scholarly journals Genomic taxonomic assignment of individual root-knot nematodes

2021 ◽  
Author(s):  
Graham S Sellers ◽  
Daniel C Jeffares ◽  
Bex Lawson ◽  
Tom Prior ◽  
David H Lunt
Keyword(s):  
Plant Pathogens ◽  
Low Cost ◽  
Hybrid Origin ◽  
Taxonomic Identification ◽  
Taxonomic Assignment ◽  
Root Knot Nematodes ◽  
Oxford Nanopore ◽  
Marker Loci ◽  
Long Read ◽  
Individual Root

Root-knot nematodes (RKN; genus Meloidogyne) are polyphagous plant pathogens of great economic importance to agriculturalists globally. These species are small, diverse, and can be challenging for accurate taxonomic identification. Many of the most important crop pests confound analysis with simple genetic marker loci as they are polyploids of likely hybrid origin. Here we take a low-coverage, long-read genome sequencing approach to characterisation of individual root-knot nematodes. We demonstrate library preparation for Oxford Nanopore Technologies Flongle sequencing of low input DNA from individual juveniles and immature females, multiplexing up to twelve samples per flow cell. Taxonomic identification with Kraken 2 (a k-mer-based taxonomic assignment tool) is shown to reliably identify individual nematodes to species level, even within the very closely related Meloidogyne incognita group. Our approach forms a robust, low-cost, and scalable method for accurate RKN species diagnostics.

scholarly journals Identification of individual root-knot nematodes using low coverage long-read sequencing

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0253248
Author(s):  
Graham S. Sellers ◽  
Daniel C. Jeffares ◽  
Bex Lawson ◽  
Tom Prior ◽  
David H. Lunt
Keyword(s):  
Plant Pathogens ◽  
Low Cost ◽  
Hybrid Origin ◽  
Taxonomic Identification ◽  
Taxonomic Assignment ◽  
Root Knot Nematodes ◽  
Marker Loci ◽  
Long Read ◽  
Low Coverage ◽  
Individual Root

Root-knot nematodes (RKN; genus Meloidogyne) are polyphagous plant pathogens of great economic importance to agriculturalists globally. These species are small, diverse, and can be challenging for accurate taxonomic identification. Many of the most important crop pests confound analysis with simple genetic marker loci as they are polyploids of likely hybrid origin. Here we take a low-coverage, long-read genome sequencing approach to characterisation of individual root-knot nematodes. We demonstrate library preparation for Oxford Nanopore Technologies Flongle sequencing of low input DNA from individual juveniles and immature females, multiplexing up to twelve samples per flow cell. Taxonomic identification with Kraken 2 (a k-mer-based taxonomic assignment tool) is shown to reliably identify individual nematodes to species level, even within the very closely related Meloidogyne incognita group. Our approach forms a robust, low-cost, and scalable method for accurate RKN species diagnostics.

scholarly journals Genome Resources for the Ex-type of Phytophthora citricola, and well-authenticated isolates of P. hibernalis, P. nicotianae and P. syringae

2021 ◽  
Author(s):  
Subodh K. Srivastava ◽  
Leandra M. Knight ◽  
Mark K. Nakhla ◽  
Z. Gloria Abad
Keyword(s):  
Plant Pathogens ◽  
High Throughput Sequencing ◽  
Economic Losses ◽  
Biological Research ◽  
Type Specimens ◽  
High Coverage ◽  
Oxford Nanopore ◽  
Long Read ◽  
Basic And Applied Research ◽  
Diagnostic Applications

Phytophthora is one of the most important genera of plant pathogens with many members causing high economic losses world-wide. To build robust molecular identification systems, it is very important to have information from well-authenticated specimens and in preference the ex-type specimens. The reference genomes of well-authenticated specimens form a critical foundation for genetics, biological research, and diagnostic applications. In this study, we describe four draft Phytophthora genomes resources for the Ex-type of P. citricola BL34 (P0716 WPC) (118 contigs for 50 Mb), and well-authenticated specimens of P. syringae BL57G (P10330 WPC) (591 contigs for 75 Mb), P. hibernalis BL41G (P3822 WPC) (404 contigs for 84 Mb), and P. nicotianae BL162 (P6303 WPC) (3984 contigs for 108 Mb) generated with MinION long-read High-Throughput Sequencing (HTS) technology (Oxford Nanopore Technologies, ONT). Using the quality reads we assembled high coverage genomes of P. citricola with 291X coverage and 16,662 annotated genes; P. nicotianae with 205X coverage and 29,271 annotated genes; P. syringae with 76X coverage and 23,331 annotated genes, and P. hibernalis with 42X coverage and 21,762 annotated genes. With the availability of genomes sequences and its annotations, we predict that these draft genomes will be accommodating for various basic and applied research including diagnostics to protect global agriculture.

