Biology subjects

Wong, J.

Publications and source records attributed to Wong, J..

108 records · Page 6Linked to original sources

bioRxiv2021

MinION barcodes: biodiversity discovery and identification by everyone, for everyone

BackgroundDNA barcodes are a useful tool for discovering, understanding, and monitoring biodiversity which are critical tasks at a time of rapid biodiversity loss. However, widespread adoption of barcodes requires cost-effective and simple barcoding methods. We here present a workflow that satisfies these conditions. It was developed via "innovation through subtraction" and thus requires minimal lab equipment, can be learned within days, reduces the barcode sequencing cost to <10 cents, and allows fast turnaround from specimen to sequence by using the portable, real-time sequencer MinION. ResultsWe describe cost-effective and rapid procedures for barcoding individual specimens with MinION sequencing. We illustrate how tagged amplicons can be obtained and sequenced with the portable, real-time MinION sequencer in many settings (field stations, biodiversity labs, citizen science labs, schools). We also provide amplicon coverage recommendations that are based on several runs of the latest generation of MinION flow cells ("R10.3") which suggest that each run can generate barcodes for >10,000 specimens. Next, we present a novel software, ONTbarcoder, which overcomes the bioinformatics challenges posed by MinION reads. The software is compatible with Windows 10, Macintosh, and Linux, has a graphical user interface (GUI), and can generate thousands of barcodes on a standard laptop within hours based on only two input files (FASTQ, demultiplexing file). We document that MinION barcodes are virtually identical to Sanger and Illumina barcodes for the same specimens (>99.99%) and provide evidence that MinION flow cells and reads have improved rapidly since 2018. ConclusionsWe propose that barcoding with MinION is the way forward for government agencies, universities, museums, and schools because it combines low consumable and capital cost with scalability. Small projects can use the flow cell dongle ("Flongle") while large projects can rely on MinION flow cells that can be stopped and re-used after collecting sufficient data for a given project.

ecology↗

bioRxiv2021

Implant- and anesthesia-related factors affecting threshold intensities for vagus nerve stimulation

Vagus nerve stimulation (VNS) is used as therapy in epilepsy and depression and is tested as a potential treatment for several chronic disorders. Typically, VNS is delivered at increasing stimulus intensity until a response is observed (threshold intensity). Factors that affect threshold intensities for engagement of different fiber types and concomitant physiological responses have not been studied. We determined neural and physiological responses to increasing stimulus intensities of VNS in anesthetized and awake animals, and examined the effect of implant- and anesthesia-related factors on threshold intensities in a rodent model of VNS. In acute and long-term cervical vagus nerve implants (53 and 14 rats, respectively) VNS was delivered under isoflurane, ketamine-xylazine, or awake at different intensities. Stimulus-evoked compound action potentials (eCAPs) were recorded, elicited physiological responses were registered, including changes heart rate (HR), breathing, and blood pressure (BP), and threshold intensities were determined. The intensity that elicits eCAPs ("neural threshold") is significantly lower than what elicits a physiological response ("physiological threshold", PT) (25 A {+/-}1.8 vs. 70 A {+/-}5.2, respectively; Mean {+/-}SEM). Changes in BP occur at the lowest stimulus intensities (80 A {+/-}7), followed by changes in HR (105 A {+/-}8.4) and finally in breathing (310 A {+/-}32.5). PT is lower with than without electrode insulation (60 A {+/-}12, vs. 700 A {+/-}123). PT and electrode impedance are correlated in long-term (r=0.47; p<0.001) but not in acute implants (r=-0.34; p NS); both PT and impedance increase with implant age (Pearson correlation r=0.44; p<0.001 and r=0.64; p<0.001, respectively). PT is lowest when animals are awake (210 A {+/-}33; Mean {+/-}SEM), followed by ketamine-xylazine (630 A {+/-}154), and isoflurane (1075 A {+/-}131). The sequence of physiological responses with increasing VNS intensity is similar in both anesthetized and awake states. Implant age, electrical impedance and the type of anesthesia affect VNS threshold and should be accounted for when determining stimulation dose.

bioengineering↗

bioRxiv2020

Applications of genetic-epigenetic tissue mapping for plasma DNA in prenatal testing, transplantation and oncology

