Abstract
Metagenome profiling research using next-generation sequencing (NGS), a technique widely used to analyze the diversity and composition of microorganisms living in the human body, especially the gastrointestinal tract, has been actively conducted, and there is a growing interest in the quantitative and diagnostic technology for specific microorganisms. According to recent trends, quantitative real-time PCR (qRT-PCR) is still a considerable technique in detecting and quantifying bacteria associated with the human oral and nasal cavities, due to the analytical cost and time burden of NGS technology. Here, based on NGS metagenome profiling data produced by utilizing 100 gut microbiota samples, we conducted a comparative analysis for the identification and quantification of five bacterial genera (Akkermansia, Bacteroides, Bifidobacterium, Phascolarctobacterium, and Roseburia) within same metagenomic DNA samples through qRT-PCR assay in parallel. Genus-specific primers, targeting the particular gene of each genus for qRT-PCR assay, allowed a statistically consistent quantification pattern with the metagenome profiling data. Furthermore, results of bacterial identification through Sanger validation demonstrated the high genus-specificity of each primer set. Therefore, our study suggests that an approach to quantifying specific microorganisms by applying the qRT-PCR method can compensate for the concerns (potential issues) of NGS while also providing efficient benefits to various microbial industries.
Highlights
As a high-throughput nucleic acid sequencing technology, next-generation sequencing (NGS) has developed rapidly over the past 15 years
This study confirmed the consistency of the relative proportion pattern within samples of five particular bacterial genera between 16S metagenome profiling data and quantitative real-time PCR (qRT-PCR)
BLAST results of Bifidobacterium_low groups were undefined with National Center for Biotechnology Information Database (NCBI DB), we found that the relative proportion in 100 people calculated from the NGS frequency was almost similar to that based on qRT-PCR Ct value
Summary
As a high-throughput nucleic acid sequencing technology, next-generation sequencing (NGS) has developed rapidly over the past 15 years. Microbial identification was possible through conventional Sanger sequencing. Conventional Sanger sequencing can only be used for culture-dependent microorganisms. NGS technology can be used to obtain genomes of multiple microbes (called metagenome), including culturable and unculturable microorganisms [10]. This approach for classifying microbiomes through metagenome sequencing technology (e.g., whole-metagenome sequencing and 16S rRNA sequencing) has been used to understand complex microbial communities and biological interactions [11,12]. Among several metagenome sequencing technologies, 16S rRNA gene sequencing is generally used because the 16S rRNA gene can be used as a phylogenetic marker to classify bacteria [13]
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