Biology subjects

Vines, J.

Publications and source records attributed to Vines, J..

5 recordsLinked to original sources

bioRxiv2023

Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides

Phosphoinositides are a small family of phospholipids, acting as signalling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organisation and is most commonly done by over-expression of biosensors. However, this leads to perturbations of phosphoinositide signalling and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of all 8 phosphoinositides using a unifying staining approach for fixed cells and tissue, based on recombinant biosensors with self-labelling SNAP tags. These recombinant biosensors are highly specific, and reliably visualise the subcellular distributions of phosphoinositides across scales, ranging from 2D or 3D cell culture to Drosophila tissue. Using stimulated emission depletion (STED) microscopy, we reveal the nanoscale organisation of PI(3)P on endosomes and PI(4)P on the Golgi and confirm the preservation of subcellular membranes. Multiplex staining enables the investigation of phosphoinositide conversions and reveals an unexpected presence of residual PI(3,5)P2 positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualisation of multiple phosphoinositide species in an unprecedented manner.

cell biology↗

bioRxiv2021

Novel canine high-quality metagenome-assembled genomes, prophages, and host-associated plasmids by long-read metagenomics together with Hi-C proximity ligation

Long-read metagenomics facilitates the assembly of high-quality metagenome-assembled genomes (HQ MAGs) out of complex microbiomes. It provides highly contiguous assemblies by spanning repetitive regions, complete ribosomal genes, and mobile genetic elements. Hi-C proximity ligation data bins the long contigs and their associated extra-chromosomal elements to their bacterial host. Here, we characterized a canine fecal sample combining a long-read metagenomics assembly with Hi-C data, and further correcting frameshift errors. We retrieved 27 HQ MAGs and seven medium-quality (MQ) MAGs considering MIMAG criteria. All the long-read canine MAGs improved previous short-read MAGs from public datasets regarding contiguity of the assembly, presence, and completeness of the ribosomal operons, and presence of canonical tRNAs. This trend was also observed when comparing to representative genomes from a pure culture (short-read assemblies). Moreover, Hi-C data linked six potential plasmids to their bacterial hosts. Finally, we identified 51 bacteriophages integrated into their bacterial host, providing novel host information for eight viral clusters that included Gut Phage Database viral genomes. Even though three viral clusters were species-specific, most of them presented a broader host range. In conclusion, long-read metagenomics retrieved long contigs harboring complete assembled ribosomal operons, prophages, and other mobile genetic elements. Hi-C binned together the long contigs into HQ and MQ MAGs, some of them representing closely related species. Long-read metagenomics and Hi-C proximity ligation are likely to become a comprehensive approach to HQ MAGs discovery and assignment of extra-chromosomal elements to their bacterial host.

microbiology↗

bioRxiv2019

Applying Nanopore sequencing to a One-Health scenario for colistin resistance transmission among pigs, cows and the farmer

One-Health studies applying massive-parallel and single-molecule sequencing are a suitable approximation to try to understand how antibiotic resistances flow between the human-animal-environment scenario. Colistin has been withdrawn in human medicine due to its toxicity, limiting its usage as a last-resort treatment option for multidrug-resistant Gram-negative bacteria. However, it is still used orally to treat Enterobacteriaceae infections in veterinary medicine. Since 2015, colistin resistance appeared to be located in mobile genetic elements, raising the concern of the likelihood of transmission by horizontal gene transfer between animals and humans. In this study, 202 faecal samples were collected in a mixed farm from pigs, calves, and the farmer. PCR for the mcr-1 gene was positive for 18 of the isolates, and Nanopore sequencing allowed us to determine the location of the gene, either on the chromosome or in plasmids. Three types of replicons were found within the positive isolates harbouring the mcr-1: IncX4, IncI2, and IncHI2. Four different genetic contexts probably indicate different stages of gene stabilization, either in the chromosome or plasmid, with ISApl1 as the main insertion element flanking the gene. Moreover, 43 other resistance genes were found in our samples, related to more than six different antibiotic families (e.g. aminoglycosides, lincosamides, beta-lactams, macrolides, trimethoprim, phenicols, and sulphonamides). We found resistance genes against colistin and that six antibiotic families together in at least one of the isolates from human, swine, and bovine. Isolate 15B-22 harboured one plasmid with seven resistance genes related to four families of antibiotics other than polymyxins, meaning that there are more chances to maintain colistin resistance even with the withdrawn of colistin. Nanopore long reads allowed us to assemble the DNA elements within the isolates easily and determine the genetic context of the mcr-1 gene. Furthermore, they allowed us to describe and locate more antimicrobial resistance genes to other antibiotic families and antiseptic compounds.

microbiology↗

bioRxiv2018

Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon

BackgroundProfiling microbiome on low biomass samples is challenging for metagenomics since these samples are prone to present DNA from other sources, such as the host or the environment. The usual approach is sequencing specific hypervariable regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. Here, we aim to assess long-amplicon PCR-based approaches for assigning taxonomy at the genus and species level. We use Nanopore sequencing with two different markers: full-length 16S rRNA ([~]1,500 bp) and the whole rrn operon (16S rRNA gene - ITS - 23S rRNA gene; 4,500 bp).\n\nMethodsWe sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities (HM-783D, Bei Resources; D6306, ZymoBIOMICS) and two pools of lowbiomass samples (dog skin). Nanopore sequencing was performed on MinION (Oxford Nanopore Technologies) using 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using WIMP workflow on EPI2ME (ONT) or Minimap2 software with rrn database.\n\nResultsFull-length 16S rRNA and the rrn operon retrieved the microbiota composition from the bacterial isolate, the mock communities and the complex skin samples, even at the genus and species level. For Staphylococcus pseudintermedius isolate, when using EPI2ME, the amplicons were assigned to the correct bacterial species in [~]98% of the cases with rrn operon as the marker, and [~]68% of the cases with 16S rRNA gene respectively. In both skin microbiota samples, we detected many species with an environmental origin. In chin, we found different Pseudomonas species in high abundance, whereas in dorsal skin there were more taxa with lower abundances.\n\nConclusionsBoth full-length 16S rRNA and the rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, rrn operon would be the best choice.

microbiology↗

bioRxiv2017

Using MinION to characterize dog skin microbiota through full-length 16S rRNA gene sequencing approach

The most common strategy to assess microbiota is sequencing specific hypervariable regions of 16S rRNA gene using 2nd generation platforms (such as MiSeq or Ion Torrent PGM). Despite obtaining high-quality reads, many sequences fail to be classified at the genus or species levels due to their short length. This pitfall can be overcome sequencing the full-length 16S rRNA gene (1,500bp) by 3rd generation sequencers.\n\nWe aimed to assess the performance of nanopore sequencing using MinION on characterizing microbiota complex samples. First set-up step was performed using a staggered mock community (HM-783D). Then, we sequenced a pool of several dog skin microbiota samples previously sequenced by Ion Torrent PGM. Sequences obtained for full-length 16S rRNA with degenerated primers retrieved increased richness estimates at high taxonomic level (Bacteria and Archaea) that were missed with short-reads. Besides, we were able to obtain taxonomic assignments down to species level, although it was not always feasible due to: i) incomplete database; ii) primer set chosen; iii) low taxonomic resolution of 16S rRNA gene within some genera; and/or iv) sequencing errors. Nanopore sequencing of the full-length 16S rRNA gene using MinION with 1D sequencing kit allowed us inferring microbiota composition of a complex microbial community to lower taxonomic levels than short-reads from 2nd generation sequencers.

microbiology↗