16S rRNA gene-based amplicon sequencing is less costly and is commonly used to characterise the gut microbiota of FMT donors and recipients

16S sequences do not contain sufficient resolution to inform whether the same microbial species or strains are found in both donors and recipients

Breadth of sequences covering entire genomes provides additional information, such as gene content and nucleotide polymorphisms, to more accurately infer movement of microbial populations from donor to recipient.

Assembly of DNA sequences into contiguous sequences and reconstruction of bacteria genomes (termed binning) can be used to identify exact microbial strains transferred between donor and recipients and their genetic potential.

Ability to compare genome content across studies, assessment of metabolic functions to predict engraftment, and tracking the transfer of microbial genomes in pooled multi-donor FMTs

Sequence counts are used as a proxy for functionality.14

There are few metagenome-based studies of FMT gut communities,15–17 but none specifically compares gene content between donors and recipients before and after FMT. For example, increased concentrations of gamma-aminobutyric acid in plasma,12 short-chain fatty acids18 and secondary bile acids19 have been demonstrated in recipients following FMT for CDI.

Gene sequence data can be verified against measurable parameters to confirm whether metagenomics data corroborates changes in metabolites associated with FMT, and provide support for (or against) the use of DNA sequencing in studying mechanisms of FMT.

Genome-centric metagenomics aims to reconstruct microbial population genomes from sequence data (binning) without relying on reference genomes.15

Genomes recovered this way, commonly termed metagenome-assembled genomes or MAGs, represent sample-specific microbial populations and can be analysed for their gene sequence and content, metabolic potential, correlated with patient-specific clinical data or serve as mapping references for downstream transcriptomic studies.

This approach has been applied in two separate studies to identify the donor microbial populations that establish in recipients. The authors reported establishment by Bacteroidetes and Firmicutes taxa.15 17 Lee and colleagues reconstructed population genomes from donor and recipient metagenomes and used read mappings to detect their presence across subjects.15 Using a slightly different approach, Smillie and colleagues mapped metagenome sequence reads to reference microbial genomes from the Human Microbiome Project and inferred single-nucleotide polymorphisms in read alignments to detect strains shared by donors and recipients post-FMT.17 Both studies reported variability in the outcomes of donor micro-organisms engrafting in recipients, with different species or strains showing establishment in some recipients and not others. The amount of engraftment also varied, with donor strains constituting between 1% and 80% of the recipient gut community.17 Smillie and colleagues built a statistical model based on their data to predict patterns of engraftment, reporting that bacterial taxonomy, abundance in donors/recipients and elapsed time post-FMT were the major determining factors.17 Some donor-derived strains could be detected in recipients for only 1 day post-FMT, and others persisted for longer than 1 month.17 Although the exact ecological drivers for engraftment and persistence are not yet resolved, it was demonstrated that it is possible to derive genomic information with enough resolution to track microbial engraftment in FMT through metagenomics.