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"Clark, Matthew D."
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Nanopore adaptive sampling: a tool for enrichment of low abundance species in metagenomic samples
Adaptive sampling is a method of software-controlled enrichment unique to nanopore sequencing platforms. To test its potential for enrichment of rarer species within metagenomic samples, we create a synthetic mock community and construct sequencing libraries with a range of mean read lengths. Enrichment is up to 13.87-fold for the least abundant species in the longest read length library; factoring in reduced yields from rejecting molecules the calculated efficiency raises this to 4.93-fold. Finally, we introduce a mathematical model of enrichment based on molecule length and relative abundance, whose predictions correlate strongly with mock and complex real-world microbial communities.
Journal Article
Superman : President Luthor
His fame bolstered after helping to rebuild Gotham City after an earthquake, billionaire Lex Luthor decides to run for the highest office in the land, the American presidency.
Book
Plant gene editing through de novo induction of meristems
Plant gene editing is typically performed by delivering reagents such as Cas9 and single guide RNAs to explants in culture. Edited cells are then induced to differentiate into whole plants by exposure to various hormones. The creation of edited plants through tissue culture is often inefficient, time-consuming, works for only limited species and genotypes, and causes unintended changes to the genome and epigenome. Here we report two methods to generate gene-edited dicotyledonous plants through de novo meristem induction. Developmental regulators and gene-editing reagents are delivered to somatic cells of whole plants. This induces meristems that produce shoots with targeted DNA modifications, and gene edits are transmitted to the next generation. The de novo induction of gene-edited meristems sidesteps the need for tissue culture and promises to overcome a bottleneck in plant gene editing.
Methods to induce edited somatic plant cells to form meristems circumvent tissue culture and enable genome editing of a wider set of plant species.
Journal Article
Accelerated cloning of a potato late blight–resistance gene using RenSeq and SMRT sequencing
A method for rapid cloning of plant disease-resistance genes could provide sustainable genetic solutions to crop pests and pathogens in place of agrichemicals.
Global yields of potato and tomato crops have fallen owing to potato late blight disease, which is caused by
Phytophthora infestans
. Although most commercial potato varieties are susceptible to blight, many wild potato relatives show variation for resistance and are therefore a potential source of
Resistance
to
P. infestans
(
Rpi
) genes. Resistance breeding has exploited
Rpi
genes from closely related tuber-bearing potato relatives, but is laborious and slow
1
,
2
,
3
. Here we report that the wild, diploid non-tuber-bearing
Solanum americanum
harbors multiple
Rpi
genes. We combine resistance (R) gene sequence capture (RenSeq)
4
with single-molecule real-time (SMRT) sequencing (SMRT RenSeq) to clone
Rpi-amr3i
. This technology should enable
de novo
assembly of complete nucleotide-binding, leucine-rich repeat receptor (NLR) genes, their regulatory elements and complex multi-NLR loci from uncharacterized germplasm. SMRT RenSeq can be applied to rapidly clone multiple R genes for engineering pathogen-resistant crops.
Journal Article
Grapevine trunk diseases of cold-hardy varieties grown in Northern Midwest vineyards coincide with canker fungi and winter injury
Grapevine trunk diseases make up a disease complex associated with several vascular fungal pathogenic species. Surveys to characterize the composition of grapevine trunk diseases have been conducted for most major grape growing regions of the world. This study presents a similar survey characterizing the fungi associated with grapevine trunk diseases of cold-hardy interspecific hybrid grape varieties grown nearly exclusively in the atypical harsh winter climate of Northern Midwestern United states vineyards. From the 172 samples collected in 2019, 640 isolates obtained by culturing were identified by ITS sequencing and represent 420 sample-unique taxa. From the 420 representative taxa, opportunistic fungi of the order Diaporthales including species of Cytospora and Diaporthe were most frequently identified. Species of Phaeoacremonium , Paraconiothyrium , and Cadophora were also prevalent. In other milder Mediterranean growing climates, species of Xylariales and Botryosphaeriales are often frequently isolated but in this study they were isolated in small numbers. No Phaeomoniellales taxa were isolated. We discuss the possible compounding effects of winter injury, the pathogens isolated, and management strategies. Additionally, difficulties in researching and understanding the grapevine trunk disease complex are discussed.
