Biology subjects

Cozzuto, L.

Publications and source records attributed to Cozzuto, L..

10 recordsLinked to original sources

bioRxiv2026

Systematic benchmarking of commercial workflows for isoform-resolved single-nucleus transcriptomics

Short-read sequencing-based single-cell transcriptomics represents the current gold standard for studying cellular transcriptomes but remains limited in its ability to resolve full-length transcript isoforms and splicing patterns. Long-read single-cell and single-nucleus RNA sequencing (LR sc/snRNA-seq) enables the transcriptome-wide characterization of full-length isoforms at cellular resolution, yet the relative performance of commercially available workflows remains insufficiently explored. Here, using nuclei extracted from a standardized multi-species benchmark sample and Oxford Nanopore Technologies long-read sequencing, we systematically benchmarked four LR snRNA-seq strategies: 10x Genomics 3', 10x Genomics 5', ArgenTag, and Parse Biosciences. Comparing transcriptome features qualitatively and quantitatively, as well as the concordance with matched short-read data and the ability to resolve cellular heterogeneity, we identified substantial method-specific differences in read length and yield, transcript coverage, isoform detection, and recovery of sample-specific biological information, with the 10x Genomics 3' and 5' assays emerging as the most balanced approaches for comprehensive isoform-resolved single-nucleus transcriptomics. Altogether, our study provides a systematic assessment of four commercially available workflows for performing LR snRNA-seq and highlights key methodological trade-offs related to distinct library preparation strategies, thus providing practical guidance for future isoform-resolved transcriptome studies at the single-nucleus level.

genomics↗

bioRxiv2026

AMaNITA: an end-to-end workflow for native tRNA nanopore sequencing data analysis

Transfer RNA (tRNA) molecules serve as essential adapters during protein translation. While direct RNA sequencing (DRS) via Oxford Nanopore Technologies has emerged as a powerful platform for systematic tRNAome profiling, we currently lack a simple and robust statistical framework for nanopore tRNA data analyses. Here, we address this gap by developing AMaNITA (Abundance, Modifications, and Nanopore Intensity Toolbox Application), an end-to-end bioinformatic workflow that enables simplified, robust, and scalable analyses of nanopore native tRNA sequencing datasets. AMaNITA streamlines the entire analytical trajectory: from upstream processing (basecalling, mapping, filtering, batch effect correction) to downstream assessment of differential tRNA abundance and modification stoichiometry. The workflow generates an interactive HTML report for data exploration and analysis, allowing the user to download the source data files and resulting plots. AMaNITA can be executed using Singularity from the command line, without requiring installation of dependencies.

bioinformatics↗

bioRxiv2024

SeqTagger, a rapid and accurate tool to demultiplex direct RNA nanopore sequencing datasets

Nanopore direct RNA sequencing (DRS) enables direct measurement of RNA molecules, including their native RNA modifications, without prior conversion to cDNA. However, commercial methods for molecular barcoding of multiple DRS samples are lacking, and community-driven efforts, such as DeePlexiCon, are not compatible with newer RNA chemistry flowcells and the latest-generation GPU cards. To overcome these limitations, we introduce SeqTagger, a rapid and robust method that can demultiplex direct RNA sequencing datasets with 99% precision and 95% recall. We demonstrate the applicability of SeqTagger in both RNA002/R9.4 and RNA004/RNA chemistries and show its robust performance both for long and short RNA libraries, including custom libraries that do not contain standard poly-(A) tails, such as Nano-tRNAseq libraries. Finally, we demonstrate that increasing the multiplexing up to 96 barcodes yields highly accurate demultiplexing models. SeqTagger can be executed in a standalone manner or through the MasterOfPores NextFlow workflow. The availability of an efficient and simple multiplexing strategy improves the cost-effectiveness of this technology and facilitates the analysis of low-input biological samples.

genomics↗

bioRxiv2023

Nuclear MTHFD2 secures mitosis progression by preserving centromere integrity

Subcellular compartmentalization of metabolic enzymes may elicit specific cellular functions by establishing a unique metabolic environment. Indeed, the nuclear translocation of certain metabolic enzymes is required for epigenetic regulation and gene expression control. Here, we reveal that, in cancer cells, the mitochondrial enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) localizes in the nucleus during the G2-M phase of the cell cycle to secure mitosis progression. Nuclear MTHFD2 interacts with proteins involved in mitosis regulation and centromere stability, including the methyltransferases KMT5A and DNMT3B. Loss of MTHFD2 induces centromere overexpression and severe methylation defects and impedes correct mitosis completion. As a consequence, MTHFD2 deficient cells accumulate chromosomal aberrations arising from chromosome congression and segregation defects. Blocking the catalytic nuclear function of MTHFD2 recapitulates the phenotype observed in MTHFD2 deficient cells, attributing to nuclear MTHFD2 an enzymatic active role in controlling mitosis. Our discovery uncovers a nuclear moonlighting role for the cancer target MTHFD2, and emphasizes that cancer metabolism rewiring may encompass the relocation of metabolic enzymes to alternative subcellular compartments.

