Biology subjects

Piazza, P.

Publications and source records attributed to Piazza, P..

11 recordsLinked to original sources

bioRxiv2026

Multi-omics reveals a monocyte-macrophage-fibroblast axis in post-COVID-19 fibroinflammatory lung remodelling

Post-COVID-19 residual lung abnormalities (RLA) are associated with persistent respiratory symptoms and radiological changes, yet the underlying mechanisms remain unclear. We performed integrated multi-omic profiling of paired bronchoalveolar lavage and blood samples from patients with post-COVID-19 RLA and healthy controls, combining single-cell RNA sequencing, CITE-seq, single-cell T cell receptor sequencing, bronchoalveolar lavage fluid proteomics and functional fibroblast assays. In post-COVID-19 RLA lungs, we identified an increased abundance of profibrotic SPP1hi monocyte-derived alveolar macrophages, arising from an expanded circulating HLA-DRlowCD163+PDE4Dhi classical monocyte progenitor population, supporting a blood-lung myeloid axis. Cell-cell communication modelling positioned macrophages as central hubs of immune-stromal crosstalk, promoting monocyte recruitment with profibrotic priming, and fibroblast activation. Proteomic analysis of bronchoalveolar lavage fluid from post-COVID-19 RLA and idiopathic pulmonary fibrosis, compared with healthy controls, revealed shared and distinct signatures. These alveolar proteins in post-COVID-19 RLA were predominantly attributed to myeloid cells and predicted to engage fibroblast receptors. Bronchoalveolar lavage fluid induced fibroblast proliferation, differentiation and collagen deposition in vitro, with proliferation attenuated by the antifibrotic drug nintedanib. We also identified compartment-specific lymphoid dysregulation, including depletion of mucosal-associated invariant T (MAIT) cells in both the lung and blood, decreased natural killer (NK) cells with oligoclonal T cell expansion in the lung, and expansion of regulatory and cytotoxic T cells in the blood. These findings support a persistent monocyte-macrophage-fibroblast axis linking immune dysregulation to fibroproliferative remodelling after COVID-19 and highlights candidate therapeutic targets for post-viral lung fibrosis. We provide a publicly available atlas (on publication).

systems biology↗

bioRxiv2024

Castanet: a pipeline for rapid analysis of targeted multi-pathogen genomic data

MotivationTarget enrichment strategies generate genomic data from multiple pathogens in a single process, greatly improving sensitivity over metagenomic sequencing and enabling cost-effective, high throughput surveillance and clinical applications. However, uptake by research and clinical laboratories is constrained by an absence of computational tools that are specifically designed for the analysis of multi-pathogen enrichment sequence data. Here we present the Castanet pipeline: an analysis pipeline for end-to-end processing and consensus sequence generation for use with multi-pathogen enrichment sequencing data. Castanet is designed to work with short-read data produced by existing targeted enrichment strategies, but can be readily deployed on any BAM file generated by another methodology. It is packaged with usability features, including graphical interface and installer script. ResultsIn addition to genome reconstruction, Castanet reports method-specific metrics that enable quantification of capture efficiency, estimation of pathogen load, differentiation of low-level positives from contamination, and assessment of sequencing quality. Castanet can be used as a traditional end-to-end pipeline for consensus generation, but its strength lies in the ability to process a flexible, pre-defined set of pathogens of interest directly from multi-pathogen enrichment experiments. In our tests, Castanet consensus sequences were accurate reconstructions of reference sequences, including in instances where multiple strains of the same pathogen were present. Castanet performs effectively on standard laptop computers and can process the entire output of a 96-sample enrichment sequencing run (50M reads) using a single batch process command, in < 2 h. Availability and ImplementationSource code freely available under GPL-3 license at https://github.com/MultipathogenGenomics/castanet, implemented in Python 3.10 and supported in Ubuntu Linux 22.04 and other Bash-like environments. The data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB77004.

bioinformatics↗

bioRxiv2023

Pan-genotypic probe-based enrichment to improve efficiency of Hepatitis B virus sequencing

Hepatitis B Virus (HBV) genome sequencing can be used to provide more complete genetic information at the population and individual level to shed light on the limitations of current interventions, and inform new strategies for elimination. HBV sequencing is challenging due to the partially dsDNA genome, high diversity, low viral loads and presence of large amounts of host genetic material in clinical samples. Here we describe the design and use of a pan-genotypic panel of 74 HBV specific capture-probes and nuclease treatment in improving sequencing efficiency. We processed 20 plasma samples (viral loads 1.98 to 4.07 log10, genotypes A-E) and three positive controls (human total brain RNA and bacteriophage lambda DNA) in triplicate to compare DNAse vs. RNAse vs. no nuclease treatment. We prepared libraries using the Takara Bio SMARTer Stranded Total RNA-Seq Kit v3, split the library in two, enriching half with the custom-designed probe panel and xGen Hybridization and Wash Kit (IDT), the other half was not enriched. Both libraries were sequenced on the NovaSeq6000 platform with 2x150nt paired-end reads. Capture resulted in a 47,970 fold increase in the number of reads mapped to the HBV genome in the "no nuclease" arm (243 HBV reads per million reads sequenced in the capture pool vs. 5x10-3 reads per million in the no-capture pool). Out of 20 samples, only 1 without capture generated HBV reads (viral load 3.89 log10 IU/ml) vs. 19 samples with capture. HBV sequence yield was increased in the capture arm and resulted in 2.30 log10 (95% confidence interval 1.99 - 2.48 log10) increase in HBV reads (per million reads sequenced) per log10 increase in viral load. The proportion of HBV reads increased a median of 12 fold with RNAse treatment. We developed a targeted pan-genotypic sequencing method using a custom panel of biotinylated oligos that increases the sequencing efficacy of HBV. This method will allow us to gain a better insight into HBV diversity.

