ORCID Profile
0000-0002-7368-1666
Current Organisation
Australian National University
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Publisher: Springer Science and Business Media LLC
Date: 02-11-2017
DOI: 10.1038/S41467-017-01306-9
Abstract: Better mitigation of anthropogenic stressors on marine ecosystems is urgently needed to address increasing bio ersity losses worldwide. We explore opportunities for stressor mitigation using whole-of-systems modelling of ecological resilience, accounting for complex interactions between stressors, their timing and duration, background environmental conditions and biological processes. We then search for ecological windows, times when stressors minimally impact ecological resilience, defined here as risk, recovery and resistance. We show for 28 globally distributed seagrass meadows that stressor scheduling that exploits ecological windows for dredging c aigns can achieve up to a fourfold reduction in recovery time and 35% reduction in extinction risk. Although the timing and length of windows vary among sites to some degree, global trends indicate favourable windows in autumn and winter. Our results demonstrate that resilience is dynamic with respect to space, time and stressors, varying most strongly with: (i) the life history of the seagrass genus and (ii) the duration and timing of the impacting stress.
Publisher: ZappyLab, Inc.
Date: 16-08-2019
DOI: 10.17504/PROTOCOLS.IO.6KAHCSE
Abstract: 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.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 02-2013
DOI: 10.1016/J.PAIN.2012.10.024
Abstract: Human unmyelinated (C) tactile afferents signal the pleasantness of gentle skin stroking on hairy (nonglabrous) skin. After neuronal injury, that same type of touch can elicit unpleasant sensations: tactile allodynia. The prevailing pathophysiological explanation is a spinal cord sensitization, triggered by nerve injury, which enables Aβ afferents to access pain pathways. However, a recent mouse knockout study demonstrates that C-tactile afferents are necessary for allodynia to develop, suggesting a role for not only Aβ but also C-tactile afferent signaling. To examine the contribution of C-tactile afferents to the allodynic condition in humans, we applied the heat/capsaicin model of tactile allodynia in 43 healthy subjects and in 2 sensory neuronopathy patients lacking Aβ afferents. Healthy subjects reported tactile-evoked pain, whereas the patients did not. Instead, patients reported their C-touch percept (faint sensation of pleasant touch) to be significantly weaker in the allodynic zone compared to untreated skin. Functional magnetic resonance imaging in 18 healthy subjects and in 1 scanned patient indicated that stroking in the allodynic and control zones evoked different responses in the primary cortical receiving area for thin fiber signaling, the posterior insular cortex. In addition, reduced activation in the medial prefrontal cortices, key areas for C-tactile hedonic processing, was identified. These findings suggest that dynamic tactile allodynia is associated with reduced C-tactile mediated hedonic touch processing. Nevertheless, because the patients did not develop allodynic pain, this seems dependent on Aβ signaling, at least under these experimental conditions.
Publisher: ZappyLab, Inc.
Date: 14-10-1970
DOI: 10.17504/PROTOCOLS.IO.UNBEVAN
Abstract: Illumina® short-read DNA sequencing has become an integral tool in biology for genome-wide studies. Offering accurate base-pair resolution at the most competitive price, the technology has become widespread. However, the generation of multiplexed DNA libraries remains costly and cumbersome. Here, we present a streamlined cost-conscious protocol for generating multiplexed short read DNA libraries using a transposase from Illumina®. By implementing small volumes that use 1/25th the amount of transposase compared to Illumina® NexteraTM protocols, the cost of library preparation can be significantly reduced, by 1/10th or more. Furthermore, we optimised the protocol to minimise carboxylate bead-based cleanups between steps, further reducing cost, time and DNA input. By developing our own indicies to multiplex nine 96-well plates, up to 864 s les can be placed on a single flow cell. This enables efficient usage of monolithic sequencing platforms that can offer over three terabases of sequencing per flow cell.
Publisher: Springer Science and Business Media LLC
Date: 15-09-2021
DOI: 10.1186/S12915-021-01123-Z
Abstract: Silencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive. We use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici ( Pgt ), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (~250 kb) that are highly erse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally ersified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5′ uracil derived from genes. In contrast, the late wave sRNAs are mainly 22-nt sRNAs with a 5′ adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes. We conclude that rust fungi use an epigenetic silencing pathway that might have similarity with RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.
