Small amounts of misassembly can have disproportionate effects on pangenome-based metagenomic analyses.

Individual genes from microbiomes can drive host-level phenotypes. To help identify such candidate genes, several recent tools estimate microbial gene copy numbers directly from metagenomes. These tools rely on alignments to pangenomes, which in turn are derived from the set of all individual genomes from one species. While large-scale metagenomic assembly efforts have made pangenome estimates more complete, mixed communities can also introduce contamination into assemblies, and it is unknown how robust pangenome-based metagenomic analyses are to these errors. To gain insight into this problem, we re-analyzed a case-control study of the gut microbiome in cirrhosis, focusing on commensal Clostridia previously implicated in this disease. We tested for differentially prevalent genes in the Importance: Metagenome-assembled genomes, or MAGs, can be constructed without pure cultures of microbes. Large scale efforts to build MAGs have yielded more complete pangenomes (i.e., sets of all genes found in one species). Pangenomes allow us to measure strain variation in gene content, which can strongly affect phenotype. However, because MAGs come from mixed communities, they can contaminate pangenomes with unrelated DNA, and how much this impacts downstream analyses has not been studied. Using a metagenomic study of gut microbes in cirrhosis as our test case, we investigate how contamination affects analyses of microbial gene content. Surprisingly, even small, typical amounts of MAG contamination (<5%) result in disproportionately high levels of false positive associations (38%). Fortunately, we show that most contaminants can be automatically flagged, and provide a simple method for doing so. Furthermore, applying this method reveals a new association between cirrhosis and gut microbial motility.