genomics↗

bioRxiv2022

Viral burdens are associated with age and viral variant in a population-representative study of SARS-CoV-2 that accounts for time-since-infection related sampling bias.

In this study, we evaluated the impact of viral variant, in addition to other variables, on within-host viral burdens, by analysing cycle threshold (Ct) values derived from nose and throat swabs, collected as part of the UK COVID-19 Infection Survey. Because viral burden distributions determined from community survey data can be biased due to the impact of variant epidemiology on the time-since-infection of samples, we developed a method to explicitly adjust observed Ct value distributions to account for the expected bias. Analysing the adjusted Ct values using partial least squares regression, we found that among unvaccinated individuals with no known prior infection, the average Ct value was 0.94 lower among Alpha variant infections, compared those with the predecessor strain, B.1.177. However, among vaccinated individuals, it was 0.34 lower among Delta variant infections, compared to those with the Alpha variant. In addition, the average Ct value decreased by 0.20 for every 10 year age increment of the infected individual. In summary, within-host viral burdens are associated with age, in addition to the interplay of vaccination status and viral variant.

genetics↗

bioRxiv2019

Targeted metagenomic sequencing enhances the identification of pathogens associated with acute infection.

The routine identification of pathogens during infection remains challenging because it relies on multiple modalities such as culture and nucleic acid amplification and tests that tend to be specific for very few of an enormous number of possible infectious agents. Metagenomics promises single-test identification, but shotgun sequencing remains unwieldy and expensive or in many cases insufficiently sensitive to detect the amount of pathogen material in a clinical sample. Here we present the validation and application of Castanet, a method for metagenomic sequencing with enrichment that exploits clinical knowledge to construct a broad panel of relevant organisms for detection at low cost with sensitivity comparable to PCR. Castanet targets both DNA and RNA, works with small sample volumes, and can be implemented in a high-throughput diagnostic setting. We used Castanet to analyse plasma samples from 573 patients from the GAinS sepsis cohort and CSF samples from 243 patients from the ChiMES meningitis cohort that had been evaluated using standard clinical microbiology methods, identifying relevant pathogens in many cases where no pathogen had previously been detected. Castanet is intended for use in defining the distribution of pathogens in samples, diseases and populations, for large-scale clinical studies and for verifying the performance of routine testing regimens. By providing sequence as output, Castanet combines pathogen identification directly with subtyping and phylo-epidemiology.

microbiology↗

bioRxiv2018

Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV)

Advancing interventions to tackle the huge global burden of hepatitis B virus (HBV) infection depends on improved insights into virus epidemiology, transmission, within-host diversity, drug resistance and pathogenesis, all of which can be facilitated by the large-scale generation of full-length virus genome data. Here we describe advances to a protocol to exploit the circular HBV genome structure, using isothermal rolling-circle amplification to enrich for HBV DNA and to generate concatemeric amplicons containing multiple successive copies of the same genome. We show that this product is suitable for Nanopore sequencing as single reads, as well as for generating short-read Illumina sequences. Nanopore reads can be used to implement a straightforward method for error correction that reduces the per-read error rate, by comparing multiple genome copies combined into a single concatemer and by comparing reads generated from plus and minus strands. Thus we can achieve improved consensus sequencing accuracy of 99.7% and resolve intra-sample sequence variants to form whole-genome haplotypes. The combination of isothermal amplification and Nanopore sequencing offers the longer-term potential to develop point-of-care tests for HBV, which could also be adapted for other viruses.

genomics↗

bioRxiv2018

Single cell RNA-seq reveals profound transcriptional similarity between Barretts esophagus and esophageal glands

Barretts esophagus is a precursor of esophageal adenocarcinoma. In this common condition, squamous epithelium in the esophagus is replaced by columnar epithelium in response to acid reflux. Barretts esophagus is highly heterogeneous and its relationships to normal tissues are unclear. We investigated the cellular complexity of Barretts esophagus and the upper gastrointestinal tract using RNA-sequencing of 2895 single cells from multiple biopsies from four patients with Barretts esophagus and two patients without esophageal pathology. We found that uncharacterised cell populations in Barretts esophagus, marked by LEFTY1 and OLFM4, exhibit a profound transcriptional overlap with a subset of esophageal cells, but not with gastric or duodenal cells. Additionally, SPINK4 and ITLN1 mark cells that precede morphologically identifiable goblet cells in colon and Barretts esophagus, potentially aiding the identification of metaplasia. Our findings reveal striking transcriptional relationships between normal tissue populations and cells in a premalignant condition, with implications for clinical practice.

genetics↗

bioRxiv2018

Interferon lambda 4 impacts broadly on hepatitis C virus diversity.