We developed Genetic-Epigenetic Tissue Mapping (GETMap) to determine the tissue composition of plasma DNA carrying genetic variants not present in the constitutional genome through comparing their methylation profiles with relevant tissues. We validated this approach by showing that, in pregnant women, circulating DNA carrying fetal-specific alleles was entirely placenta-derived. In lung-transplant recipients, we showed that, at 72 hours after transplantation, the lung contributed only a median of 17% to the plasma DNA carrying donor-specific alleles and hematopoietic cells contributed a median of 78%. In hepatocellular cancer patients, the liver was identified as the predominant source of plasma DNA carrying tumor-specific mutations. In a pregnant woman with lymphoma, plasma DNA molecules carrying cancer mutations and fetal-specific alleles were accurately shown to be derived from the lymphocytes and placenta, respectively. Analysis of tissue origin for plasma DNA carrying genetic variants is potentially useful for noninvasive prenatal testing, transplantation monitoring and cancer screening.

physiology↗

bioRxiv2020

OmpK36 and TraN facilitate conjugal transfer of the Klebsiella pneumoniae carbapenem resistance plasmid pKpQIL

We investigated the mechanism of conjugal transfer of the endemic Klebsiella pneumoniae carbapenem resistance plasmid, pKpQIL. Transfer efficiency of this plasmid was found to be dependent on the expression of the major outer membrane porin, OmpK36, in recipient cells. We also found that conjugal uptake is reduced in recipients expressing an OmpK36 isoform associated with the globally pervasive K. pneumoniae ST258 clade (OmpK36ST258). This reduction was attributed to a glycine-aspartate insertion in loop 3 of OmpK36ST258, which constricts the pore by 26%. Deletion of finO, which encodes an RNA-binding protein, derepressed transfer of pKpQIL and enabled visualisation of the conjugation pilus and OmpK36-dependent conjugation in real time. While deletion of traN abolished pKpQIL conjugation, substituting traN in pKpQIL with its homologue from R100-1 circumvented OmpK36 dependency. These results suggest that OmpK36 in recipient K. pneumoniae and the pKpQIL-encoded TraN in donor bacteria cooperate to facilitate plasmid transfer. This is the first report since 1998 to suggest a novel recipient cell receptor for IncF plasmid transfer and supports the idea that TraN mediates receptor specificity for plasmids belonging to this incompatibility group.

microbiology↗

bioRxiv2020

A versatile system to introduce clusters of genomic double-strand breaks in large cell populations

In vitro assays for clustered DNA lesions will facilitate the analysis of the mechanisms underlying complex genome rearrangements such as chromothripsis, including the recruitment of repair factors to sites of DNA double-strand breaks. We present a novel method generating localized DNA double-strand breaks using UV-irradiation with photomasks. The size of the damage foci and the spacing between lesions are fully adjustable, making the assay suitable for different cell types and targeted areas. We validated this set-up with genomically stable epithelial cells, normal fibroblasts, pluripotent stem cells and patient-derived primary cultures. Our method does not require a specialized device such as a laser, making it accessible to a broad range of users. Sensitization by BrdU incorporation is not required, which enables analyzing the DNA damage response in post-mitotic cells. Irradiated cells can be cultivated further, followed by time-lapse imaging or used for downstream biochemical analyses, thanks to the high-throughput of the system. Importantly, we showed genome rearrangements in the irradiated cells, providing a proof of principle for the induction of structural variants by localized DNA lesions.

genomics↗

bioRxiv2020

Genetic analysis identifies the Ostrea stentina/aupouria/equestris oyster species complex in Hawai'i, and resolves its lineage as the western Pacific O. equestris

BackgroundExtensive phenotypic plasticity in oysters makes them difficult to identify based on morphology alone, but their identities can be resolved by applying genetic and genomic technologies. In this study, we collected unknown oyster specimens from Hawaiian waters for genetic identification. MethodsWe sequenced two partial gene fragments, mitochondrial 16S ribosomal RNA (16S) and cytochrome c oxidase subunit I (COI), in 48 samples: 27 unidentified oyster specimens collected from two locations on O ahu, 13 known specimens from a hatchery in Hilo, Hawai i Island, and 8 known specimens from Hilo Bay, Hawai i Island. ResultsMolecular data identified approximately 85% of unknown samples as belonging to the Ostrea stentina/aupouria/equestris species complex, a globally distributed group with a history of uncertain and controversial taxonomic status. The remaining unknown samples were the native Dendostrea sandvichensis (G. B. Sowerby II, 1871), and nonnative Crassostrea gigas (Thunberg, 1793), the latter of which is a commercial species that was introduced to Hawai I from multiple sources during the 20th century. Phylogenetic analysis placed Hawai i Ostrea alongside samples from China, Japan, and New Zealand, grouping them within the recently classified western Pacific O. equestris. Until now, four extant species of true oyster have been documented in Hawai i. This study expands the known range of O. equestris by providing the first verification of its occurrence in Hawai i.