Publisher: Cold Spring Harbor Laboratory
Date: 20-03-2020
DOI: 10.1101/2020.03.18.996108
Abstract: Austropuccinia psidii , originating in South America, is a globally invasive fungal plant pathogen that causes rust disease on Myrtaceae. Several biotypes are recognized, with the most widely distributed pandemic biotype spreading throughout the Asia-Pacific and Oceania regions over the last decade. Austropuccinia psidii has a broad host range with more than 480 myrtaceous species. Since first detected in Australia in 2010, the pathogen has caused the near extinction of at least three species and negatively affected commercial production of several Myrtaceae. To enable molecular and evolutionary studies into A. psidii pathogenicity, we assembled a highly contiguous genome for the pandemic biotype. With an estimated haploid genome size of just over 1 Gb (gigabases), it is the largest assembled fungal genome to date. The genome has undergone massive expansion via distinct transposable element (TE) bursts. Over 90% of the genome is covered by TEs predominantly belonging to the Gypsy superfamily. These TE bursts have likely been followed by deamination events of methylated cytosines to silence the repetitive elements. This in turn led to the depletion of CpG sites in transposable elements and a very low overall GC content of 33.8%. The overall gene content is highly conserved, when compared to other closely related Pucciniales, yet the intergenic distances are increased by an order of magnitude indicating a general insertion of TEs between genes. Overall, we show how transposable elements shaped the genome evolution of A. psidii and provide a greatly needed resource for strategic approaches to combat disease spread.
Publisher: Wiley
Date: 17-11-2019
DOI: 10.1111/MEC.15287
Publisher: ZappyLab, Inc.
Date: 21-03-2022
DOI: 10.17504/PROTOCOLS.IO.B6I5RCG6
Abstract: With the advancement of long-read sequencing technologies and associated bioinformatics tools, it has now become possible to de novo assemble complex plant genomes with unrivalled contiguity, completeness and correctness. As read lengths can surpass repeat lengths, the ability to assemble genomes de novo has dramatically improved, whereby complex plant genomes of widely variable sizes and repeat content have highly benefited. Despite these improvements, challenges remain in performing de novo assembly, namely in developing a reliable workflow and in tool choice. Here we present a protocol collection of bioinformatic workflows detailing plant genome assembly using Oxford Nanopore Technologies long-reads with a de novo assembler (Canu), syntenic or Hi-C scaffolding, and RNA and/or gene homology-based annotation. We have developed and tested these protocols on multiple plant genomes. Using these protocols with sufficient coverage of long-reads, a highly contiguous, complete, and correct plant genome can be assembled. These genomes can further genomic research into structural variation among groups, and SNP genotyping and association studies among populations.
Publisher: ZappyLab, Inc.
Date: 19-10-2020
DOI: 10.17504/PROTOCOLS.IO.BNJHMCJ6
Abstract: 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.
Publisher: Oxford University Press (OUP)
Date: 27-11-2021
DOI: 10.1093/G3JOURNAL/JKAA015
Abstract: Austropuccinia psidii, originating in South America, is a globally invasive fungal plant pathogen that causes rust disease on Myrtaceae. Several biotypes are recognized, with the most widely distributed pandemic biotype spreading throughout the Asia-Pacific and Oceania regions over the last decade. Austropuccinia psidii has a broad host range with more than 480 myrtaceous species. Since first detected in Australia in 2010, the pathogen has caused the near extinction of at least three species and negatively affected commercial production of several Myrtaceae. To enable molecular and evolutionary studies into A. psidii pathogenicity, we assembled a highly contiguous genome for the pandemic biotype. With an estimated haploid genome size of just over 1 Gb (gigabases), it is the largest assembled fungal genome to date. The genome has undergone massive expansion via distinct transposable element (TE) bursts. Over 90% of the genome is covered by TEs predominantly belonging to the Gypsy superfamily. These TE bursts have likely been followed by deamination events of methylated cytosines to silence the repetitive elements. This in turn led to the depletion of CpG sites in TEs and a very low overall GC content of 33.8%. Compared to other Pucciniales, the intergenic distances are increased by an order of magnitude indicating a general insertion of TEs between genes. Overall, we show how TEs shaped the genome evolution of A. psidii and provide a greatly needed resource for strategic approaches to combat disease spread.
Publisher: ZappyLab, Inc.
