MeStanG—Resource for High-Throughput Sequencing Standard Data Sets Generation for Bioinformatic Methods Evaluation and Validation
2025
Daniel Ramos Lopez | Francisco J. Flores | Andres S. Espindola
Metagenomics analysis has enabled the measurement of the microbiome diversity in environmental samples without prior targeted enrichment. Functional and phylogenetic studies based on microbial diversity retrieved using HTS platforms have advanced from detecting known organisms and discovering unknown species to applications in disease diagnostics. Robust validation processes are essential for test reliability, requiring standard samples and databases deriving from real samples and in silico generated artificial controls. We propose a MeStanG as a resource for generating HTS Nanopore data sets to evaluate present and emerging bioinformatics pipelines. MeStanG allows samples to be designed with user-defined organism abundances expressed as number of reads, reference sequences, and predetermined or custom errors by sequencing profiles. The simulator pipeline was evaluated by analyzing its output mock metagenomic samples containing known read abundances using read mapping, genome assembly, and taxonomic classification on three scenarios: a bacterial community composed of nine different organisms, samples resembling pathogen-infected wheat plants, and a viral pathogen serial dilution sampling. The evaluation was able to report consistently the same organisms, and their read abundances as provided in the mock metagenomic sample design. Based on this performance and its novel capacity of generating exact number of reads, MeStanG can be used by scientists to develop mock metagenomic samples (artificial HTS data sets) to assess the diagnostic performance metrics of bioinformatic pipelines, allowing the user to choose predetermined or customized models for research and training.
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