Journal Article
Rapid MinION profiling of preterm microbiota and antimicrobial-resistant pathogens
The MinION sequencing platform offers near real-time analysis of DNA sequence; this makes the tool attractive for deployment in fieldwork or clinical settings. We used the MinION platform coupled to the NanoOK RT software package to perform shotgun metagenomic sequencing and profile mock communities and faecal samples from healthy and ill preterm infants. Using Nanopore data, we reliably classified a 20-species mock community and captured the diversity of the immature gut microbiota over time and in response to interventions such as probiotic supplementation, antibiotic treatment or episodes of suspected sepsis. We also performed rapid real-time runs to assess gut-associated microbial communities in critically ill and healthy infants, facilitated by NanoOK RT software package, which analysed sequences as they were generated. Our pipeline reliably identified pathogenic bacteria (that is,
Klebsiella pneumoniae
and
Enterobacter cloacae
) and their corresponding antimicrobial resistance gene profiles within as little as 1 h of sequencing. Results were confirmed using pathogen isolation, whole-genome sequencing and antibiotic susceptibility testing, as well as mock communities and clinical samples with known antimicrobial resistance genes. Our results demonstrate that MinION (including cost-effective Flongle flow cells) with NanoOK RT can process metagenomic samples to a rich dataset in < 5 h, which creates a platform for future studies aimed at developing these tools and approaches in clinical settings with a focus on providing tailored patient antimicrobial treatment options.
Shotgun metagenomic sequencing (using the MinION platform) of mock microbial communities and faecal samples from healthy and ill preterm infants can be used to identify pathogens and their antimicrobial resistance gene profiles in real time, indicating the potential for translation into clinical settings.
Journal Article
Comparative analysis of targeted long read sequencing approaches for characterization of a plant’s immune receptor repertoire
Background
The Oxford Nanopore Technologies MinION™ sequencer is a small, portable, low cost device that is accessible to labs of all sizes and attractive for in-the-field sequencing experiments. Selective breeding of crops has led to a reduction in genetic diversity, and wild relatives are a key source of new genetic resistance to pathogens, usually via NLR immune receptor-encoding genes. Recent studies have demonstrated how crop NLR repertoires can be targeted for sequencing on Illumina or PacBio (RenSeq) and the specific gene conveying pathogen resistance identified.
Results
Sequence yields per MinION run are lower than Illumina, making targeted resequencing an efficient approach. While MinION generates long reads similar to PacBio it doesn’t generate the highly accurate multipass consensus reads, which presents downstream bioinformatics challenges. Here we demonstrate how MinION data can be used for RenSeq achieving similar results to the PacBio and how novel NLR gene fusions can be identified via a Nanopore RenSeq pipeline.
Conclusion
The described library preparation and bioinformatics methods should be applicable to other gene families or any targeted long DNA fragment nanopore sequencing project.
Journal Article
A world of opportunities with nanopore sequencing
Oxford Nanopore Technologies’ MinION sequencer was launched in pre-release form in 2014 and represents an exciting new sequencing paradigm. The device offers multi-kilobase reads and a streamed mode of operation that allows processing of reads as they are generated. Crucially, it is an extremely compact device that is powered from the USB port of a laptop computer, enabling it to be taken out of the lab and facilitating previously impossible in-field sequencing experiments to be undertaken. Many of the initial publications concerning the platform focused on provision of tools to access and analyse the new sequence formats and then demonstrating the assembly of microbial genomes. More recently, as throughput and accuracy have increased, it has been possible to begin work involving more complex genomes and metagenomes. With the release of the high-throughput GridION X5 and PromethION platforms, the sequencing of large genomes will become more cost efficient, and enable the leveraging of extremely long (>100 kb) reads for resolution of complex genomic structures. This review provides a brief overview of nanopore sequencing technology, describes the growing range of nanopore bioinformatics tools, and highlights some of the most influential publications that have emerged over the last 2 years. Finally, we look to the future and the potential the platform has to disrupt work in human, microbiome, and plant genomics.
Journal Article