scholarly journals Consistent ultra-long DNA sequencing with automated slow pipetting

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Trent M. Prall ◽  
Emma K. Neumann ◽  
Julie A. Karl ◽  
Cecilia G. Shortreed ◽  
David A. Baker ◽  
...  
Keyword(s):  
Low Cost ◽  
Volumetric Flow Rate ◽  
Read Length ◽  
Great Length ◽  
Nanopore Sequencing ◽  
Library Preparation ◽  
Average Read Length ◽  
Oxford Nanopore ◽  
Long Read ◽  
Significant Length

Abstract Background Oxford Nanopore Technologies’ instruments can sequence reads of great length. Long reads improve sequence assemblies by unambiguously spanning repetitive elements of the genome. Sequencing reads of significant length requires the preservation of long DNA template molecules through library preparation by pipetting reagents as slowly as possible to minimize shearing. This process is time-consuming and inconsistent at preserving read length as even small changes in volumetric flow rate can result in template shearing. Results We have designed SNAILS (Slow Nucleic Acid Instrument for Long Sequences), a 3D-printable instrument that automates slow pipetting of reagents used in long read library preparation for Oxford Nanopore sequencing. Across six sequencing libraries, SNAILS preserved more reads exceeding 100 kilobases in length and increased its libraries’ average read length over manual slow pipetting. Conclusions SNAILS is a low-cost, easily deployable solution for improving sequencing projects that require reads of significant length. By automating the slow pipetting of library preparation reagents, SNAILS increases the consistency and throughput of long read Nanopore sequencing.

The effect of ultra high-diluted drugs on plant-nematode interaction

2021 ◽  
Vol 14 (2) ◽  
pp. 09-10
Author(s):  
Natalia Rodiuc
Keyword(s):  
Plant Pathogens ◽  
Low Cost ◽  
Genetically Modified Crops ◽  
Root Knot Nematodes ◽  
Physiological Variability ◽  
Resistant Varieties ◽  
Plant Nematode ◽  
High Dilutions ◽  
Model Plant Arabidopsis Thaliana ◽  
Alternative Routes

Among plant pathogens, sedentary endoparasitic nematodes are one of the most damaging pests in global agriculture. Within this group, root-knot nematodes (RKN) Meloidogyne spp. is one of the most specialized phytoparasitic nematodes according to the complexity of the induced feeding sites. These phytopathogenic worms are highly resistant due to the large physiological variability, therefore difficult to fight against. The traditional methods of control, such as the crop rotation and the use of resistant varieties by classical selection are not always effective, time consuming and costly. The modern methods including the application of genetically modified crops (GMO), synthetic pesticides and bionematicides could be dangerous for human health and ecologically hazardous (Thomason, 1987; Malatesta et al. 2002a,b, 2008a,b; Spiroux de Vendômois et al., 2010; Seralini et al., 2012). Therefore, alternative routes must be taken in order to obtain plants resistant to nematodes. The ultra high dilutions (HDs) approach is largely in charge to dissect the infective process. HDs method is eco-friendly, low cost and leaves no residue in the environment. The classical phytopathological methods combined with modern microscopy approaches allowed to characterize the effect of different HD drugs on compatible interaction between model plant Arabidopsis thaliana and root-knot nematodes Meloidogyne incognita.

scholarly journals A Highly Contiguous Genome for the Golden-Fronted Woodpecker (Melanerpes aurifrons) via Hybrid Oxford Nanopore and Short Read Assembly

2020 ◽  
Vol 10 (6) ◽  
pp. 1829-1836 ◽  
Author(s):  
Graham Wiley ◽  
Matthew J. Miller
Keyword(s):  
Molecular Evolution ◽  
Transposable Elements ◽  
Comparative Studies ◽  
Low Cost ◽  
Bird Species ◽  
Hybrid Zones ◽  
Sequencing Data ◽  
Short Read ◽  
Oxford Nanopore ◽  
Long Read