Date: 21-07-2021
DOI: 10.17504/PROTOCOLS.IO.BWR8PD9W
Abstract: Extracting pure high-molecular weight DNA from fungi is often 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, making them difficult to remove from solution. Some contaminants may even be undetected by spectrophotometers or fluorometric devices, however they still substantially interferes with long-read DNA sequencing. In attempt to resolve these challenges, a protocol is presented that utilses cetrimonium bromide (CTAB) for lysis and precipitation. CTAB in the lysis solution acts as a cationic detergent to dissolve cell membranes and remove polysaccharides by the formation of CTAB-polysaccharide complexes that co-precipitate during chloroform phase separation. Using CTAB for DNA precipitation under low salt, low ionic conditions, CTAB-DNA complexes form while leaving polysaccharides in solution, resulting in significantly less co-precipitation than alcohol methods. The presented protocol also includes some updates to current strategies and incorporates two different options for DNA clean-up and size selection. Using this protocol, we have been successfully sequencing various fungi with a MinION (Oxford Nanopore Technologies). For wheat stripe rust Puccinia striiformis and wheat leaf rust Puccinia triticina, we performed the presented method followed by size selection with an automated gel purification system, with sequencing yielding 5.88 Gbp with an N50 of 34.79 kb and 4.88 Gbp with an N50 of 27.61 kb, respectively for the two fungi. These fungi are notorious for being recalcitrant and these results have been positive improvements on many other methods. To increase sequencing output for these s les, more work is needed to identify and remove the elusive contaminants. Other fungi, such as sorghum rot fungus Macrophomina phaseolina, we chose an alternative size selection method, precipitation with a polymer and salt solution. This sequencing yielded 13.71 Gbp with an N50 of 21.75 kb, while another strain yielded 9.72 Gbp with an N50 of 43.50 kb.
Publisher: ZappyLab, Inc.
Date: 21-03-2022
DOI: 10.17504/PROTOCOLS.IO.B6HHRB36
Abstract: With the advancement of long-read sequencing technologies and associated bioinformatics tools, it has now become possible to de novo assemble complex plant genomes with unrivalled contiguity, completeness and correctness. As read lengths can surpass repeat lengths, the ability to assemble genomes de novo has dramatically improved, whereby complex plant genomes of widely variable sizes and repeat content have highly benefited. Despite these improvements, challenges remain in performing de novo assembly, namely in developing a reliable workflow and in tool choice. Here we present a protocol collection of bioinformatic workflows detailing plant genome assembly using Oxford Nanopore Technologies long-reads with a de novo assembler (Canu), syntenic or Hi-C scaffolding, and RNA and/or gene homology-based annotation. We have developed and tested these protocols on multiple plant genomes. Using these protocols with sufficient coverage of long-reads, a highly contiguous, complete, and correct plant genome can be assembled. These genomes can further genomic research into structural variation among groups, and SNP genotyping and association studies among populations.
Publisher: Cold Spring Harbor Laboratory
Date: 21-10-2023
Publisher: ZappyLab, Inc.
Date: 15-11-1970
DOI: 10.17504/PROTOCOLS.IO.VMEE43E
Abstract: Evolution has driven genetic ersity 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 and detergent based nuclei extraction to 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, the 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 have been obtaining gigabases of sequencing from a single MinION flow cell, including reads over 100 kb in length. Such ultra-long reads assist the assembly of high quality genomes, from telomere to telomere.
Publisher: Wiley
Date: 03-02-2015
DOI: 10.1002/HBM.22757
Publisher: ZappyLab, Inc.
Date: 28-05-2020
DOI: 10.17504/PROTOCOLS.IO.BGW5JXG6
Abstract: 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 small fragment removal step using Polyethylene Glycol. Using this protocol, we have been successfully sequencing the lentil pathogen Ascochyta lentis with a MinION (Oxford Nanopore Technologies). Sequencing yields have surpassed 13 gigabases with an N50 of approximately 15 kb. To increase sequencing output, more work is needed to identify and remove the elusive contaminants. DNA extraction modified from: www.protocols.io/view/high-molecular-weight-dna-extraction-from-challeng-5isg4ee PEG small fragment elimination after: www.protocols.io/view/size-selective-precipitation-of-dna-using-peg- -7erhjd6
Publisher: ZappyLab, Inc.
Date: 09-11-2019
DOI: 10.17504/PROTOCOLS.IO.87EHZJE
Abstract: 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.
Publisher: ZappyLab, Inc.
Date: 26-02-2021
DOI: 10.17504/PROTOCOLS.IO.BSS7NEHN
Abstract: Rapid advancements in long-read sequencing technologies have transformed sequencing read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles, insects and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale at low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30-50 kb, with reads exceeding 200 kb and outputs ranging from 15-30 Gbp. 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.
Publisher: Future Medicine Ltd
Date: 11-2015
DOI: 10.2217/PMT.15.44
Publisher: Public Library of Science (PLoS)
Date: 15-07-2021
DOI: 10.1371/JOURNAL.PONE.0253830
Abstract: Rapid advancements in long-read sequencing technologies have transformed read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale in low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Oxford Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30–50 kb, with reads exceeding 200 kb and outputs ranging from 15–30 Gbp. This has been routinely achieved with various plant, fungi, animal and bacteria s les.
Publisher: ZappyLab, Inc.
