Biology subjects

Auxillos, J. Y.

Publications and source records attributed to Auxillos, J. Y..

5 recordsLinked to original sources

bioRxiv2026

Systematic benchmark of demultiplexing workflows for direct RNA sequencing

Direct RNA nanopore sequencing offers the capacity to directly profile native RNAs, enabling detection of diverse epitranscriptomic modifications based on current changes as the RNA passes through a pore. Innovations in chemistry, hardware and bioinformatic tools have increased sequencing capacity and accuracy, creating demand for sequencing multiple samples within individual runs. Two recent solutions enable barcoding and demultiplexing of samples, but independent comparisons of their performance is limited. Here we benchmark WarpDemuX and SeqTagger using orthogonal ground truths derived from species identity and matched IVT spike-ins across four RNA004 flow cells. Both achieve high demultiplexing precision but differ in their trade-offs in precision and retention. SeqTagger provided strong performance at its recommended settings, whilst WarpDemuX performance varied by model, barcode and read quality. IVT-derived score cutoffs allowed dataset-specific calibration and increased WarpDeMux assignment yield, although precision achieved on IVTs was not consistently reproduced in species reads. These results support matched IVTs as internal controls for guiding choice of demultiplexing settings, while demonstrating the need to validate demultiplexing precision in biological RNA populations.

genomics↗

bioRxiv2026

SurpHer: a genetically encoded ratiometric sensor for dynamic extracellular pH imaging

Extracellular pH is a key microenvironmental factor shaping cell physiology and disease, creating a need for quantitative biosensors that can capture dynamic changes in pHe at the surface of individual living cells. Here, we develop a genetically encoded, ratiometric extracellular pH biosensor through systematic screening of a modular library of membrane-display designs that combine SEpHluorin with a pH-stable reference fluorophore. Screening identified a cell-surface-localised mKate2-SEpHluorin construct, named SurpHer, that exhibits dynamic ratiometric responses across the pHe range of 6 - 7.8. SurpHer shows robust membrane localisation and extracellular pH responsiveness across diverse human cell types including HEK293T, PANC-1 and MDA-MB231 cells. Following stable integration in MDA-MB-231 cells, SurpHer enabled time-course imaging of pHe gradients in a microfluidic platform for modelling tumour microenvironments. SurpHer enables real-time interrogation of the pericellular pH environment of tumor cells and, more broadly, provides a strategy to probe microenvironmental pH dynamics across diverse biological contexts.

cell biology↗

bioRxiv2025

An epitranscriptomic map of RNA modifications on integrated HPV18 expressed from HeLa cells

Human papillomavirus (HPV)-driven cancers remain a major global health burden, and understanding how post-transcriptional regulation shapes viral gene expression may inform new therapeutic strategies. Here, we reanalysed independently generated public HeLa datasets to identify candidate RNA modification sites on integrated HPV18 transcripts expressed from the HeLa genome. Using Oxford Nanopore direct RNA sequencing of native RNAs and in vitro transcribed controls, together with GLORI, eTAM-seq and staged 4sU-GLORI datasets, we identified a set of candidate m6A sites on HPV18 early transcripts. m6A levels at a subset of these sites were reduced following perturbation of the m6A pathway, most clearly after METTL3 inhibition, WTAP knockdown or FTO overexpression. We further show that the E6*I-proximal m6A site at position 224 is enriched on unspliced transcripts in direct RNA sequencing, eTAM-seq and staged 4sU-GLORI data. In contrast, we find no convincing evidence for m5C or pseudouridine within the HPV18 regions covered by these datasets. Together, our analyses provide a candidate map of RNA modifications on HeLa-expressed HPV18 early transcripts.

genomics↗

bioRxiv2021

Probing 3'UTRs as modular regulators of gene expression

Genes are commonly abstracted into a coding sequence and cis-regulatory elements (CREs), such as promoters and terminators, and short sequence motifs within these regions. Modern cloning techniques allow easy assembly of synthetic genetic constructs from discrete cis-regulatory modules. However, it is unclear how much the contributions of CREs to gene expression depend on other CREs in the host gene. Using budding yeast, we probe the extent of composability, or independent effects, of distinct CREs. We confirm that the quantitative effect of a terminator on gene expression depends on both promoter and coding sequence. We then explore whether individual cisregulatory motifs within terminator regions display similar context dependence, using transcriptomewide datasets of mRNA decay. To test the extent of composability, we construct reporter genes consisting of combinations of motifs within various terminator contexts, paired with different promoters. Our results show that the effect of a motif on RNA abundance depends both on its host terminator, and also on the associated promoter sequence. This emphasises the need for improved motif inference that includes both local and global context effects, which in turn could aid in the accurate use of CREs for the engineering of synthetic genetic constructs.

systems biology↗

bioRxiv2021

Multiplexed activation in mammalian cells using dFnCas12a-VPR

The adoption of CRISPR systems for the generation of synthetic transcription factors has greatly simplified the process for upregulating endogenous gene expression, with a plethora of applications in cell biology, bioproduction and cell reprogramming. In particular the recently discovered Cas12a systems offer extended potential, as Cas12a is capable of processing its own crRNA array to provide multiple individual crRNAs for subsequent targeting from a single transcript. Here we show the application of dFnCas12a-VPR in mammalian cells, with FnCas12a possessing a shorter PAM sequence than As or Lb variants, enabling denser targeting of genomic loci. We observe that synergistic activation and multiplexing can be achieved using crRNA arrays but also show that crRNAs expressed towards the 5 of 6-crRNA arrays show evidence of enhanced activity. This not only represents a more flexible tool for transcriptional modulation but further expands our understanding of the design capabilities and limitations when considering longer crRNA arrays for multiplexed targeting.

synthetic biology↗