Woodpeckers are found in nearly every part of the world and have been important for studies of biogeography, phylogeography, and macroecology. Woodpecker hybrid zones are often studied to understand the dynamics of introgression between bird species. Notably, woodpeckers are gaining attention for their enriched levels of transposable elements (TEs) relative to most other birds. This enrichment of TEs may have substantial effects on molecular evolution. However, comparative studies of woodpecker genomes are hindered by the fact that no high-contiguity genome exists for any woodpecker species. Using hybrid assembly methods combining long-read Oxford Nanopore and short-read Illumina sequencing data, we generated a highly contiguous genome assembly for the Golden-fronted Woodpecker (Melanerpes aurifrons). The final assembly is 1.31 Gb and comprises 441 contigs plus a full mitochondrial genome. Half of the assembly is represented by 28 contigs (contig L50), each of these contigs is at least 16 Mb in size (contig N50). High recovery (92.6%) of bird-specific BUSCO genes suggests our assembly is both relatively complete and relatively accurate. Over a quarter (25.8%) of the genome consists of repetitive elements, with 287 Mb (21.9%) of those elements assignable to the CR1 superfamily of transposable elements, the highest proportion of CR1 repeats reported for any bird genome to date. Our assembly should improve comparative studies of molecular evolution and genomics in woodpeckers and allies. Additionally, the sequencing and bioinformatic resources used to generate this assembly were relatively low-cost and should provide a direction for development of high-quality genomes for studies of animal biodiversity.

Nematode management through genome editing.

2021 ◽  
pp. 408-413
Author(s):  
Shahid Siddique ◽  
Sebastian Eves-van den Akker
Keyword(s):  
Rna Interference ◽  
Recent Progress ◽  
Plant Pathogens ◽  
Low Cost ◽  
Parasitic Nematodes ◽  
Plant Parasitic Nematodes ◽  
Cyst Nematodes ◽  
Root Knot Nematodes ◽  
Plant Parasitic

Abstract Plant parasitic nematodes are among the most destructive plant pathogens, causing an estimated US$78 billion yield losses globally. Although approximately 3000 species of plant parasitic nematodes have been described, most of the damage is caused by a small group of root-infecting sedentary endoparasitic nematodes that include root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera spp.). Given that previous literature amply reviews the breadth of biotechnological methods for the control of plant parasitic nematodes, this chapter will briefly touch on long-standing biotechnological methods but focus on recent progress in, and long-term promise of, the use of CRISPR technology for introducing targeted modifications into host genomes with the goal of enhancing resistance against plant parasitic nematodes. It is predicted that expanding reverse genetic approaches beyond RNA interference, using low-cost, technically simple and efficient transformation (transient or stable) will be the single most important advance in the field in some years.

scholarly journals Consistent ultra-long DNA sequencing with automated slow pipetting

2020 ◽  
Author(s):  
Trent M. Prall ◽  
Emma K. Neumann ◽  
Julie A. Karl ◽  
Cecilia G. Shortreed ◽  
David A. Baker ◽  
...  
Keyword(s):  
Low Cost ◽  
Volumetric Flow Rate ◽  
Read Length ◽  
Great Length ◽  
Nanopore Sequencing ◽  
Library Preparation ◽  
Average Read Length ◽  
Oxford Nanopore ◽  
Long Read ◽  
Significant Length

AbstractBackgroundOxford Nanopore Technologies’ instruments can sequence reads of great length. Long reads improve sequence assemblies by unambiguously spanning repetitive elements of the genome. Sequencing reads of significant length requires the preservation of long DNA template molecules through library preparation by pipetting reagents as slowly as possible in order to minimizing shearing. This process is time consuming and inconsistent at preserving read length as even small changes in volumetric flow rate can result in template shearing.ResultsWe have designed SNAILS (Slow Nucleic Acid Instrument for Long Sequences), a 3D-printable instrument that automates slow pipetting of reagents used in long read library preparation for Oxford Nanopore sequencing. Across six sequencing libraries, SNAILS preserved more reads exceeding one hundred kilobases in length and increased the average read length of its libraries over manual slow pipetting.ConclusionsSNAILS is a low-cost, easily deployable solution for improving sequencing projects that require reads of significant length. By automating the slow pipetting of library preparation reagents, SNAILS both increases the consistency and throughput of long read Nanopore sequencing.

scholarly journals Genome-guided discovery of natural products through multiplexed low coverage whole-genome sequencing of soil Actinomycetes on Oxford Nanopore Flongle

2021 ◽  
Author(s):  
Rahim Rajwani ◽  
Shannon I Ohlemacher ◽  
Gengxiang Zhao ◽  
Hong-Bing Liu ◽  
Carole A. Bewley
Keyword(s):  
Natural Products ◽  
High Resolution Mass Spectrometry ◽  
Low Cost ◽  
Genome Mining ◽  
Cost Effective ◽  
Gene Clusters ◽  
Mass Spectrometry Data ◽  
Human Pathogens ◽  
Oxford Nanopore ◽  
Long Read