Date: 08-04-2020
DOI: 10.17504/PROTOCOLS.IO.BETDJEI6
Abstract: 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.
Publisher: ZappyLab, Inc.
Date: 14-07-2021
DOI: 10.17504/PROTOCOLS.IO.BWKDPCS6
Abstract: 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, polymer and salt based solutions are used to selectively precipitate DNA based on size, which also appears to further 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.
Publisher: Elsevier BV
Date: 10-2023
Publisher: Cold Spring Harbor Laboratory
Date: 28-09-2023
Publisher: ZappyLab, Inc.
Date: 21-03-2022
DOI: 10.17504/PROTOCOLS.IO.B6HGRB3W
Abstract: With the advancement of long-read sequencing technologies and associated bioinformatics tools, it has now become possible to de novo assemble complex plant genomes with unrivalled contiguity, completeness and correctness. As read lengths can surpass repeat lengths, the ability to assemble genomes de novo has dramatically improved, whereby complex plant genomes of widely variable sizes and repeat content have highly benefited. Despite these improvements, challenges remain in performing de novo assembly, namely in developing a reliable workflow and in tool choice. Here we present a protocol collection of bioinformatic workflows detailing plant genome assembly using Oxford Nanopore Technologies long-reads with a de novo assembler (Canu), syntenic or Hi-C scaffolding, and RNA and/or gene homology-based annotation. We have developed and tested these protocols on multiple plant genomes. Using these protocols with sufficient coverage of long-reads, a highly contiguous, complete, and correct plant genome can be assembled. These genomes can further genomic research into structural variation among groups, and SNP genotyping and association studies among populations.
Publisher: ZappyLab, Inc.
Date: 09-04-2020
DOI: 10.17504/PROTOCOLS.IO.BEUVJEW6
Abstract: The extraction of pure DNA can be challenging due to the presence of sugars, oils and other endogenous chemicals present within biological s les. 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.
Publisher: Springer Science and Business Media LLC
Date: 25-03-2022
DOI: 10.1186/S13059-022-02658-2
Abstract: Most animals and plants have more than one set of chromosomes and package these haplotypes into a single nucleus within each cell. In contrast, many fungal species carry multiple haploid nuclei per cell. Rust fungi are such species with two nuclei (karyons) that contain a full set of haploid chromosomes each. The physical separation of haplotypes in dikaryons means that, unlike in diploids, Hi-C chromatin contacts between haplotypes are false-positive signals. We generate the first chromosome-scale, fully-phased assembly for the dikaryotic leaf rust fungus Puccinia triticina and compare Nanopore MinION and PacBio HiFi sequence-based assemblies. We show that false-positive Hi-C contacts between haplotypes are predominantly caused by phase switches rather than by collapsed regions or Hi-C read mis-mappings. We introduce a method for phasing of dikaryotic genomes into the two haplotypes using Hi-C contact graphs, including a phase switch correction step. In the HiFi assembly, relatively few phase switches occur, and these are predominantly located at haplotig boundaries and can be readily corrected. In contrast, phase switches are widespread throughout the Nanopore assembly. We show that haploid genome read coverage of 30–40 times using HiFi sequencing is required for phasing of the leaf rust genome, with 0.7% heterozygosity, and that HiFi sequencing resolves genomic regions with low heterozygosity that are otherwise collapsed in the Nanopore assembly. This first Hi-C based phasing pipeline for dikaryons and comparison of long-read sequencing technologies will inform future genome assembly and haplotype phasing projects in other non-haploid organisms.
Publisher: ZappyLab, Inc.
Date: 21-03-2022
DOI: 10.17504/PROTOCOLS.IO.B6HFRB3N
Abstract: With the advancement of long-read sequencing technologies and associated bioinformatics tools, it has now become possible to de novo assemble complex plant genomes with unrivalled contiguity, completeness and correctness. As read lengths can surpass repeat lengths, the ability to assemble genomes de novo has dramatically improved, whereby complex plant genomes of widely variable sizes and repeat content have highly benefited. Despite these improvements, challenges remain in performing de novo assembly, namely in developing a reliable workflow and in tool choice. Here we present a protocol collection of bioinformatic workflows detailing plant genome assembly using Oxford Nanopore Technologies long-reads with a de novo assembler (Canu), syntenic or Hi-C scaffolding, and RNA and/or gene homology-based annotation. We have developed and tested these protocols on multiple plant genomes. Using these protocols with sufficient coverage of long-reads, a highly contiguous, complete, and correct plant genome can be assembled. These genomes can further genomic research into structural variation among groups, and SNP genotyping and association studies among populations.
