Metagenomic Sequencing

About

Detection and characterization of microbial pathogens are essential elements of many health and environmental applications, including clinical diagnostics, food safety protection, public health surveillance, and environmental monitoring. Although recent advancements in molecular methods for gene detection and quantification have significantly broadened the coverage and throughput of detection and characterization, these methods still require a priori knowledge of the target gene markers, and their throughput levels are still very limited in comparison to the tremendous diversity of microbial pathogens and their genetic traits. Shotgun metagenomic sequencing provides the much-needed capabilities of detecting a large number of genes simultaneously, including known genetic targets and novel elements of significance. Although shotgun metagenomics has the potential to revolutionize many the abovementioned application fields, it in current forms can only provide qualitative detection or provide relative quantification of genes in the sequencing outputs. Absolute concentrations of genetic markers in clinical, food, and environmental samples can provide more comprehensive and quantitative understanding of samples and enable new applications. Researchers at the University of Hawaii, College of Engineering have developed a quantitative metagenomic sequencing approach for high throughput gene detection and quantification. This new approach includes a novel design of metagenomic sequencing internal DNA standards that are nearly identical to some natural DNA sequences in nature yet completely xenobiotic because of several short in-frame insertions. The numerous internal DNA standards of different concentration are spiked into DNA samples and undergo the same metagenomic sequencing processes. A set of computation and bioinformatic algorithms are used to establish the quantitative relationship between spiked concentrations of the internal standards and numbers of sequences detected through metagenomic sequencing. The relationship is mathematically described by a new process parameter called sequencing yield, which can then be used to calculate the absolute abundance of all sequence reads in the sample DNA based on number of sequences detected in the metagenomic sequencing experiment. Further Details Li et al. Applied Environmental Microbiology https://journals.asm.org/doi/10.1128/AEM.00871-21?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed