Biology subjects

Zeglinski, K.

Publications and source records attributed to Zeglinski, K..

6 recordsLinked to original sources

bioRxiv2025

alpseq: an open-source workflow to turbocharge nanobody discovery with high-throughput sequencing

Nanobodies have emerged as promising tools for many biotechnological applications due to their small size, high stability, and remarkable binding specificity. Next-Generation Sequencing (NGS) enables deep profiling of large nanobody libraries and panning campaigns, however the scale and diversity of nanobody NGS datasets presents a significant bioinformatic challenge. To this end, we have developed alpseq, an optimised, open-source software pipeline designed specifically for the efficient and accurate processing of NGS data from nanobody libraries and panning campaigns. alpseq is also paired with a PCR-free sequencing library preparation protocol to allow researchers to easily generate their own data while avoiding biases. The alpseq software pipeline is composed of two parts: a pre-processing module written in Nextflow efficiently handles raw nanobody reads in a single line of code. These results are then fed into the analysis module, which contains a comprehensive suite of functions for quality control, diversity analysis, identification of enriched sequences and clustering. alpseq also creates a user-friendly interactive report which empowers scientists to explore their data without the need for extensive bioinformatic experience. Sophisticated panning campaign designs are supported, such as replicates and comparisons between different pans to find cross-binding leads. alpseq thus generates insights into the nanobody selection process and delivers a list of lead candidates for further experimental validation and downstream applications. alspeq is available at https://github.com/kzeglinski/alpseq.

bioinformatics↗

bioRxiv2025

Igniting full-length isoform analysis in single-cell and spatial RNA-seq data with FLAMESv2

Long-read single-cell RNA-sequencing enables the profiling of RNA isoform expression and alternative splicing at single cell resolution. However, diverse single-cell technologies and sparse isoform data demand flexible and accurate analysis tools. We introduce FLAMESv2, a highly modular and protocol-agnostic R/Bioconductor package for long-read single-cell RNA-seq data analysis. FLAMESv2 supports a wide range of single-cell and spatial protocols, is highly configurable, scales to allow multi-sample analysis and provides versatile visualisation and analysis outputs. We demonstrate its compatibility with both droplet-based and combinatorial barcoding single-cell methods, as well as spatial transcriptomics workflows. Benchmarking confirms FLAMESv2 achieves field-leading performance across key analysis tasks. Applying FLAMESv2 to in vitro differentiation of stem cells into neurons, we identify cell-types, differentiation trajectories, expression of annotated and novel isoforms and isoform expression diversity and heterogeneity within individual cells. FLAMESv2 provides a comprehensive, flexible approach to analysing long-read single-cell RNA-sequencing, unlocking this powerful methodology for RNA isoform characterisation.

bioinformatics↗

bioRxiv2025

Benchmarking long-read RNA-sequencing technologies with LongBench: a cross-platform reference dataset profiling cancer cell lines with bulk and single-cell approaches

Long-read RNA sequencing enables full-length transcript profiling and improved isoform resolution, but variable platforms and evolving chemistries demand careful benchmarking for reliable application. We present LongBench, a matched, multi-platform reference dataset spanning bulk, single-cell, and single-nucleus transcriptomics across eight human lung cancer cell lines with synthetic spike-in controls. LongBench in-corporates three state-of-the-art long-read protocols alongside Illumina short reads: Oxford Nanopore Technologies (ONT) PCR-cDNA, ONT direct RNA, and PacBio Kinnex. We systematically evaluate transcript capture, quantification accuracy, differential expression, isoform usage, variant detection, and allele-specific analyses. Our results show high concordance in gene-level differential analyses across protocols, but reduced consistency for transcript-level and isoform analyses due to length- and platform-dependent biases. Single-cell long-read data are highly concordant with bulk for high-confidence features, though single-nuclei data show reduced feature detection. LongBench provides one of the largest publicly available long-read benchmarking resources, enabling rigorous cross-platform evaluation and guiding technology selection for transcriptomic research.