Publisher: Elsevier BV
Date: 04-2018
DOI: 10.1016/J.MCP.2018.03.001
Abstract: Early diagnosis of leptospirosis is important for ensuring better clinical management and achieving better outcomes. Currently, serological assays suffer from inconsistent performance and are less useful for early diagnosis of leptospirosis. As an alternative, qPCR is more sensitive, specific and able to detect the presence of leptospiral DNA during the acute phase of the infection. Meanwhile, most molecular assays do not detect the non-pathogenic group of Leptospira, even though these groups may also infect humans, although less frequently and less severely. A set of primers and probe targeting rrs genes of 22 Leptospira spp. were designed and evaluated on 31 Leptospira isolates, 41 other organisms and 65 clinical s les from suspected patients. The developed assay was able to detect as low as 20 fg Leptospira DNA per reaction (equivalent to approximately 4 copies) and showed high specificity against the tested leptospiral strains. No cross lification was observed with the other organisms. During the evaluation of the confirmed clinical specimens, the developed assay was able to correctly identify all positive s les (n = 10/10). One lification was observed in a negative s le (n = 1/55). The sequencing of the PCR product of the discordant s le revealed that the sequences were similar to those of L. interrogans and L. kirschneri. The findings suggest that the developed Taqman qPCR assay is sensitive, specific and has potential to be applied in a larger subsequent study.
Publisher: Oxford University Press (OUP)
Date: 20-07-2012
Abstract: Brain changes associated with the loss of a sensory modality such as vision and audition have previously been reported. Here, we examined the effect of loss of discriminative touch and proprioception on cortical thickness and functional connectivity. We performed structural magnetic resonance imaging and resting-state functional magnetic resonance imaging scans on a 60-year-old female who at age 31 suffered a selective loss of large-diameter myelinated primary afferents and, therefore, relies mainly on her intact thin-fiber senses (temperature, pain, itch, and C-fiber touch) and vision to negotiate her environment. The patient showed widespread cortical thinning compared with 12 age-matched female controls. In contrast, her right anterior insula was significantly thick. Seed-based resting-state analysis revealed that her right anterior insula had increased connectivity to bilateral posterior insula. A separate independent component analysis revealed the increased connectivity between the insula and visual cortex in the patient. As the insula is an important processing area for temperature and C-fiber tactile information, the increased intrainsular and insular-visual functional connectivity could be related to the patient's use of C-fiber (gentle) touch and temperature information in conjunction with visual information to navigate her environment. We, thus, demonstrated plasticity in networks involving the insular cortex following denervation of large-diameter somatosensory afferents.
Publisher: ZappyLab, Inc.
Date: 21-03-2022
DOI: 10.17504/PROTOCOLS.IO.81WGB6ZK3LPK/V1
Abstract: With the advancement of long-read sequencing technologies and associated bioinformatics tools, it has now become possible to de novo assemble complex plant genomes with unrivalled contiguity, completeness and correctness. As read lengths can surpass repeat lengths, the ability to assemble genomes de novo has dramatically improved, whereby complex plant genomes of widely variable sizes and repeat content have highly benefited. Despite these improvements, challenges remain in performing de novo assembly, namely in developing a reliable workflow and in tool choice. Here we present a protocol collection of bioinformatic workflows detailing plant genome assembly using Oxford Nanopore Technologies long-reads with a de novo assembler (Canu), syntenic or Hi-C scaffolding, and RNA and/or gene homology-based annotation. We have developed and tested these protocols on multiple plant genomes. Using these protocols with sufficient coverage of long-reads, a highly contiguous, complete, and correct plant genome can be assembled. These genomes can further genomic research into structural variation among groups, and SNP genotyping and association studies among populations.
Publisher: ZappyLab, Inc.
Date: 21-03-2022
DOI: 10.17504/PROTOCOLS.IO.B6HIRB4E
Abstract: With the advancement of long-read sequencing technologies and associated bioinformatics tools, it has now become possible to de novo assemble complex plant genomes with unrivalled contiguity, completeness and correctness. As read lengths can surpass repeat lengths, the ability to assemble genomes de novo has dramatically improved, whereby complex plant genomes of widely variable sizes and repeat content have highly benefited. Despite these improvements, challenges remain in performing de novo assembly, namely in developing a reliable workflow and in tool choice. Here we present a protocol collection of bioinformatic workflows detailing plant genome assembly using Oxford Nanopore Technologies long-reads with a de novo assembler (Canu), syntenic or Hi-C scaffolding, and RNA and/or gene homology-based annotation. We have developed and tested these protocols on multiple plant genomes. Using these protocols with sufficient coverage of long-reads, a highly contiguous, complete, and correct plant genome can be assembled. These genomes can further genomic research into structural variation among groups, and SNP genotyping and association studies among populations.
