Biology subjects

Owusu, M.

Publications and source records attributed to Owusu, M..

5 recordsLinked to original sources

bioRxiv2025

EnviroAmpDesigner, a tool for designing high specificity multiplex PCR primer panels for detecting and subtyping a target organism in environmental surveillance samples

BackgroundAmplicon sequencing is a popular method for understanding the diversity of bacterial communities in environmental or similar samples as exemplified by 16S rRNA sequencing. This approach has been extended into multiplex amplicon sequencing in which multiple targets are amplified in the same PCR reaction such as virus sequencing using tiled amplicons. Multiple tools exist to design PCR primers, and some support design of multiplex panels. However, despite increasing interest in the use of environmental or wastewater sampling for detecting and typing specific antimicrobial resistant (AMR) bacteria such as typhoid or cholera agents, we were unable to find a tool for designing a multiplex PCR panel for environmental samples that not only focused on detection of a specific organism, but also on amplifying lineage-specific or AMR-associated alleles of the target organism while minimising amplification of off-target genomes present in the sample. We found that existing tools either depend on the target organism being very distinct from the rest of the organisms in the sample, or focus on detection rather than genotyping of the organism of interest. We have developed EnviroAmpDesigner (v0.1.3, DOI: 10.5281/zenodo.14967337) to fill this gap, which we used to design a multiplex amplicon panel for detection, genotyping and AMR profiling of the typhoidal pathogens Salmonella enterica serovars Typhi and Paratyphi A. EnviroAmpDesigner can design amplicons for both short and long-read sequencing. The software first identifies single nucleotide polymorsphisms (SNPs) that distinguish individual genotypes of the target pathogen (in our case, S. Typhi). It then identifies SNPs that distinguish the target organism from others. Finally, the software designs primers that simultaneously target genotyping SNPs and have at least one primer with a 3 end at each SNP that distinguishes the target organism. The purpose of the latter condition is to increase the specificity of the primers, to minimise amplification of homologous sequences in non-target organisms. While our use case focuses on S. Typhi and Paratyphi A, the tool is organism agnostic and should work for any haploid organism. The tool is freely available via Bioconda and GitHub (https://github.com/AntonS-bio/EnviroAmpDesigner). Impact statementWe have developed EnviroAmpDesigner (v 0.1.3) software to design a multiplexed panel of long-read amplicons for detection and lineage typing of bacteria in environmental samples. We apply this tool to Salmonella Typhi and Paratyphi A, the bacterial agents of typhoid fever, but the tool is organism agnostic. The tool is computationally efficient and can be used on mid-range laptop. It has potential to support more widespread deployment of sequencing-based surveillance for bacterial pathogens in environmental and wastewater samples. Data SummaryFor the purposes of designing primers for S. Typhi and Paratyphi A we used 13,134 samples from the Global Typhoid Genomics Consortium data (target organisms) (1) and 1,810 publicly available Enterobacterales genomes from NCBIs RefSeq database (representing off-target related organisms) (2). To validate the primers, we applied an amplicon sequencing protocol (3) to DNA extracted from two S. Typhi isolates (NCBI accessions SRR5949979 and SRR7165748) provided by Satheesh Nair (UKHSA) (4), and a pooled DNA sample of American Type Culture Collection (ATCC) isolates S. Paratyphi A (ATCC 9150D), S. Paratyphi B (ATCC-BAA-1250D), S. Paratyphi C (ATCC-BAA-1715D), Aeromonas hydrophila (ATCC-7965D), Klebsiella pneumoniae (ATCC-BAA-1706D), and Citrobacter freundii (ATCC-8090D) chosen for their close relationship to S. Typhi. The resulting amplicon sequence data was deposited in European Nucleotide Archive (project accession: PRJEB81565).

bioinformatics↗

bioRxiv2025

Dissecting DNA-mismatch-repair-driven mutational processes in human cells

Mismatch repair (MMR) is an essential DNA repair pathway that preserves genome stability by correcting replication errors and other DNA lesions. MMR deficiency (MMRd) leads to increased mutation rates and microsatellite instability, and contributes to tumorigenesis in multiple cancer types, yet the full spectrum of mutational patterns caused by MMRd remains incompletely defined. Here, using CRISPR-Cas9 knockouts in human isogenic cell lines, we systematically profile mutations arising in MMR-deficient cells. We observe a marked increase in mutation burden and the appearance of established MMRd-associated mutational signatures. Unexpectedly, we also detect signatures SBS57 and DBS14 and connect them to germline single-nucleotide polymorphisms that co-occur with MMR-driven indels. In addition, we provide experimental genomic evidence supporting a role of MMR in preventing mutations underlying the widespread SBS1 signature, and we link MMR deficiency to the indel signature ID12, which currently lacks a known aetiology. Finally, comparing in vitro MMRd profiles from rapidly dividing cells without exogenous mutagen exposure to in vivo tumour genomes that reflect lifelong mutation accumulation reveals pronounced differences in signature composition, underscoring the context dependence of MMR-linked mutagenesis. Together, our results broaden the known mutational footprint of MMR deficiency and illuminate previously uncharacterised mechanisms relevant to cancer development.

genetics↗

bioRxiv2025

AmpliconTyper tool for analysing ONT multiplex PCR data from environmental and other mixed sources