cell biology↗

bioRxiv2023

Native RNA nanopore sequencing reveals antibiotic-induced loss of rRNA modifications in the A- and P-sites

The biological relevance and dynamics of mRNA modifications have been extensively studied in the past few years, revealing their key roles in major cellular processes, such as cellular differentiation or sex determination. However, whether rRNA modifications are dynamically regulated, and under which conditions, remains largely unclear. Here, we performed a systematic characterization of bacterial rRNA modification dynamics upon exposure to diverse antibiotics using native RNA nanopore sequencing. To identify significant rRNA modification changes, we developed NanoConsensus, a novel pipeline that integrates the estimates from multiple RNA modification detection algorithms, predicting differentially modified rRNA sites with very low false positive rates and high replicability. We showed that NanoConsensus is robust across RNA modification types, stoichiometries and coverage, and outperforms all individual algorithms tested. Using this approach, we identified multiple rRNA modifications that are lost upon the presence of antibiotics, showing that rRNA modification profiles are altered in an antibiotic-specific manner. We found that significantly altered rRNA modified sites upon antibiotic exposure are located in the vicinity of the A and P-sites of the ribosome, possibly contributing to antibiotic resistance. We then systematically examined whether loss of antibiotic-sensitive rRNA modifications may be sufficient to confer antibiotic resistance, finding that depletion of some rRNA modification enzymes guiding dysregulated rRNA modifications confers increased antibiotic resistance. Altogether, our work reveals that rRNA modification profiles can be rapidly altered in response to environmental exposures, and that nanopore sequencing can accurately identify dysregulated rRNA modifications, contributing to the mechanistic dissection of antibiotic resistance. Moreover, we provide a novel, robust workflow to study rRNA modification dynamics in any species using nanopore sequencing in a scalable and reproducible manner.

microbiology↗

bioRxiv2021

Selective cleavage of ncRNA and antiviral activity by human RNase2/EDN in a macrophage infection model

RNase2, also named the Eosinophil derived Neurotoxin (EDN), is one of the main proteins secreted by the eosinophil secondary granules. RNase2 is also expressed in other leukocyte cells and is the member of the human ribonuclease A family most abundant in macrophages. The protein is endowed with a high ribonucleolytic activity and participates in the host antiviral activity. Although RNase2 displays a broad antiviral activity, it is mostly associated to the targeting of single stranded RNA viruses. To explore RNase2 mechanism of action in antiviral host defence we knocked out RNase2 expression in the THP1 monocyte cell line and characterized the cell response to human Respiratory Syncytial Virus (RSV). We observed that RSV infection induced the RNase2 expression and protein secretion in THP1 macrophage-derived cells, whereas the knockout (KO) of RNase2 resulted in higher RSV burden and reduced cell viability. Next, by means of the cP-RNAseq methodology, which uniquely amplifies the RNA 23cyclic-phosphate-end products released by an endonuclease cleavage, we compared the ncRNA population in native and RNase2-KO cell lines. Among the ncRNAs accumulated in WT versus KO cells, we found mostly tRNA-derived fragments and secondly miRNAs. Analysis of the differential sequence coverage of tRNAs molecules in native and KO cells identified fragments derived from only few parental tRNAs, revealing a predominant cleavage at anticodon loops and secondarily at D-loops. Inspection of cleavage region identified U/C and A, at 5 and 3 sides of cleavage sites respectively (namely RNase B1 and B2 base binding subsites). Likewise, only few selected miRNAs were significantly more abundant in WT versus RNase2-KO cells, with cleavage sites located at the end of stem regions with predominance for pyrimidines at B1 but following an overall less defined nucleotide specificity. Complementarily, by screening of a tRF&tiRNA PCR array we identified an enriched population of tRNA-derived fragments associated to RNase2 expression and RSV infection. The present results confirm the contribution of the protein in macrophage response against virus infection and provide the first evidence of its cleavage selectivity against ncRNA population. A better understanding of the mechanism of action of RNase2 recognition of cellular RNA during the antiviral host defence should pave the basis for the design of novel antiviral drugs.

cell biology↗

bioRxiv2021

A temporally controlled sequence of X-chromosomeinactivation and reactivation defines female mouse in vitro germ cells with meiotic potential

