Effectiveness of multi-region 16S rRNA gene sequencing in studying the microbiome of gastric cancer tissues

Abstract

BACKGROUND

The gastric microbiome is closely associated with gastric cancer, and single-region 16S rRNA sequencing has limitations in analyzing its characteristics, necessitating the search for a better sequencing method.

AIM

To evaluate the effectiveness of multi-region 16S rRNA gene sequencing in studying the microbiome of gastric cancer tissues.

METHODS

Patients with gastric cancer (n = 118) who underwent surgery at Liyang People's Hospital from January 2022 to December 2024 were enrolled. Fifty-nine paraffin-embedded and 59 fresh tissue samples were obtained. The ZymoBIOMICS^TM microbial community standard and Escherichia coli ATCC 25922 were used as positive controls. Multi-region and single-region 16S rRNA gene sequencing were performed. Species identification, detection rates at varying microbial abundances, operational taxonomic unit (OTU) counts, and alpha diversity indices in gastric cancer tissues were compared between the two methods.

RESULTS

Multi-region 16S rRNA sequencing identified more species (eight species and eight genera) in the positive controls compared with single-region sequencing (one species and six genera). Detection rates at concentrations of 10³, 10², and 10 CFU/mg were significantly higher using multi-region sequencing (P < 0.05). Multi-region sequencing also revealed significantly higher OTU counts and alpha diversity indices (Shannon, Simpson, and Chao1) in gastric cancer tissues (P < 0.05).

CONCLUSION

Compared with single-region sequencing, multi-region 16S rRNA gene sequencing demonstrates superior species resolution and detection sensitivity, providing a more comprehensive profile of microbial diversity in gastric cancer tissues.

Keywords: 16S rRNA gene; Multi-region sequencing; Gastric cancer; Species resolution; Microbiome; Alpha diversity

Core Tip: This study demonstrated that multi-region 16S rRNA gene sequencing provides significantly improved species resolution and detection rates compared with traditional single-region method. In both positive controls and gastric cancer tissue samples, it revealed richer and more diverse microbial communities. These findings suggest that compared to single-region sequencing, multi-region sequencing is a more accurate and effective approach for profiling the microbiome in gastric cancer research.