Comparative analysis of short-read and full-length 16S rRNA sequencing for circulating microbiota profiling in cirrhosis

Although current next-generation 16S rRNA sequencing (NGS) offers high throughput and low cost, its short read length severely limits resolution at the species level, particularly for closely related taxa. However, full-length 16S rRNA sequencing (FL) methods such as PacBio SMRT generate ultra-long reads spanning the complete 16S rRNA gene, granting precise identification at the species levels, and, in selected cases, at the subspecies levels. The higher resolution of full-length 16S rRNA sequencing could be critical for studying circulating microbiota in diseases prone to spontaneous infections, such as cirrhosis. However, direct comparisons between the two approaches in the blood of cirrhotic patients are lacking. This study compares Illumina short-read and PacBio long-read sequencing of the 16S rRNA gene in 30 blood samples from patients undergoing transjugular intrahepatic portosystemic shunt (TIPS) procedures. Blood samples from peripheral, hepatic, and portal veins were collected. Subsequently, V3-V4 (Illumina) and V1-V9 (PacBio) regions of the 16S rRNA gene were amplified and sequenced. Raw data were processed using QIIME2 software. Denoising and the generation of the amplicon sequence variants (ASV) table were performed using DADA2. Subsequent data visualization was carried out using R software (version 4.4.2). Full-length 16S rRNA gene sequencing demonstrated higher resolution in detecting the circulating microbiome at the species level. Both methods showed robust detection sensitivity, largely unaffected by sequencing depth. Moreover, both α- and β-diversity metrics generated by these two methods demonstrated strong concordance across the three vascular compartments. We investigated the circulating microbiome in patients undergoing TIPS and demonstrated the complementary utility of short-read (V3-V4) and full-length 16S rRNA sequencing for circulating microbiota profiling. These results could lay the groundwork for developing circulating microbiota-based biomarkers and targeted therapies for cirrhotic patients.