Publisher: Springer Science and Business Media LLC
Date: 14-12-2022
DOI: 10.1186/S13007-022-00971-2
Abstract: Long-read sequencing platforms offered by Oxford Nanopore Technologies (ONT) allow native DNA containing epigenetic modifications to be directly sequenced, but can be limited by lower per-base accuracies. A key step post-sequencing is basecalling, the process of converting raw electrical signals produced by the sequencing device into nucleotide sequences. This is challenging as current basecallers are primarily based on mixtures of model species for training. Here we utilise both ONT PromethION and higher accuracy PacBio Sequel II HiFi sequencing on two plants, Phebalium stellatum and Xanthorrhoea johnsonii , to train species-specific basecaller models with the aim of improving per-base accuracy. We investigate sequencing accuracies achieved by ONT basecallers and assess accuracy gains by training single-species and species-specific basecaller models. We also evaluate accuracy gains from ONT’s improved flowcells (R10.4, FLO-PRO112) and sequencing kits (SQK-LSK112). For the truth dataset for both model training and accuracy assessment, we developed highly accurate, contiguous diploid reference genomes with PacBio Sequel II HiFi reads. Basecalling with ONT Guppy 5 and 6 super-accurate gave almost identical results, attaining read accuracies of 91.96% and 94.15%. Guppy’s plant-specific model gave highly mixed results, attaining read accuracies of 91.47% and 96.18%. Species-specific basecalling models improved read accuracy, attaining 93.24% and 95.16% read accuracies. R10.4 sequencing kits also improve sequencing accuracy, attaining read accuracies of 95.46% (super-accurate) and 96.87% (species-specific). The use of a single mixed-species basecaller model, such as ONT Guppy super-accurate, may be reducing the accuracy of nanopore sequencing, due to conflicting genome biology within the training dataset and study species. Training of single-species and genome-specific basecaller models improves read accuracy. Studies that aim to do large-scale long-read genotyping would primarily benefit from training their own basecalling models. Such studies could use sequencing accuracy gains and improving bioinformatics tools to improve study outcomes.
Publisher: ZappyLab, Inc.
Date: 04-02-2021
DOI: 10.17504/PROTOCOLS.IO.14EGNX27ZL5D/V2
Abstract: Massively parallel, second-generation short-read DNA sequencing has become an integral tool in biology for genomic studies. Offering highly accurate base-pair resolution at the most competitive price, the technology has become widespread. However, high-throughput generation of multiplexed DNA libraries can be costly and cumbersome. Here, we present a cost-conscious protocol for generating multiplexed short-read DNA libraries using a bead-linked transposome from Illumina. By preparing libraries in high-throughput with small reaction volumes that use 1/50th the amount of transposome compared to Illumina DNA Prep tagmentation protocols, the cost per library can be substantially reduced, by approximately 1/20th. Furthermore, we optimised the protocol to minimise magnetic bead-based clean-ups between steps, further reducing cost, time and DNA input requirements. By developing our own dual index primers to multiplex nine 96-well microplates, up to 864 s les can be placed on a single flow cell. This enables efficient usage of large-scale sequencing platforms, such as the Illumina NovaSeq 6000, which offers up to three terabases of sequencing per S4 flow cell.
Publisher: Wiley
Date: 21-07-2022
DOI: 10.1111/MEC.16608
Abstract: Synteny, the ordering of sequences within homologous chromosomes, must be maintained within the genomes of sexually reproducing species for the sharing of alleles and production of viable, reproducing offspring. However, when the genomes of closely related species are compared, a loss of synteny is often observed. Unequal homologous recombination is the primary mechanism behind synteny loss, occurring more often in transposon rich regions, and resulting in the formation of chromosomal rearrangements. To examine patterns of synteny among three closely related, interbreeding, and wild Eucalyptus species, we assembled their genomes using long‐read DNA sequencing and de novo assembly. We identify syntenic and rearranged regions between these genomes and estimate that ~48% of our genomes remain syntenic while ~36% is rearranged. We observed that rearrangements highly fragment microsynteny. Our results suggest that synteny between these species is primarily lost through small‐scale rearrangements, not through sequence loss, gain, or sequence ergence. Further examination of identified rearrangements suggests that rearrangements may be altering the phenotypes of Eucalyptus species. Our study also underscores that the use of single reference genomes in genomic variation studies could lead to reference bias, especially given the scale at which we show potentially adaptive loci have highly erged, deleted, duplicated and/or rearranged. This study provides an unbiased framework to look at potential speciation and adaptive loci among a rapidly radiating foundation species of woodland trees that are free from selective breeding seen in most crop species.
Publisher: ZappyLab, Inc.