genetics↗

bioRxiv2019

Ctcf Haploinsufficiency Mediates Intron Retention in A Tissue-specific Manner

CTCF is a master regulator of gene transcription and chromatin organization with occupancy at thousands of DNA target sites. CTCF is essential for embryonic development and somatic cell viability and has been characterized as a haploinsufficient tumor suppressor. Increasing evidence demonstrates CTCF as a key player in several alternative splicing (AS) regulatory mechanisms, including transcription elongation, regulation of splicing factors, and epigenetic regulation. However, the genome-wide impact of Ctcf dosage on AS has not been investigated. We examined the effect of Ctcf haploinsufficiency on gene expression and AS in multiple tissues from Ctcf hemizygous (Ctcf+/-) mice. Distinct tissue-specific differences in gene expression and AS were observed in Ctcf+/- mice compared to wildtype mice. We observed a surprisingly large number of increased intron retention (IR) events in Ctcf+/- liver and kidney, specifically in genes associated with cytoskeletal organization, splicing and metabolism. This study provides further evidence for Ctcf dose-dependent and tissue-specific regulation of gene expression and AS. Our data provide a strong foundation for elucidating the mechanistic role of CTCF in AS regulation and its biological consequences.

molecular biology↗

bioRxiv2019

Rag1D600A, a novel catalytically inactive RAG mouse model

The RAG complex (RAG1 and RAG2) can bind to recombination signal sequences of antigen receptor loci gene segments and coordinate V(D)J recombination which is the primary method of generating antigen receptor diversity. Previous biochemistry studies discovered RAG1 D600, D708 and E962 residues as essential for catalytic DNA nicking and hairpin forming activity of the RAG complex. Neutralization of each of the acidic residues does not impair DNA binding to recombination signal sequence containing DNA substrates, but cleavage of the substrates is severely compromised. These three acidic residues are thought to comprise a DDE motif that is responsible for binding to a divalent cation that is necessary for cleavage activity. Although a Rag1-/-; RAG1-D708A transgenic mouse model system has been used to study dynamics of RAG activity, transgenic expression may not precisely mimic expression from the endogenous locus. In order to improve upon this model, we created Rag1D600A mice that lack B and T cells and demonstrate a developmental block at the pro-B and DN stages, respectively. Thus, Rag1D600A mice provide a novel mouse model system for studying the poorly understood noncanonical functions of RAG1.

immunology↗

bioRxiv2019

PI3K/AKT inhibition in tumor propagating cells of DLBCL reverses R-CHOP resistance by destabilizing SOX2

Drug resistance is a major obstacle for the success of conventional anticancer therapy, and the development of drug resistance is at least partly attributed to tumor propagating cells (TPCs). Up to one-third of diffuse large B cell lymphoma (DLBCL) patients eventually develop resistance to R-CHOP regimen. We found that the TPC proportion was remarkably increased in resistant germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL subtypes. Elevated SOX2 was the determinant for resistance development, and SOX2 was phosphorylated by activated PI3K/AKT1 signaling, thus preventing ubiquitin-mediated SOX2 degradation. Furthermore, multiple factors, including BCR, integrins, chemokines and FGFR1/2 signaling, regulated PI3K/AKT1 activation. CDK6 in the GCB subtype and FGFR1/2 in the ABC subtype were SOX2 targets in the PI3K/AKT1 pathway. Chemical inhibition of PI3K/AKT1 in both subtypes, CDK6 in the GCB subtype, and FGFR1/2 in the ABC subtype significantly enhanced the susceptibility of resistant cells to CHO treatment. More importantly, PI3K and FGFR1/2 inhibitors but not a CDK6 inhibitor effectively suppressed the tumor growth of R-CHO-resistant DLBCL cells, most likely by converting TPCs to chemo-sensitive differentiated cells. Therefore, this pro-differentiation therapy against TPCs warrants further study in clinical trials for the treatment of resistant DLBCL.

cancer biology↗

bioRxiv2019

Genomic signatures of somatic hybrid vigor due to heterokaryosis in the oomycete pathogen, Bremia lactucae.