Genome-mining is an important tool for discovery of new natural products; however, the number of publicly available genomes for natural product-rich microbes such as Actinomycetes, relative to human pathogens with smaller genomes, is small. To obtain contiguous DNA assemblies and identify large (ca. 10 to greater than 100 Kb) biosynthetic gene clusters (BGCs) with high-GC (>70%) and -repeat content, it is necessary to use long-read sequencing methods when sequencing Actinomycete genomes. One of the hurdles to long-read sequencing is the higher cost. In the current study, we assessed Flongle, a recently launched platform by Oxford Nanopore Technologies, as a low-cost DNA sequencing option to obtain contiguous DNA assemblies and analyze BGCs. To make the workflow more cost-effective, we multiplexed up to four samples in a single Flongle sequencing experiment while expecting low-sequencing coverage per sample. We hypothesized that contiguous DNA assemblies might enable analysis of BGCs even at low sequencing depth. To assess the value of these assemblies, we collected high-resolution mass-spectrometry data and conducted a multi-omics analysis to connect BGCs to secondary metabolites. In total, we assembled genomes for 20 distinct strains across seven sequencing experiments. In each experiment, 50% of the bases were in reads longer than 10 Kb, which facilitated the assembly of reads into contigs with an average N50 value of 3.5 Mb. The programs antiSMASH and PRISM predicted 629 and 295 BGCs, respectively. We connected BGCs to metabolites for N,N-dimethyl cyclic-ditryptophan, a novel lassopeptide and three known Actinomycete-associated siderophores, namely mirubactin, heterobactin and salinichelin.

scholarly journals Improved assembly of noisy long reads by k-mer validation

2016 ◽  
Author(s):  
A. Bernardo Carvalho ◽  
Eduardo G Dupim ◽  
Gabriel Nassar
Keyword(s):  
Low Cost ◽  
Error Rates ◽  
Read Length ◽  
Celera Assembler ◽  
Short Reads ◽  
Sequencing Errors ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Validation Procedure

Genome assembly depends critically on read length. Two recent technologies, PacBio and Oxford Nanopore, produce read lengths above 20 kb, which yield genome assemblies that are vastly superior to those based on Sanger or short-reads. However, the very high error rates of both technologies (around 15%-20%) makes assembly computationally expensive and imprecise at repeats longer than the read length. Here we show that the efficiency and quality of the assembly of these noisy reads can be significantly improved at a minimal cost, by leveraging on the low error rate and low cost of Illumina short reads. Namely, k-mers from the PacBio raw reads that are not present in the Illumina reads (which account for ~95% of the distinct k-mers) are deemed as sequencing errors and ignored at the seed alignment step. By focusing on ~5% of the k-mers which are error-free, read overlap sensitivity is dramatically increased. Equally important, the validation procedure can be extended to exclude repetitive k-mers, which avoids read miscorrection at repeats and further improve the resulting assemblies. We tested the k-mer validation procedure in one long-read technology (PacBio) and one assembler (MHAP/ Celera Assembler), but is likely to yield analogous improvements with alternative long-read technologies and overlappers, such as Oxford Nanopore and BLASR/DAligner.

scholarly journals Draft Assembly of Phytophthora capsici from Long-Read Sequencing Uncovers Complexity

2019 ◽  
Vol 32 (12) ◽  
pp. 1559-1563 ◽  
Author(s):  
Chenming Cui ◽  
John H. Herlihy ◽  
Aureliano Bombarely ◽  
John M. McDowell ◽  
David C. Haak
Keyword(s):  
Plant Pathogen ◽  
Low Cost ◽  
Phytophthora Capsici ◽  
Broad Host Range ◽  
Evolutionary Analysis ◽  
Short Read ◽  
Oxford Nanopore ◽  
Selective Agents ◽  
Long Read ◽  
Complex Life History

Resolving complex plant pathogen genomes is important for identifying the genomic shifts associated with rapid adaptation to selective agents such as hosts and fungicides, yet assembling these genomes remains challenging and expensive. Phytophthora capsici is an important, globally distributed plant pathogen that exhibits widespread fungicide resistance and a broad host range. As with other pathogenic oomycetes, P. capsici has a complex life history and a complex genome. Here, we leverage Oxford Nanopore Technologies and existing short-read resources to rapidly generate a low-cost, improved assembly. We generated 10 Gbp from a single MinION flow cell resulting in >1.25 million reads with an N50 of 13 kb. The resulting assembly is 95.2 Mbp in 424 scaffolds with an N50 length of 313 kb. This assembly is approximately 30 Mbp bigger than the current reference genome of 64 Mbp. We confirmed this larger genome size using flow cytometry, with an estimated size of 110 Mbp. BUSCO analysis identified 97.4% complete orthologs (19.2% duplicated). Evolutionary analysis supports a recent whole-genome duplication in this group. Our work provides a blueprint for rapidly integrating benchtop long-read sequencing with existing short-read data, to dramatically improve assembly quality and integrity of complex genomes and offer novel insights into pathogen genome function and evolution.

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