Biology subjects

Payne, A.

Publications and source records attributed to Payne, A..

26 records · Page 2Linked to original sources

bioRxiv2020

Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems

AbstractMethods for highly multiplexed RNA imaging are limited in spatial resolution, and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to mouse brain, yielding readout of thousands of genes, including splice variants and novel transcripts. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in neurons of the mouse hippocampus, revealing patterns across multiple cell types; layer-specific cell types across mouse visual cortex; and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus ExSeq enables highly multiplexed mapping of RNAs, from nanoscale to system scale. One Sentence SummaryIn situ sequencing of physically expanded specimens enables multiplexed mapping of RNAs at nanoscale, subcellular resolution.

genomics↗

bioRxiv2020

Nanopore adaptive sequencing for mixed samples, whole exome capture and targeted panels.

Nanopore sequencers enable selective sequencing of single molecules in real time by individually reversing the voltage across specific nanopores. Thus DNA molecules can be rejected and replaced with new molecules enabling targeted sequencing to enrich, deplete or achieve specific coverage in a set of reads to address a biological question. We previously demonstrated this method worked using dynamic time warping mapping signal to reference, but required significant compute and did not scale to gigabase references. Using direct base calling with GPU we can now scale to gigabase references. We enrich for specific chromosomes mapping against the human genome and we develop pipelines enriching low abundance organisms from mixed populations without prior knowledge of sample composition. Finally, we enrich panels including 25,600 exon targets from 10,000 human genes and 717 genes implicated in cancer. Using this approach we identify PML-RARA fusions in the NB4 cell line in under 15 hours sequencing. These methods can be used to efficiently screen any target panel of genes without specialised sample preparation using a single computer and suitably powerful GPU.

genomics↗

bioRxiv2019

A multi-tissue transcriptome analysis of human metabolites guides the interpretability of associations based on multi-SNP models for gene expression

There is particular interest in transcriptome-wide association studies (TWAS) - gene-level tests based on multi-SNP predictive models of gene expression - for identifying causal genes at loci associated with complex traits. However, interpretation of TWAS associations may be complicated by divergent effects of model SNPs on trait phenotype and gene expression. We developed an iterative modelling scheme for obtaining multi-SNP models of gene expression and applied this framework to generate expression models for 43 human tissues from the Genotype-Tissues Expression (GTEx) Project. We characterized the performance of single- and multi-SNP TWAS models for identifying causal genes in GWAS data for 46 circulating metabolites. We show that: (a) multi-SNP models captured more variation in expression than the top cis-eQTL (median 2 fold improvement); (b) predicted expression based on multi-SNP models was associated (FDR<0.01) with metabolite levels for 826 unique gene-metabolite pairs, but, after step-wise conditional analyses, 90% were dominated by a single eQTL SNP; (c) amongst the 35% of associations where a SNP in the expression model was a significant cis-eQTL and metabolomic-QTL (met-QTL), 92% demonstrated colocalization between these signals, but interpretation was often complicated by incomplete overlap of QTLs in multi-SNP models; (d) using a \"truth\" set of causal genes at 61 met-QTLs, the sensitivity was high (67%), but the positive predictive value was low, as only 8% of TWAS associations at met-QTLs involved true causal genes. These results guide the interpretation of TWAS and highlight the need for corroborative data to provide confident assignment of causality.

genomics↗

bioRxiv2019

Increased temperatures reduce the vectorial capacity of Aedes mosquitoes for Zika virus

Rapid and significant range expansion of both ZIKV and its Aedes host species has resulted in ZIKV being declared a global health threat. Mean temperatures are projected to increase globally, likely resulting in alterations of the transmission potential of mosquito-borne pathogens. The relationship between temperature and ZIKV transmission has not been well characterised for Aedes aegypti and Aedes albopictus.\n\nTo understand the effect of diurnal temperature range on the vectorial capacity of Aedes aegypti and Aedes albopictus for ZIKV, factors contributing to transmission potential were measured at different temperature regimens. Their longevity and blood feeding rates were assessed, and vector competence was determined following feeding on blood meals with 8.3 log10 PFU/ml ZIKV.\n\nHigher temperature resulted in decreased longevity of Ae. aegypti [Log-rank (Mantel-Cox) Test, Chi-square, df 35.66, 5 P (<0.0001)] and a significant decrease in blood feeding rates across groups [Z score (-5.8478) P (0.0444)]. Temperature had a population and species-specific impact on ZIKV infection rates. Overall, Ae. albopictus reared at the lowest temperature regimen demonstrated the highest vectorial capacity (1.63) and the highest transmission efficiency (57%). Temperature increases decreased vectorial capacity across groups, yet the largest decreases were measured for Ae. aegypti.\n\nThe results of this study suggest that future climate change could significantly impact vector competence, blood feeding behavior and longevity, and therefore decrease the overall vectorial capacity of Aedes mosquitoes. It is also clear that this impact is likely to be both species and population-specific.

microbiology↗

bioRxiv2019

Zika virus infection and temperature significantly impact Aedes albopictus gut microbiota