Lettuce downy mildew caused by Bremia lactucae is the most important disease of lettuce globally. This oomycete pathogen is highly variable and has rapidly overcome resistance genes and fungicides deployed in attempts to control it. The described high-quality genome assembly of B. lactucae provides the foundation for detailed understanding of this economically important pathogen. The biotrophic nature of B. lactucae coupled with high levels of heterozygosity and the recently expanded repeat content made genome assembly challenging. The combined use of multiple read types, including synthetic long reads, single molecule sequences, and Hi-C, resulted in a high-quality, chromosome-scale, consensus assembly of this diploid organism. Phylogenetic analysis supports polyphyly in the downy mildews consistent with the biotrophic mode of pathogenesis evolving more than once in the Peronosporaceae. Flow cytometry plus resequencing of 30 field isolates as well as sexual offspring and asexual derivatives from multinucleate single sporangia demonstrated a high incidence of heterokaryosis in B. lactucae. Heterokaryons have phenotypic differences and increased fitness compared to homokaryotic derivatives. Consequently, B. lactucae exhibits somatic hybrid vigor and selection should be considered as acting on a population of nuclei within coenocytic mycelia. This provides evolutionary flexibility to the pathogen enabling rapid adaptation to different repertoires of host resistance genes and other challenges. The advantages of asexual persistence of heterokaryons may have been one of the drivers of selection that resulted in the loss of uninucleate zoospores in multiple downy mildews.

genomics↗

bioRxiv2018

How People Initiate Energy Optimization and Converge on Their Optimal Gaits

A central principle in motor control is that the coordination strategies learned by our nervous system are often optimal. Here we combined human experiments with computational reinforcement learning models to study how the nervous system navigates possible movements to arrive at an optimal coordination. Our experiments used robotic exoskeletons to reshape the relationship between how participants walk and how much energy they consume. We found that while some participants used their relatively high natural gait variability to explore the new energetic landscape and spontaneously initiate energy optimization, most participants preferred to exploit their originally preferred, but now suboptimal, gait. We could nevertheless reliably initiate optimization in these exploiters by providing them with the experience of lower cost gaits suggesting that the nervous system benefits from cues about the relevant dimensions along which to re-optimize its coordination. Once optimization was initiated, we found that the nervous system employed a local search process to converge on the new optimum gait over tens of seconds. Once optimization was completed, the nervous system learned to predict this new optimal gait and rapidly returned to it within a few steps if perturbed away. We model this optimization process as reinforcement learning and find behavior that closely matches these experimental observations. We conclude that the nervous system optimizes for energy using a prediction of the optimal gait, and then refines this prediction with the cost of each new walking step.

neuroscience↗

bioRxiv2018

Modeling RNA-binding protein specificity in vivo by precisely registering protein-RNA crosslink sites

RNA-binding proteins (RBPs) regulate post-transcriptional gene expression by recognizing short and degenerate sequence elements in their target transcripts. Despite the expanding list of RBPs with in vivo binding sites mapped genomewide using crosslinking and immunoprecipitation (CLIP), defining precise RBP binding specificity remains challenging. We previously demonstrated that the exact protein-RNA crosslink sites can be mapped using CLIP data at single-nucleotide resolution and observed that crosslinking frequently occurs at specific positions in RBP motifs. Here we have developed a computational method, named mCross, to jointly model RBP binding specificity while precisely registering the crosslinking position in motif sites. We applied mCross to 112 RBPs using ENCODE eCLIP data and validated the reliability of the resulting motifs by genome-wide analysis of allelic binding sites also detected by CLIP. We found that the prototypical SR protein SRSF1 recognizes GGA clusters to regulate splicing in a much larger repertoire of transcripts than previously appreciated.

molecular biology↗

bioRxiv2018

High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate.\n\nIn two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed E. coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains.\n\nThere are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

microbiology↗

bioRxiv2018

Defective DNA damage repair leads to frequent catastrophic genomic events in murine and human tumors

