Circulating microbiome profiling in transjugular intrahepatic portosystemic shunt patients: 16S rRNA vs. shotgun sequencing

The study focused on comparing the performance of 16S rRNA sequencing and shotgun metagenomic sequencing for profiling the circulating microbiome in patients undergoing transjugular intrahepatic portosystemic shunt (TIPS) procedures. Below is a detailed breakdown of the findings and implications:

### **Clinical Context**

• Profiling the circulating microbiome in blood samples is inherently challenging due to **low microbial biomass** and **high host DNA contamination**, which can obscure microbial signals.
• The TIPS procedure, which creates a shunt between the portal and systemic circulation, provides a unique opportunity to simultaneously collect blood samples from the **portal vein**, **hepatic vein**, and **peripheral vein**, reducing inter-individual variability and allowing for more robust comparisons of the circulating microbiome.

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### **Comparison of 16S rRNA Sequencing vs. Shotgun Metagenomic Sequencing**

#### **1. Sensitivity and Detection**

• **16S rRNA sequencing** demonstrated **greater sensitivity** in detecting microbial signals in blood samples compared to shotgun metagenomics.
• It was particularly effective in identifying **low-abundance and rare microbial taxa**, which are often missed by shotgun sequencing.
• Shotgun metagenomics faced significant limitations due to **high host DNA interference**, which reduced its ability to detect microbial DNA effectively.

#### **2. Taxonomic Coverage**

• **16S rRNA sequencing** provided **broader taxonomic coverage**, identifying a wider range of microbial taxa.
• Shotgun sequencing was limited in its ability to detect a diverse microbial community, partly due to its susceptibility to host DNA contamination.

#### **3. Method-Specific Bias**

• There was **low concordance** between the taxonomic profiles generated by the two methods, indicating method-dependent biases.
• Many microbial genera were uniquely detected by 16S rRNA sequencing, while some were exclusively identified by shotgun sequencing, though these were sporadic and inconsistently present across samples.
• Primer mismatches in 16S rRNA sequencing explained why certain taxa detected by shotgun sequencing were absent in 16S datasets.

#### **4. Detection Depth**

• 16S rRNA sequencing reached sufficient detection depth more efficiently than shotgun metagenomics, making it more practical for analyzing low-biomass samples like blood.

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### **Microbial Diversity and Composition**

#### **1. Alpha Diversity (Within-Sample Diversity)**

• Microbial diversity within blood samples remained **stable across different vascular compartments** (portal, hepatic, and peripheral veins).

#### **2. Beta Diversity (Between-Sample Diversity)**

• The composition of microbial communities did not significantly differ between the portal, hepatic, and peripheral blood compartments, suggesting a **homogeneous circulating microbiome** across these regions.

#### **3. Core Microbiota**

• Both sequencing methods consistently detected a **shared core microbiota**, further supporting the stability and uniformity of the circulating microbiome across different blood compartments.

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### **Peripheral Blood as a Representative Sample**

• Peripheral blood was found to reliably represent the **systemic circulating microbiota**, making it a convenient and accessible sample source for future microbiome studies.

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### **Clinical and Translational Implications**

#### **1. Feasibility for Clinical Use**

• **16S rRNA sequencing** emerged as a more **practical, cost-effective, and sensitive** method for clinical studies of the circulating microbiome, particularly in low-biomass samples like blood.
• Shotgun metagenomics, while comprehensive in other contexts, was less suitable for blood microbiome studies due to its susceptibility to host DNA contamination and lower sensitivity.

#### **2. Future Research Directions**

• The findings support the use of **16S rRNA sequencing** and **peripheral blood sampling** in future studies of the circulating microbiome.
• The ability of 16S rRNA sequencing to detect low-abundance taxa and provide broader taxonomic coverage makes it especially valuable for investigating the role of the microbiome in systemic diseases and conditions associated with TIPS patients.

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### **Conclusion**

The study highlights the superiority of **16S rRNA sequencing** over shotgun metagenomics for profiling the circulating microbiome in TIPS patients. It offers greater sensitivity, broader taxonomic coverage, and better feasibility for clinical applications. Peripheral blood sampling was validated as a reliable and representative approach for systemic microbiome studies, paving the way for translational research into the role of the circulating microbiome in health and disease.