Amplicon sequencing is a popular method for understanding the diversity of bacterial communities in mixed samples as exemplified by 16S rRNA metagenome sequencing. This approach has been extended into multiplex amplicon sequencing in which multiple targets are amplified in the same polymerase chain reaction (PCR). Multiple tools exist to process the sequencing data produced via the short-read Illumina platform, but there are fewer options for long-read Oxford Nanopore Technologies (ONT) sequencing, or for processing data from environmental surveillance or other sources with many different organisms. We have developed AmpliconTyper (v0.1.28, DOI: 10.5281/zenodo.14621928) for analysing multiplex amplicon sequencing data from environmental (e.g. wastewater) or similarly contaminated samples, generated using ONT devices. The software tool uses machine learning to classify sequencing reads into target and non-target organisms with very high specificity and sensitivity. The user can train models using public and/or user-generated data, which can subsequently be applied to analyse new data. The tool can also generate amplicon consensus sequences, as well as identify single nucleotide polymorphisms (SNPs) and report their genotype implications, such as association with lineages or antimicrobial resistance (AMR). The tool is freely available via Bioconda and GitHub (https://github.com/AntonS-bio/AmpliconTyper). AmpliconTyper allows robust identification of target organism reads in ONT sequenced environmental samples, and can identify user-specified lineage or AMR markers. Impact statementAmpliconTyper (v0.1.28) is a software package that enables users to analyse amplicon sequences generated by targeted amplification followed by ONT sequencing, for environmental or other similarly contaminated samples. The analysis includes mapping of reads to target amplicon sequences, classification of each sequenced read as either originating from a target or non-target organism, followed by identification of user-specified SNPs and generation of an interactive report summarising the findings. The strength of AmpliconTyper lies in its ability to train a machine learning model using public data to create sequencing read classification models tailored to a users application. AmpliconTyper is designed specifically to work with extremely noisy data that includes a large share of off-target amplification reads such as those encountered in environmental surveillance applications. Data SummaryFor the purpose of designing and testing AmpliconTyper we have used two datasets. The first consisted of 69 Salmonella enterica serovar. Typhi (S. Typhi) and 10,303 other Enterobacteriaceae WGS ONT nanopore sequencing libraries (Supp. Data 1) from NCBI Sequence Read Archive (SRA) (1). We used these data to evaluate the performance of different classifier models and to train a model for our use-case, i.e. amplicon-based detection of S. Typhi from environmental surveillance samples (2). In addition, to further evaluate the performance of AmpliconTyper in our use case, we have applied an amplicon sequencing protocol (2) to generate amplicon data for S. Typhi using two S. Typhi isolates (NCBI accessions SRR5949979 and SRR7165748) provided by Satheesh Nair (UKHSA) (3). We also used same protocol to the pooled sample of American Type Culture Collection (ATCC) consisting of S. Paratyphi A (ATCC 9150D), S. Paratyphi B (ATCC-BAA-1250D), S. Paratyphi C (ATCC-BAA-1715D), Aeromonas hydrophila (ATCC-7965D), Klebsiella pneumoniae (ATCC-BAA-1706D), and Citrobacter freundii (ATCC-8090D) chosen for their close relationship to S. Typhi. The test data for classification is available at https://github.com/AntonS-bio/AmpliconTyper/tree/main/test_data. Newly generated data was deposited in European Nucleotide Archive project PRJEB81565.

genomics↗

bioRxiv2023

High-density sampling reveals volume growth in human tumours

In growing cell populations such tumours, mutations can serve as markers that allow tracking the past evolution from current samples. The genomic analyses of bulk samples and samples from multiple regions have shed light on the evolutionary forces acting on tumours. However, little is known empirically on the spatio-temporal dynamics of tumour evolution. Here, we leverage published data from resected hepatocellular carcinomas, each with several hundred samples taken in two and three dimensions. Using spatial metrics of evolution, we find that tumour cells grow predominantly uniformly within the tumour volume instead of at the surface. We determine how mutations and cells are dispersed throughout the tumour and how cell death contributes to the overall tumour growth. Our methods shed light on the early evolution of tumours in vivo and can be applied to high-resolution data in the emerging field of spatial biology.

cancer biology↗

bioRxiv2018

Mapping the human kinome in response to DNA damage

We provide a catalog for the effects of the human kinome on cell survival in response to DNA damaging agents, selected to cover all major DNA repair pathways. By treating 313 kinase-deficient cell lines with ten diverse DNA damaging agents, including seven commonly used chemotherapeutics, we were able to identify kinase specific vulnerabilities and resistances. In order to identify novel synthetic lethal interactions, we investigate the cellular response to carmustine for 25 cell lines, by establishing a phenotypic FACS assay designed to mechanistically investigate and validate gene-drug interactions. We show apoptosis, cell cycle, DNA damage and proliferation after alkylation or crosslink-induced damage for selected cell lines and rescue the cellular sensitivity of DYRK4, EPHB6, MARK3, PNCK as a proof of principle for our study. Our data suggest that some cancers with inactivated DYRK4, EPHB6, MARK3 or PNCK gene could be particularly vulnerable to treatment by alkylating chemotherapeutic agents carmustine or temozolomide.

systems biology↗