Type III interferons (IFN-{lambda}) are part of the innate immune response to hepatitis C virus (HCV) infection however the specific role of IFN-{lambda}4 and the nature of the viral adaption to this pressure have not been defined. Here we use paired genome-wide human and viral genetic data in 485 patients infected with HCV genotype 3a to explore the role of IFN-{lambda}4 on HCV evolution during chronic infection. We show that genetic variations within the host IFNL4 locus have a broad and systematic impact on HCV amino acid diversity. We also demonstrate that this impact is larger in patients producing a more active form of IFN-{lambda}4 protein compared to the less active form. A similar observation was noted for viral load. We conclude that IFN-{lambda}4 protein is a likely causal agent driving widespread HCV amino acid changes and associated with viral load and possibly other clinical and biological outcomes of HCV infection.

genomics↗

bioRxiv2017

Insights into platypus population structure and history from whole-genome sequencing

The platypus is an egg-laying mammal which, alongside the echidna, occupies a unique place in the mammalian phylogenetic tree. Despite widespread interest in its unusual biology, little is known about its population structure or recent evolutionary history. To provide new insights into the dispersal and demographic history of this iconic species, we sequenced the genomes of 57 platypuses from across the whole species range in eastern mainland Australia and Tasmania. Using a highly-improved reference genome, we called over 6.7M SNPs, providing an informative genetic data set for population analyses. Our results show very strong population structure in the platypus, with our sampling locations corresponding to discrete groupings between which there is no evidence for recent gene flow. Genome-wide data allowed us to establish that 28 of the 57 sampled individuals had at least a third-degree relative amongst other samples from the same river, often taken at different times. Taking advantage of a sampled family quartet, we estimated the de novo mutation rate in the platypus at 7.0x10-9/bp/generation (95% CI 4.1x10-9 - 1.2x10-8/bp/generation). We estimated effective population sizes of ancestral populations and haplotype sharing between current groupings, and found evidence for bottlenecks and long-term population decline in multiple regions, and early divergence between populations in different regions. This study demonstrates the power of whole-genome sequencing for studying natural populations of an evolutionarily important species.

genomics↗

bioRxiv2017

A high throughput screen for active human transposable elements

Transposable elements (TEs) are mobile genetic sequences that randomly propagate within their hosts genome. This mobility has the potential to affect gene transcription and cause disease. However, TEs are technically challenging to identify, which complicates efforts to assess the impact of TE insertions on disease. Here we present a targeted sequencing protocol and computational pipeline to identify polymorphic and novel TE insertions using next-generation sequencing: TE-NGS. The method simultaneously targets the three subfamilies that are responsible for the majority of recent TE activity (L1HS, AluYa5/8, and AluYb8/9) thereby obviating the need for multiple experiments and reducing the amount of input material required. Here we describe the laboratory protocol and detection algorithm, and a benchmark experiment for the reference genome NA12878. We demonstrate a substantial enrichment for on-target fragments, and high sensitivity and precision to both reference and NA12878-specific insertions. We report 17 previously unreported loci for this individual which are supported by orthogonal long-read evidence, and we identify 1,471 polymorphic and novel TEs in 12 additional samples that were previously undocumented in databases of insertion polymorphisms. We anticipate that future applications of TE-NGS alongside exome sequencing of patients with sporadic disease will reduce the number of unresolved cases, and improve estimates of the contribution of TEs to human genetic disease.

genomics↗

bioRxiv2017

A practical solution for preserving single cells for RNA sequencing

The design and implementation of single-cell experiments is often limited by their requirement for fresh starting material. We have adapted a method for histological tissue fixation using dithio-bis(succinimidyl propionate) (DSP), or Lomants Reagent, to stabilise cell samples for single-cell transcriptomic applications. DSP is a reversible cross-linker of free amine groups that has previously been shown to preserve tissue integrity for histology while maintaining RNA integrity and yield in bulk RNA extractions. Although RNA-seq data from DSP-fixed single cells appears to be prone to characteristic artefacts, such as slightly reduced yield of cDNA and a detectable 3 bias in comparison with fresh cells, cell preservation using DSP does not appear to substantially reduce RNA complexity at the gene level. In addition, there is evidence that instantaneous fixation of cells can reduce inter-cell technical variability. The ability of DSP-fixed cells to retain commonly used dyes, such as propidium iodide, enables the tracking of experimental sub-populations and the recording of cell viability at the point of fixation. Preserving cells using DSP will remove several barriers in the staging of single-cell experiments, including the transport of samples and the scheduling of shared equipment for downstream single-cell isolation and processing.

genomics↗