Chromothripsis and chromoanasynthesis are catastrophic events leading to clustered genomic rearrangements. Whole-genome sequencing revealed frequent chromothripsis or chromoanasynthesis (n= 16/26) in brain tumors developing in mice deficient for factors involved in homologous-recombination-repair or non-homologous-end-joining. Catastrophic events were tightly linked to Myc/Mycn amplification, with increased DNA damage and inefficient apoptotic response already observable at early postnatal stages. Inhibition of repair processes and comparison of the mouse tumors with human medulloblastomas (n=68) and glioblastomas (n=32) identified chromothripsis as associated with MYC/MYCN gains and with DNA repair deficiencies, pointing towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements.

cancer biology↗

bioRxiv2017

bioSyntax: Syntax Highlighting For Computational Biology

Computational biology requires the reading and comprehension of biological data files. Plain-text formats such as SAM, VCF, GTF, PDB and FASTA, often contain critical information that is obfuscated by the complexity of the data structures. bioSyntax (http://bioSyntax.org) is a freely available suite of syntax highlighting packages for vim, gedit, Sublime, and less, which aids computational scientists to parse and work with their data more efficiently.

bioinformatics↗

bioRxiv2017

Pathway enrichment analysis of -omics data

Pathway enrichment analysis helps gain mechanistic insight into large gene lists typically resulting from genome scale (-omics) experiments. It identifies biological pathways that are enriched in the gene list more than expected by chance. We explain pathway enrichment analysis and present a practical step-by-step guide to help interpret gene lists resulting from RNA-seq and genome sequencing experiments. The protocol comprises three major steps: define a gene list from genome scale data, determine statistically enriched pathways, and visualize and interpret the results. We focus on differentially expressed genes and mutated cancer genes, however the described principles can be applied to diverse -omics data. The protocol is designed for biologists with no prior bioinformatics training and uses freely available software including g:Profiler, GSEA, Cytoscape and Enrichment Map.

bioinformatics↗

bioRxiv2017

Selenzyme: Enzyme selection tool for pathway design

Synthetic biology applies the principles of engineering to biology in order to create biological functionalities not seen before in nature. One of the most exciting applications of synthetic biology is the design of new organisms with the ability to produce valuable chemicals including pharmaceuticals and biomaterials in a greener; sustainable fashion. Selecting the right enzymes to catalyze each reaction step in order to produce a desired target compound is, however, not trivial. Here, we present Selenzyme, a free online enzyme selection tool for metabolic pathway design. The user is guided through several decision steps in order to shortlist the best candidates for a given pathway step. The tool graphically presents key information about enzymes based on existing databases and tools such as: similarity of sequences and of catalyzed reactions; phylogenetic distance between source organism and intended host species; multiple alignment highlighting conserved regions, predicted catalytic site, and active regions; and relevant properties such as predicted solubility and transmembrane regions. Selenzyme provides bespoke sequence selection for automated workflows in biofoundries. The tool is integrated as part of the pathway design stage into the design-build-test-learn SYNBIOCHEM pipeline. The Selenzyme web server is available at http://selenzyme.synbiochem.co.uk.

bioinformatics↗

bioRxiv2017

Pan-cancer analysis reveals complex tumor-specific alternative polyadenylation

Alternative polyadenylation (APA) of 3 untranslated regions (3 UTRs) has been implicated in cancer development. Earlier reports on APA in cancer primarily focused on 3 UTR length modifications, and the conventional wisdom is that tumor cells preferentially express transcripts with shorter 3 UTRs. Here, we analyzed the APA patterns of 114 genes, a select list of oncogenes and tumor suppressors, in 9,939 tumor and 729 normal tissue samples across 33 cancer types using RNA-Seq data from The Cancer Genome Atlas, and we found that the APA regulation machinery is much more complicated than what was previously thought. We report 77 cases (gene-cancer type pairs) of differential 3 UTR cleavage patterns between normal and tumor tissues, involving 33 genes in 13 cancer types. For 15 genes, the tumor-specific cleavage patterns are recurrent across multiple cancer types. While the cleavage patterns in certain genes indicate apparent trends of 3 UTR shortening in tumor samples, over half of the 77 cases imply 3 UTR length change trends in cancer that are more complex than simple shortening or lengthening. This work extends the current understanding of APA regulation in cancer, and demonstrates how large volumes of RNA-seq data generated for characterizing cancer cohorts can be mined to investigate this process.

genomics↗