Amplicon sequencing is a cutting-edge technique that involves sequencing PCR products or captured fragments of specific lengths, enabling the analysis of sequence variations. Among the diverse applications of this technology, the 16S rRNA gene has emerged as a fundamental tool for sequence-based bacterial analysis, boasting decades of significance. The process of 16S/18S/ITS amplicon sequencing entails DNA extraction from environmental samples, careful selection of universal primers to amplify the target region of 16S/18S/ITS, and the subsequent detection of sequence variations and abundance in the target region. This facilitates the study of differences in environmental microbial diversity and community composition.
Technical Advantages
High Throughput: Amplicon sequencing is well-suited for investigating specific genomic regions across a large number of samples, making it highly efficient for research purposes.
Short Cycle: Its rapid turnaround time accelerates clinical applications and facilitates prompt article publication, contributing to the swift advancement of scientific knowledge.
Rich Information Content: Amplicon sequencing enables in-depth research on particular genomic regions of interest, as it facilitates the identification of genetic variation sites within the genome.
Microbial Amplicon Sequencing Analysis of Microbial Populations
The risk of lung cancer is known to be associated with exposure to cigarette smoke carcinogens and alterations in intestinal microbiota. However, the impact of NNK and BaP—two crucial components of cigarette smoke carcinogens—on the intestinal microbiota of lung cancer remains inadequately explored. A recent study delves into this subject, describing the effects of NNK and BaP exposure on lung cancer, fecal metabolite composition, and gut microbiota in mice. To accomplish this, the researchers administered NNK and BaP to A/J mice and employed 16S rRNA gene sequencing and metabolomics to characterize the changes in gut microbiota and fecal metabolism profiles, respectively.
The results, supported by light microscopy and histopathology evaluations, revealed that the exposure to the mixture of NNK and BaP triggered the occurrence of lung cancer. Moreover, 16S rRNA sequencing of the gut microbiota demonstrated alterations in the composition of fecal bacteria upon exposure to NNK and BaP. The increased levels of actinomycetes, bifidobacteria, and Enterobacteriaceae, alongside the decreased levels of physiologic bacteria, visceral odor bacteria, and Acetatifactor bacteria, were associated with NNK and BaP-induced lung cancer. These findings provide valuable insights into utilizing gut microbiota as biomarkers to evaluate the progression of lung cancer and offer potential intervention targets for future disease control strategies.
Amplicon Sequencing Assists in Microbial Resistance Research
Neonicotinoid insecticides, widely applied to control agricultural insect pests, face challenges due to the evolution of neonicotinoid resistance, leading to the failure of pest control in the field. Research has indicated that insect symbiotic bacteria can directly metabolize toxic substances or indirectly mediate the expression of host detoxification enzymes or related genes, thereby influencing the detoxification metabolic function of insects. Through 16S Amplicon sequencing, the study revealed a significant increase in the abundance of sphingosine spp. in the gut of imidacloprid-resistant cotton aphids compared to sensitive cotton aphids.
Further investigation demonstrated that the removal of sphingosine from the intestinal tract of the imidacloprid-resistant strain of Aphis gossypii increased its sensitivity to imidacloprid. Conversely, the addition of sphingosine to the imidacloprid-sensitive strain of Aphis gossypii significantly reduced its sensitivity to the insecticide. These findings highlight the crucial role of Amplicon sequencing technology in unraveling the mechanisms underlying microbial resistance, shedding light on potential strategies to combat insecticide resistance in agricultural settings.
16S Sequencing Technology Analysis of Intestinal Microbiota to Assist in Precise Drug Use
Irritable Bowel Syndrome (IBS) is commonly believed to be an intestinal disease resulting from disorders in the brain-gut axis, with multifactorial etiology that includes changes in gut microbiota. To explore this further, researchers collected samples from 346 IBS patients and 170 healthy control (HC) individuals, who completed dietary inventory surveys to reflect their most frequently consumed diets. Subsequently, fecal samples were collected from 171 IBS patients and 98 HC individuals for 16S rRNA gene sequencing and microbial composition analysis.
The 16S sequencing results indicated higher abundances of Rieknellaceae and Paraacteroides in the IBS group compared to the HC group. After adjusting for diet and race, only Rikenellaceae showed significantly higher abundance values in the IBS group. This study suggests that IBS patients may adopt more restricted diets to mitigate the severity of gastrointestinal symptoms, which, in turn, can influence the composition of their fecal microbiota. These alterations in the gut microbiota can cause physiological changes in brain-gut interactions, potentially contributing to IBS symptoms. Although the microbiota of IBS patients displays variability, the study highlights the role of dietary influences on gut microbiota, elucidating the differences between the irritable bowel syndrome and healthy control groups.
Conclusion
Amplicon sequencing technology has proven to be an invaluable tool for exploring the intricate relationship between microorganisms and human diseases, as well as their interaction with the environment. With its remarkable advantages, Amplicon sequencing holds tremendous promise in unraveling the mysteries of microorganisms, empowering researchers to make significant advancements in various fields of study. As we look to the future, this technology is poised to continue playing a pivotal role in furthering our understanding of microbial populations, leading to new insights and breakthroughs in the scientific community.