Date: 21-03-2022
DOI: 10.17504/PROTOCOLS.IO.KQDG36D87G25/V2
Abstract: With the advancement of long-read sequencing technologies and associated bioinformatics tools, it has now become possible to de novo assemble complex plant genomes with unrivalled contiguity, completeness and correctness. As read lengths can surpass repeat lengths, the ability to assemble genomes de novo has dramatically improved, whereby complex plant genomes of widely variable sizes and repeat content have highly benefited. Despite these improvements, challenges remain in performing de novo assembly, namely in developing a reliable workflow and in tool choice. Here we present a protocol collection of bioinformatic workflows detailing plant genome assembly using Oxford Nanopore Technologies long-reads with a de novo assembler (Canu), syntenic or Hi-C scaffolding, and RNA and/or gene homology-based annotation. We have developed and tested these protocols on multiple plant genomes. Using these protocols with sufficient coverage of long-reads, a highly contiguous, complete, and correct plant genome can be assembled. These genomes can further genomic research into structural variation among groups, and SNP genotyping and association studies among populations.
Publisher: ZappyLab, Inc.
Date: 21-03-2022
DOI: 10.17504/PROTOCOLS.IO.E6NVW578ZVMK/V2
Abstract: With the advancement of long-read sequencing technologies and associated bioinformatics tools, it has now become possible to de novo assemble complex plant genomes with unrivalled contiguity, completeness and correctness. As read lengths can surpass repeat lengths, the ability to assemble genomes de novo has dramatically improved, whereby complex plant genomes of widely variable sizes and repeat content have highly benefited. Despite these improvements, challenges remain in performing de novo assembly, namely in developing a reliable workflow and in tool choice. Here we present a protocol collection of bioinformatic workflows detailing plant genome assembly using Oxford Nanopore Technologies long-reads with a de novo assembler (Canu), syntenic or Hi-C scaffolding, and RNA and/or gene homology-based annotation. We have developed and tested these protocols on multiple plant genomes. Using these protocols with sufficient coverage of long-reads, a highly contiguous, complete, and correct plant genome can be assembled. These genomes can further genomic research into structural variation among groups, and SNP genotyping and association studies among populations.
Publisher: ZappyLab, Inc.
Date: 21-03-2022
DOI: 10.17504/PROTOCOLS.IO.DM6GPBR1JLZP/V2
Abstract: With the advancement of long-read sequencing technologies and associated bioinformatics tools, it has now become possible to de novo assemble complex plant genomes with unrivalled contiguity, completeness and correctness. As read lengths can surpass repeat lengths, the ability to assemble genomes de novo has dramatically improved, whereby complex plant genomes of widely variable sizes and repeat content have highly benefited. Despite these improvements, challenges remain in performing de novo assembly, namely in developing a reliable workflow and in tool choice. Here we present a protocol collection of bioinformatic workflows detailing plant genome assembly using Oxford Nanopore Technologies long-reads with a de novo assembler (Canu), syntenic or Hi-C scaffolding, and RNA and/or gene homology-based annotation. We have developed and tested these protocols on multiple plant genomes. Using these protocols with sufficient coverage of long-reads, a highly contiguous, complete, and correct plant genome can be assembled. These genomes can further genomic research into structural variation among groups, and SNP genotyping and association studies among populations.
Publisher: Cold Spring Harbor Laboratory
Date: 20-04-2023
DOI: 10.1101/2023.04.19.537464
Abstract: Genomes have a highly organised architecture (non-random organisation of functional and non-functional genetic elements within chromosomes) that is essential for many biological functions, particularly, gene expression and reproduction. Despite the need to conserve genome architecture, a surprisingly high level of structural variation has been observed within species. As species separate and erge, genome architecture also erges, becoming increasingly poorly conserved as ergence time increases. However, within plant genomes, the processes of genome architecture ergence are not well described. Here we use long-read sequencing and de novo assembly of 33 phylogenetically erse, wild and naturally evolving Eucalyptus species, covering 1-50 million years of erging genome evolution to measure genome architectural conservation and describe architectural ergence. The investigation of these genomes revealed that following lineage ergence genome architecture is highly fragmented by rearrangements. As genomes continue to erge, the accumulation of mutations and subsequent ergence beyond recognition of rearrangements becomes the primary driver of genome ergence. The loss of syntenic regions also contribute to genome ergence, but at a slower pace than rearrangements. We hypothesise that duplications and translocations are potentially the greatest contributors to genome ergence.
