Biology subjects

Delgado-Tejedor, A.

Publications and source records attributed to Delgado-Tejedor, A..

7 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

Single-molecule m6A profiling reveals position-dependent mRNA regulation and non-canonical roles for Ythdf2 in early embryogenesis

The maternal-to-zygotic transition (MZT) requires coordinated clearance and deadenylation of maternally deposited mRNAs, yet the underlying molecular mechanisms remain poorly understood. N6-methyladenosine (m6A) has emerged as a key regulator of maternal mRNA fate, but prior studies have relied on population-averaged short-read methods that cannot resolve modification state, poly(A) tail length, or isoform identity on the same molecule. Here, we employ nanopore direct RNA sequencing on zebrafish embryos across MZT to resolve the interplay between m6A deposition, mRNA clearance, and poly(A) tail length dynamics at single-molecule resolution. We find that 78% of expressed maternal genes harbor m6A-modified isoforms, significantly exceeding prior bulk estimates. Within-isoform comparisons demonstrate that m6A promotes mRNA decay, with CDS m6A contributing more to maternal mRNA clearance than 3-UTR m6A. The positional context of m6A alone is sufficient to determine the temporal regulation of poly(A) tail lengths. CDS m6A constitutively suppresses tail length throughout MZT, while 3'-UTR m6A acquires shortening activity only after zygotic genome activation (ZGA). Transcriptomic analysis of ythdf2 knockout embryos reveals two unrecognized roles. Ythdf2 stabilizes m6A-marked maternal transcripts to set stoichiometry at MZT onset, and is also responsible for maintaining global poly(A) tail homeostasis prior to ZGA through an m6A-independent mechanism. Together, these findings define the single-molecule logic by which m6A modifications shape transcript fate during vertebrate MZT.

developmental biology↗

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

Enhanced detection of RNA modifications and mappability with high-accuracy nanopore RNA basecalling models

In recent years, nanopore direct RNA sequencing (DRS) has established itself as a valuable tool for studying the epitranscriptome, due to its ability to detect multiple modifications within the same full-length native RNA molecules. While RNA modifications can be identified in the form of systematic basecalling errors in DRS datasets, N6-methyladenosine (m6A) modifications produce relatively low errors compared to other RNA modifications, limiting the applicability of this approach to m6A sites that are modified at high stoichiometries. Here, we demonstrate that the use of alternative RNA basecalling models, trained with fully unmodified sequences, increases the error signal of m6A, leading to enhanced detection and improved sensitivity even at low stoichiometries. Moreover, we find that high-accuracy alternative RNA basecalling models can show up to 97% median basecalling accuracy, outperforming currently available RNA basecalling models, which show 91% median basecalling accuracy. Notably, the use of high-accuracy basecalling models is accompanied by a significant increase in the number of mapped reads -especially in shorter RNA fractions- and increased basecalling error signatures at pseudouridine ({Psi}) and N1-methylpseudouridine (m1{Psi}) modified sites. Overall, our work demonstrates that alternative RNA basecalling models can be used to improve the detection of RNA modifications, read mappability and basecalling accuracy in nanopore DRS datasets.

genomics↗

bioRxiv2023

N6-methyladenosine modification is not a general trait of viral RNA genomes

Despite the nuclear localization of the m6A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m6A-modified. However, these findings are mostly based on m6A-seq, an antibody-dependent technique with a high rate of false positives. Here, we addressed the presence of m6A in CHIKV and DENV RNAs. For this, we combined m6A-seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we found no evidence of m6A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m6A machinery did not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection had no effect on the m6A machinerys localization. Our results challenge the prevailing notion that m6A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.

microbiology↗

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

Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore sequencing

RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here, we develop Nanopore 3 end-capture sequencing (Nano3P-seq), a novel method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition and tail length dynamics at per-read resolution. By employing a template switching-based sequencing protocol, Nano3P-seq can sequence any given RNA molecule from its 3 end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq captures a wide diversity of RNA biotypes, providing quantitative estimates of RNA abundance and tail lengths in mRNA, lncRNA, sn/snoRNA, scaRNA, and rRNA molecules. We find that, in addition to mRNA and lncRNA, polyA tails can be identified in 16S mitochondrial rRNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we identify non-A bases within polyA tails of various lengths and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

molecular biology↗