Elizabethkingia anophelis has been the cause of four outbreaks with significant morbidity and mortality. Its transmission routes remain unknown and no point source of infection has been identified. Here we show that E. anophelis can be found in the saliva of Aedes mosquitoes, suggesting the novel possibility of vector-borne transmission of this bacterium. We additionally characterized diverse microbial communities in Aedes midguts, salivary glands and saliva. To the best of our knowledge, this represents the first description of the microbiome of Aedes saliva. Further, we demonstrate that increased abundance of E. anophelis is associated with decreased susceptibility and replication of Zika virus (ZIKV) in the midgut of Aedes mosquitoes, suggesting a novel transmission barrier for arboviruses transmitted by Aedes mosquitoes. Together, these results demonstrate the complex relationships between the mosquito, the midgut microbial community and arboviruses and offer insights into the epidemiology and control of emerging bacterial and viral pathogens. Author SummaryElizabethkingia anophelis has in the recent past caused outbreaks different parts of the world resulting both in morbidity and mortality. Until now, to the best of our knowledge, no study has been able to demonstrate that this bacterium can be transmitted by mosquitoes. We have demonstrated for the first time that Elizabethkingia anophelis is present in the saliva of both infected and non-infected Aedes mosquitoes. Further, we have shown that it confers an inhibitory effect on Zika virus establishment in the midguts of Aedes mosquitoes. Together, these results potentially display the potential for vector borne transmission of E. anophelis as well as a novel transmission barrier of ZIKV. Lastly, we have for the first time characterized salivary microbes of Aedes mosquitoes necessitating the investigation of the impact of salivary microbes in severity of disease in vertebrate hosts.

microbiology↗

bioRxiv2019

Influence of genetic variants on gene expression in human pancreatic islets - implications for type 2 diabetes

Most signals detected by genome-wide association studies map to non-coding sequence and their tissue-specific effects influence transcriptional regulation. However, many key tissues and cell-types required for appropriate functional inference are absent from large-scale resources such as ENCODE and GTEx. We explored the relationship between genetic variants influencing predisposition to type 2 diabetes (T2D) and related glycemic traits, and human pancreatic islet transcription using RNA-Seq and genotyping data from 420 islet donors. We find: (a) eQTLs have a variable replication rate across the 44 GTEx tissues (<73%), indicating that our study captured islet-specific cis-eQTL signals; (b) islet eQTL signals show marked overlap with islet epigenome annotation, though eQTL effect size is reduced in the stretch enhancers most strongly implicated in GWAS signal location; (c) selective enrichment of islet eQTL overlap with the subset of T2D variants implicated in islet dysfunction; and (d) colocalization between islet eQTLs and variants influencing T2D or related glycemic traits, delivering candidate effector transcripts at 23 loci, including DGKB and TCF7L2. Our findings illustrate the advantages of performing functional and regulatory studies in tissues of greatest disease-relevance while expanding our mechanistic insights into complex traits association loci activity with an expanded list of putative transcripts implicated in T2D development.

genomics↗

bioRxiv2018

Extensive recoding of dengue virus type 2 specifically reduces replication in primate cells without gain-of-function in Aedes aegypti mosquitoes

Dengue virus (DENV), an arthropod-borne (\"arbovirus\") virus causing a range of human maladies ranging from self-limiting dengue fever to the life-threatening dengue shock syndrome, proliferates well in two different taxa of the Animal Kingdom, mosquitoes and primates. Unexpectedly, mosquitoes and primates have distinct preferences when expressing their genes by translation, e.g. members of these taxa show taxonomic group-specific intolerance to certain codon pairs. This is called \"codon pair bias\". By necessity, arboviruses evolved to delicately balance this fundamental difference in their ORFs. Using the mosquito-borne human pathogen DENV we have undone the evolutionarily conserved genomic balance in its ORF sequence and specifically shifted the encoding preference away from primates. However, this recoding of DENV raised concerns of gain-of-function, namely whether recoding could inadvertently increase fitness for replication in the arthropod vector. Using mosquito cell cultures and two strains of Aedes aegypti we did not observe any increase in fitness in DENV2 variants codon pair deoptimized for humans. This ability to disrupt and control an arboviruss host preference has great promise towards developing the next generation of synthetic vaccines not only for DENV but for other emerging arboviral pathogens such as chikungunya virus and Zika virus.

microbiology↗

bioRxiv2018

Whale watching with BulkVis: A graphical viewer for Oxford Nanopore bulk fast5 files.

MotivationThe Oxford Nanopore Technologies (ONT) MinION is used for sequencing a wide variety of sample types with diverse methods of sample extraction. Nanopore sequencers output fast5 files containing signal data subsequently base called to fastq format. Optionally, ONT devices can collect data from all sequencing channels simultaneously in a bulk fast5 file enabling inspection of signal in any channel at any point. We sought to visualise this signal to inspect challenging or difficult to sequence samples, or where flow cell performance is modified by an external agent, such as Read Until.\n\nResultsThe BulkVis tool can load a bulk fast5 file and overlays MinKNOW classifications on the signal trace. Users can navigate to a channel and time or, given a fastq header from a read, jump to its specific position. BulkVis can export regions as Nanopore base caller compatible reads. Using BulkVis, we find long reads can be incorrectly divided by MinKNOW resulting in single DNA molecules being split into two or more reads. The longest seen to date is 2,272,580 bases in length and reported in eleven consecutive reads. We provide helper scripts that identify and reconstruct split reads given a sequencing summary file and alignment to a reference. We note that incorrect read splitting appears to vary according to input sample type and is more common in ultra long read preparations.\n\nAvailabilityThe software is available freely under an MIT license at https://github.com/LooseLab/bulkVis. The software requires python3 to run.

genomics↗