The early mammalian germ cell lineage is characterized by extensive epigenetic reprogramming, which is required for the maturation into functional eggs and sperm. In particular, the epigenome needs to be reset before parental marks can be established and then transmitted to the next generation. In the female germ line, reactivation of the inactive X-chromosome is one of the most prominent epigenetic reprogramming events, and despite its scale involving an entire chromosome affecting hundreds of genes, very little is known about its kinetics and biological function. Here we investigate X-chromosome inactivation and reactivation dynamics by employing a tailor-made in vitro system to visualize the X-status during differentiation of primordial germ cell-like cells (PGCLCs) from female mouse embryonic stem cells (ESCs). We find that the degree of X-inactivation in PGCLCs is moderate when compared to somatic cells and characterized by a large number of genes escaping full inactivation. Nevertheless, PGCLCs that fail to undergo X-inactivation show an abnormal gene expression signature and deficiencies in meiotic entry. Subsequent to X-inactivation we observe gradual step-wise X-reactivation, which is mostly completed by the end of meiotic prophase I. Cells deviating from these progressive kinetics and undergoing X-reactivation too rapidly fail to enter a meiotic trajectory. Our data reveals that a fine-tuned X-inactivation and -reactivation cycle is a critical feature of female germ cell developmental competence towards meiosis and oogenesis

developmental biology↗

bioRxiv2021

ExOrthist: a tool to infer exon orthologies at any evolutionary distance

Several bioinformatic tools have been developed for genome-wide identification of orthologous and paralogous genes among species. However, no existing tool allows the detection of orthologous/paralogous exons. Here, we present ExOrthist, a fully reproducible Nextflow-based software enabling to (i) infer exon homologs and orthogroups, (ii) visualize evolution of exon-intron structures, and (iii) assess conservation of alternative splicing patterns. ExOrthist not only evaluates exon sequence conservation but also considers the surrounding exon-intron context to derive genome-wide multi-species exon homologies at any evolutionary distance. We demonstrate its use in various evolutionary scenarios, from whole genome duplication to convergence of alternative splicing networks.

genomics↗

bioRxiv2020

Determination of primary microRNA processing in clinicalsamples by targeted pri-miR-sequencing

MicroRNA expression is important for gene regulation and deregulated microRNA expression is often observed in disease such as cancer. The processing of primary microRNA transcripts is an important regulatory step in microRNA biogenesis. Due to low expression level and association with chromatin primary microRNAs are challenging to study in clinical samples where input material is limited. Here, we present a high-sensitivity targeted method to determine processing efficiency of several hundred primary microRNAs from total RNA using as little as 500 thousand Illumina HiSeq sequencing reads. We validate the method using RNA from HeLa cells and show the applicability to clinical samples by analyzing RNA from normal liver and hepatocellular carcinoma. We identify 24 primary microRNAs with significant changes in processing efficiency from normal liver to hepatocellular carcinoma, among those the highly expressed miRNA-122 and miRNA-21, demonstrating that differential processing of primary microRNAs is occurring and could be involved in disease. With our method presented here we provide means to study pri-miRNA processing in disease from clinical samples.

molecular biology↗

bioRxiv2019

Parallel and scalable workflow for the analysis of Oxford Nanopore direct RNA sequencing datasets

The direct RNA sequencing platform offered by Oxford Nanopore Technologies allows for direct measurement of RNA molecules without the need of conversion to complementary DNA, fragmentation or amplification. As such, it is virtually capable of detecting any given RNA modification present in the molecule that is being sequenced, as well as provide polyA tail length estimations at the level of individual RNA molecules. Although this technology has been publicly available since 2017, the complexity of the raw Nanopore data, together with the lack of systematic and reproducible pipelines, have greatly hindered the access of this technology to the general user. Here we address this problem by providing a fully benchmarked workflow for the analysis of direct RNA sequencing reads, termed MasterOfPores. The pipeline converts raw current intensities into multiple types of processed data, providing metrics of the quality of the run, quality-filtering, base-calling and mapping. The output of the pipeline can in turn be used to compute per-gene counts, RNA modifications, and prediction of polyA tail length and RNA isoforms. The software is written using the NextFlow framework for parallelization and portability, and relies on Linux containers such as Docker and Singularity for achieving better reproducibility. The MasterOfPores workflow can be executed on any Unix-compatible OS on a computer, cluster or cloud without the need of installing any additional software or dependencies, and is freely available in Github (https://github.com/biocorecrg/master_of_pores). This workflow will significantly simplify the analysis of nanopore direct RNA sequencing data by non-bioinformatics experts, thus boosting the understanding of the (epi)transcriptome with single molecule resolution.

bioinformatics↗