Long-read metagenomics empowers precise tracking of bacterial strains and their genomic changes after fecal microbiota transplantation

Fecal microbiota transplantation (FMT) has revolutionized the treatment of recurrent Clostridioides difficile infection (rCDI) and is being evaluated across other diseases. Accurate tracking of bacterial strains, the functional units of the microbiota, that stably engraft in recipients is critical for understanding the determinants of strain engraftment, evaluating their correlation with clinical outcomes, and guiding the development of therapeutic bacterial consortia. While short-read sequencing has advanced our understanding of FMT, it faces challenges in the strain-level de novo metagenomic assembly of microbiome samples. In this study, we described a novel framework, LongTrack, that uses long-read metagenomic assemblies and reliable informatics tailored for FMT strain tracking. We first highlighted LongTrack advantage over short-read approaches especially when multiple strains co-exist in the same sample. We then showed LongTrack uncovered hundreds of engrafted strains across six FMT cases of rCDI and inflammatory bowel disease patients, validated by complementary bacterial culturing. Furthermore, beyond conventional strain tracking, long reads also allowed us to assess the genomic and epigenomic stability of successfully engrafted strains during the 5-year follow-ups, revealing structural variations in their genomes, which may be associated with strain adaptation in a new host environment. Combined, our study advocates the incorporation of long-read sequencing to enhance strain tracking in future FMT studies, paving the way for the development of more effective defined biotherapeutic as an alternative to FMT.