genomics↗

bioRxiv2025

Pfs48/45 nanobodies block Plasmodium falciparum transmission

Malaria parasite fertilisation occurs within the Anopheles mosquito midgut. Interventions that inhibit parasite fertilisation prevent ongoing transmission and are important for malaria elimination efforts. Pfs48/45 and Pfs230 are two leading transmission-blocking vaccine candidates. Both proteins form a complex on the surface of sexual stage parasites and are essential for male fertility. Here we have identified nanobodies against Pfs48/45 that recognise gametocytes and have strong transmission-reducing activity. The crystal structure of our most potent nanobody in complex with Pfs48/45 reveals it binds a distinct epitope to TB31F, a leading transmission-blocking monoclonal antibody. In addition, we generated bispecific nanobodies that can target both Pfs48/45 and Pfs230 simultaneously and are fused to a human Fc domain. Our results show that these bispecific nanobodies recognise both Pfs48/45 and Pfs230 and reduce malaria parasite fertilisation in Anopheles stephensi. These results demonstrate the potential of nanobodies as a versatile antibody format that can reduce malaria transmission. Author SummaryMalaria is spread when an infected Anopheles mosquito bites a human. Within the female Anopheles mosquito, malaria parasite fertilisation occurs in the mosquito midgut. If you inhibit parasite fertilisation in the mosquito, you can prevent onward transmission of the malaria parasite from mosquito to humans. Transmission blocking vaccines work by stopping parasite fertilisation and development in the mosquito and are key for malaria elimination by preventing community spread. Pfs48/45 and Pfs230 are two leading transmission-blocking vaccine candidates, and both are critical for male fertility. Here we describe the generation of nanobodies that target Pfs48/45. We show that when nanobodies against Pfs48/45 were added to infected blood meals for Anopheles mosquitoes, the nanobodies significantly reduced parasite transmission. In addition, we generated bispecific nanobodies that target both Pfs48/45 and Pfs230 and these bispecific nanobodies also significantly reduced oocyst development. Our work demonstrates the potential of nanobodies as a versatile antibody format that can reduce malaria transmission.

microbiology↗

bioRxiv2025

Cryo-EM structure of endogenous Plasmodium falciparum Pfs230 and Pfs48/45 fertilization complex

Plasmodium falciparum Pfs230 and Pfs48/45, part of a core fertilization complex, are leading malaria transmission-blocking vaccine candidates. However, how the two proteins interact is unknown. Here we report a 3.36 [A] resolution cryo-electron microscopy structure of the endogenous Pfs230-Pfs48/45 complex. We show that Pfs48/45 interacts with Pfs230 domains 13 and 14, domains that are not included in current Pfs230 vaccine immunogens. Using a transgenic parasite line with a domain 13 to 14 deletion, we show that these domains are essential for Pfs230 localization on the gamete surface. Furthermore, this line significantly reduced oocyst formation in the mosquito midgut, showing that the presence of Pfs230 domains 13 and 14 is critical for successful fertilization. Nanobodies against domains 13 and 14 inhibit Pfs230-Pfs48/45 complex formation, reduce transmission and structural analyses reveal their binding epitopes. Furthermore, domains 13 and 14 are targets of naturally acquired immunity and when delivered as mRNA-LNP immunizations induce potent immune responses and blocked transmission of malaria parasites. Our comprehensive structural insights on a core P. falciparum fertilization complex will guide the design of novel transmission-blocking vaccine candidates against malaria.

microbiology↗

bioRxiv2023

An optimised protocol for quality control of gene therapy vectors using Nanopore direct RNA sequencing

Despite recent advances made towards improving the efficacy of lentiviral gene therapies, a sizeable proportion of produced vector contains an incomplete and thus potentially nonfunctional RNA genome. This can undermine gene delivery by the lentivirus as well as increase manufacturing costs and must be improved to facilitate the widespread clinical implementation of lentiviral gene therapies. Here, we compare three long-read sequencing technologies for their ability to detect issues in vector design and determine Nanopore direct RNA sequencing to be the most powerful. We show how this approach identifies and quantifies incomplete RNA caused by cryptic splicing and polyadenylation sites, including a potential cryptic polyadenylation site in the widely used Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). Using artificial polyadenylation of the lentiviral RNA, we also identify multiple hairpin-associated truncations in the analysed lentiviral vectors, which account for most of the detected RNA fragments. Finally, we show that these insights can be used for optimization of lentiviral vector design. In summary, Nanopore direct RNA sequencing is a powerful tool for the quality control and optimisation of lentiviral vectors, which may help to improve lentivirus manufacturing and thus the development of higher quality lentiviral gene therapies.

genomics↗