Publisher: Public Library of Science (PLoS)
Date: 27-01-2023
DOI: 10.1371/JOURNAL.PONE.0280004
Abstract: Massively parallel, second-generation short-read DNA sequencing has become an integral tool in biology for genomic studies. Offering highly accurate base-pair resolution at the most competitive price, the technology has become widespread. However, high-throughput generation of multiplexed DNA libraries can be costly and cumbersome. Here, we present a cost-conscious protocol for generating multiplexed short-read DNA libraries using a bead-linked transposome from Illumina. We prepare libraries in high-throughput with small reaction volumes that use 1/50 th the amount of transposome compared to Illumina DNA Prep tagmentation protocols. By reducing transposome usage and optimising the protocol to circumvent magnetic bead-based clean-ups between steps, we reduce costs, labour time and DNA input requirements. Developing our own dual index primers further reduced costs and enables up to nine 96-well microplate combinations. This facilitates efficient usage of large-scale sequencing platforms, such as the Illumina NovaSeq 6000, which offers up to three terabases of sequencing per S4 flow cell. The protocol presented substantially reduces the cost per library by approximately 1/20 th compared to conventional Illumina methods.
Publisher: ZappyLab, Inc.
Date: 22-08-2018
DOI: 10.17504/PROTOCOLS.IO.SSVEEE6
Abstract: Evolution has driven genetic ersity 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 and detergent based nuclei extraction to 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, the 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 have been obtaining gigabases of sequencing from a single MinION flowcell, including reads over 100 kb in length. Such ultra-long reads assist the assembly of high quality genomes, from telomere to telomere.
Publisher: Wiley
Date: 16-09-2020
DOI: 10.1111/MEC.15614
Publisher: ZappyLab, Inc.
Date: 22-05-2019
DOI: 10.17504/PROTOCOLS.IO.28BGHSN
Abstract: Evolution has driven genetic ersity 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.
Publisher: ZappyLab, Inc.
Date: 16-07-2019
DOI: 10.17504/PROTOCOLS.IO.5ISG4EE
Abstract: 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.
Publisher: ZappyLab, Inc.
Date: 21-03-2022
DOI: 10.17504/PROTOCOLS.IO.EWOV14BZ7VR2/V2
Abstract: With the advancement of long-read sequencing technologies and associated bioinformatics tools, it has now become possible to de novo assemble complex plant genomes with unrivalled contiguity, completeness and correctness. As read lengths can surpass repeat lengths, the ability to assemble genomes de novo has dramatically improved, whereby complex plant genomes of widely variable sizes and repeat content have highly benefited. Despite these improvements, challenges remain in performing de novo assembly, namely in developing a reliable workflow and in tool choice. Here we present a protocol collection of bioinformatic workflows detailing plant genome assembly using Oxford Nanopore Technologies long-reads with a de novo assembler (Canu), syntenic or Hi-C scaffolding, and RNA and/or gene homology-based annotation. We have developed and tested these protocols on multiple plant genomes. Using these protocols with sufficient coverage of long-reads, a highly contiguous, complete, and correct plant genome can be assembled. These genomes can further genomic research into structural variation among groups, and SNP genotyping and association studies among populations.
Publisher: Cold Spring Harbor Laboratory
Date: 28-04-2023
DOI: 10.1101/2023.04.27.538497
Abstract: The coastal wetland tree species Melaleuca quinquenervia (Cav.) S.T.Blake (Myrtaceae), commonly named the broad-leaved paperbark, is a foundation species in eastern Australia, Indonesia, Papua New Guinea, and New Caledonia. The species has been widely grown as an ornamental, becoming invasive in areas such as Florida in the United States. Long-lived trees must respond to a wide range pests and pathogens throughout their lifespan, and immune receptors encoded by the nucleotide- binding domain and leucine-rich repeat containing (NLR) gene family play a key role in plant stress responses. Expansion of this gene family is driven largely by tandem duplication, resulting in a clustering arrangement on chromosomes. Due to this clustering and their highly repetitive domain structure, comprehensive annotation of NLR encoding genes within genomes has been difficult. Additionally, as many genomes are still presented in their haploid, collapsed state, the full allelic ersity of the NLR gene family has not been widely published for outcrossing tree species. We assembled a chromosome-level pseudo-phased genome for M . quinquenervia and describe the full allelic ersity of plant NLRs using the novel FindPlantNLRs pipeline. Analysis reveals variation in the number of NLR genes on each haplotype, differences in clusters and in the types and numbers of novel integrated domains. We anticipate that the high quality of the genome for M. quinquenervia will provide a new framework for functional and evolutionary studies into this important tree species. Our results indicate a likely role for maintenance of NLR allelic ersity to enable response to environmental stress, and we suggest that this allelic ersity may be even more important for long-lived plants.
Location: Australia
No related grants have been discovered for Ashley Jones.