Section: Methodsmentioning

confidence: 99%

High-quality chromosome-scale assembly of the walnut (Juglans regiaL) reference genome

Marrano

Britton

Zaini

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

27The release of the first reference genome of walnut (Juglans regia L.) enabled many achievements 28 in the characterization of walnut genetic and functional variation. However, it is highly 29 fragmented, preventing the integration of genetic, transcriptomic, and proteomic information to 30 fully elucidate walnut biological processes. Here we report the new chromosome-scale assembly 31 of the walnut reference genome (Chandler v2.0) obtained by combining Oxford Nanopore long-32 read sequencing with chromosome conformation capture (Hi-C) technology. Relative to the 33 previous reference genome, the new assembly features an 84.4-fold increase in N50 size, and the 34 full sequence of all 16 chromosomal pseudomolecules, nine of which present telomere sequences 35 at both ends. Using full-length transcripts from single-molecule real-time sequencing, we predicted 36 40,491 gene models, with a mean gene length higher than the previous gene annotations. Most of 37 the new protein-coding genes (90%) are full-length, which represents a significant improvement 38 compared to Chandler v1.0 (only 48%). We then tested the potential impact of the new 39 chromosome-level genome on different areas of walnut research. By studying the proteome 40 changes occurring during catkin development, we observed that the virtual proteome obtained 41 from Chandler v2.0 presents fewer artifacts than the previous reference genome, enabling the 42 identification of a new potential pollen allergen in walnut. Also, the new chromosome-scale 43 genome facilitates in-depth studies of intraspecies genetic diversity by revealing previously 44 undetected autozygous regions in Chandler, likely resulting from inbreeding, and 195 genomic 45 regions highly differentiated between Western and Eastern walnut cultivars. Overall, Chandler 46 v2.0 is a valuable resource to understand and explore walnut biology better.47 48Persian walnut (Juglans regia L.) is among the top three most-consumed nuts in the world, and 50 over the last ten years, its global production increased by 37% (International Nut and Dried Fruit 51

show abstract

“…For the doubled monoploid S. tuberosum Group Phureja clone DM1-3 516 R44 with a genome size of 844 Mb (The Potato Genome Sequencing Consortium, 2011), we successfully performed a nuclei extraction following the Workman et. al (2018) protocol.…”

Section: Resultsmentioning

confidence: 99%

“…DNA was isolated from tissue culture-grown shoots of Solanum tuberosum Group Phureja clone DM1-3 516 R44 (potato) and leaves of Lavandula angustifolia (lavender) using the Workman et. al (2018) protocol followed by the Nanobind Plant Nuclei Big DNA Kit (Circulomics, Baltimore, MD, Cat # NB-900-801-01).…”

Section: Dna Isolationmentioning

confidence: 99%

High molecular weight DNA isolation method from diverse plant species for use with Oxford Nanopore sequencing

Vaillancourt

Buell

2019

Preprint

View full text Add to dashboard Cite

The ability to generate long reads on the Oxford Nanopore Technologies sequencing platform is dependent on the isolation of high molecular weight DNA free of impurities. For some taxa, this is relatively straightforward; however, for plants, the presence of cell walls and a diverse set of specialized metabolites such as lignin, phenolics, alkaloids, terpenes, and flavonoids present significant challenges in the generation of DNA suitable for production of long reads. Success in generating long read lengths and genome assemblies of plants has been reported using diverse DNA isolation methods, some of which were tailored to the target species and/or required extensive labor. To avoid the need to optimize DNA isolation for each species, we developed a taxa-independent DNA isolation method that is relatively simple and efficient. This method expands on the Oxford Nanopore Technologies high molecular weight genomic DNA protocol from plant leaves and utilizes a conventional cetyl trimethylammonium bromide extraction followed by removal of impurities and short DNA fragments using commercially available kits that yielded robust N50 read lengths and yield on Oxford Nanopore Technologies flow cells.

show abstract

High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing v1

Cited by 39 publications

References 0 publications

Genome sequencing of four culinary herbs reveals terpenoid genes underlying chemodiversity in the Nepetoideae

Genome sequencing of four culinary herbs reveals terpenoid genes underlying chemodiversity in the Nepetoideae

High-quality chromosome-scale assembly of the walnut (Juglans regiaL) reference genome

High molecular weight DNA isolation method from diverse plant species for use with Oxford Nanopore sequencing

Contact Info

Product

Resources

About