Biology subjects

Abu-Sarkodie, Y.

Publications and source records attributed to Abu-Sarkodie, Y..

4 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

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↗

bioRxiv2020

The genomic epidemiology of multi-drug resistant nontyphoidal Salmonella causing invasive disease in sub-Saharan Africa

BackgroundInvasive nontyphoidal Salmonella (iNTS) is one of the leading causes of bacteraemia in sub-Saharan Africa. Multi-drug resistance (MDR) and further resistance to third generation cephalosporins and fluoroquinolones have emerged in multiple iNTS serotypes. Molecular epidemiological investigations of nontyphoidal Salmonella are needed to better understand the genetic characteristics and transmission dynamics associated with major MDR iNTS serotypes across the continent. MethodsA total of 166 nontyphoidal Salmonella isolates causing invasive disease were collected from a multi-centre study in eight African countries between 2010 and 2014, and whole-genome sequenced to investigate the geographical distribution, antimicrobial genetic determinants and population structure of iNTS serotypes-genotypes. Phylogeographical reconstruction was further conducted in context of the existing genomic framework of iNTS serotypes Typhimurium and Enteritidis. Population-based incidence of MDR-iNTS disease was also estimated. ResultsSalmonella enterica subsp. Enterica serotype Typhimurium (S. Typhimurium) sequence-type (ST) 313 and Salmonella enterica subsp. Enterica serotype Enteritidis (S. Enteritidis) ST11 were predominant, and both exhibited high frequencies of MDR. Salmonella enterica subsp. Enterica serotype Dublin (S. Dublin) ST10 emerged in West Africa. Mutations in the gyrA gene were identified in S. Enteritidis and S. Typhimurium in Ghana; and ST313 carrying blaCTX-M-15 was found in Kenya. Inter-country transmission of MDR ST313 lineage II and the West African Clade of MDR ST11 between Ghana and neighbouring countries including Mali, Burkina Faso, and Nigeria were evident. The incidence of MDR-iNTS disease exceeded 100/100,000-person years-of-observation (PYO) in children aged <5 years in several West African countries. ConclusionsMultiple MDR iNTS serotypes-sequence types, predominantly S. Typhimurium ST313 and S. Enteritidis ST11, are co-circulating in sub-Saharan Africa with evidence of transmission between West African countries. The development of safe and effective iNTS vaccines coupled with appropriate antimicrobial stewardship and adequate epidemiological monitoring are essential to limit the impact of these pathogens in Africa.

genomics↗

bioRxiv2020

At least seven distinct rotavirus genotype constellations in bats with evidence of reassortment and zoonotic transmissions

Bats host many viruses pathogenic to humans, and increasing evidence suggests that Rotavirus A (RVA) also belongs to this list. Rotaviruses cause diarrheal disease in many mammals and birds, and their segmented genomes allow them to reassort and increase their genetic diversity. Eighteen out of 2,142 bat fecal samples (0.8%) collected from Europe, Central America and Africa were PCR-positive for RVA and 11 of those were fully characterized using viral metagenomics. Upon contrasting their genomes with publicly available data, at least 7 distinct bat RVA genotype constellations (GCs) were identified, including evidence of reassortments and 6 novel genotypes. Some of these constellations are spread across the world, whereas others appear to be geographically restricted. Our analyses also suggest that several unusual human and equine RVA strains might be of bat RVA origin, based on their phylogenetic clustering, despite varying levels of nucleotide sequence identities between them. Although SA11 is one of the most widely used reference strains for RVA research and forms the backbone of a reverse genetics system, its origin remained enigmatic. Remarkably, the majority of the genotypes of SA11-like strains were shared with Gabonese bat RVAs, suggesting a potential common origin. Overall, our findings suggest an underexplored genetic diversity of RVAs in bats, which is likely only the tip of the iceberg. Increasing contact between humans and bat wildlife will further increase the zoonosis risk, which warrants closer attention to these viruses. ImportanceThe increased research on bat coronaviruses after SARS-CoV and MERS-CoVallowed the very rapid identification of SARS-CoV-2. This is an excellent example of the importance of knowing viruses harbored by wildlife in general and bats in particular, for global preparedness against emerging viral pathogens. The current effort to characterize bat rotavirus strains from 3 continents shed light on the vast genetic diversity of rotaviruses and also hinted at a bat origin for several atypical rotaviruses in humans and animals, implying that zoonoses of bat rotaviruses might occur more frequently than currently realized.

microbiology↗