Circulating Microbiome Profiling in Transjugular Intrahepatic Portosystemic Shunt (TIPS) Patients: 16S rRNA vs. Shotgun Sequencing

Jingxiang Zhang¹Xiaoshuang Xu¹Lei Chen¹Xincheng Yang¹Júlio Ken Matsubara²Yuyao Tian³Jiahua Liu¹Xiaoxu Jin¹He Chang¹Menghui Xu¹Chunyang Zhu¹Xin Wang¹Lingxuan Ren¹Jiale Xie¹Jiale Liu⁴Guifen Liu¹Mengyao Lu⁴Xiaowen Wang⁵Longqi Du⁵Zilin Ma⁶Xuechen Liu¹Huimin Zhao⁷Wei Chen⁷Xiaohui Huo⁸Guoqi Zheng⁹Changshun Xie¹⁰Chao Xu¹¹Xueqiang Zhang^1*Wei Qi^1*Zhijie Feng^1*

• ¹Department of Gastroenterology, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Hebei Clinical Research Center for Digestive Diseases, Second Hospital of Hebei Medical University, Shijiazhuang, China
• ²Marília Medical School, SaoPaulo, Brazil
• ³Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong, Hong Kong, SAR China
• ⁴Department of Neurology China The Key Laboratory of Neurology (Hebei Medical University), Ministry of Education, Second Hospital of Hebei Medical University, Shijiazhuang, China
• ⁵Department of Gastroenterology, Hebei General Hospital, Shijiazhuang, China
• ⁶Key Laboratory of Tumor Prevention and Precision Diagnosis and Treatment of Hebei, Clinical Oncology Research Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China
• ⁷Xingtai People's Hospital, Xingtai, China
• ⁸The First Hospital of Hebei Medical University, Shijiazhuang, China
• ⁹Cangzhou Central Hospital, Cangzhou, China
• ¹⁰The First Hospital of Qinhuangdao, Qinhuangdao, China
• ¹¹Handan Central Hospital, Handan, China

Background and aim: Current efforts to characterize the circulating microbiome are constrained by the lack of standardized protocols for isolating and sequencing microbial communities in blood. To address this challenge, our study compared 16S rRNA (V3-V4 region) and shotgun metagenomic sequencing for circulating microbiome detection. Materials and methods: After obtaining ethics committee approval and informed consent, samples were aseptically collected from 10 patients undergoing transjugular intrahepatic portosystemic shunt (TIPS) procedures. Shotgun metagenomic reads were taxonomically classified using the Kraken2-Bracken pipeline. 16S rRNA (V3-V4) data were analyzed through an ASV-based approach, with USEARCH for denoising and VSEARCH for taxonomic annotation. The results from both sequencing methods were then systematically compared. Results: Shotgun metagenomic sequencing generated 7,024,580,376 raw reads (mean depth: 234,152,679.2 reads/sample), while 16S rRNA sequencing produced 6,612,678 raw reads (mean depth: 220,422.6 reads/sample). 16S rRNA amplicon sequencing captured a broader range of microbial signals. Although the taxonomic profiles from both sequencing methods showed limited overlap, the core microbiota common to both were still identified. These conserved core microbial communities exhibited stable α- and β-diversity indices across separate vascular compartments. Conclusions: In our study, 16S rRNA amplicon sequencing captured more diverse microbial signals than shotgun metagenomics. A stable microbial community structure was observed across vascular compartments, suggesting a homogeneous microbial composition throughout the circulatory system.