Promises and pitfalls of long-read sequencing for resolving microbial complexity.

Abstract

Long-read sequencing (LRS) has driven a transition in microbial genomics, overcoming the assembly fragmentation inherent to short-read sequencing. This review elucidates the impact of LRS across isolate genomics, metagenomics, and multi-omics domains. By spanning extensive repetitive regions, LRS facilitates the reconstruction of circular chromosomes and precisely resolves mobile genetic elements (MGEs). In metagenomics, LRS enables strain-level resolution, the recovery of circular metagenome-assembled genomes, and the precise localization of MGEs within host replicons. Furthermore, the single-molecule, amplification-free properties of LRS provide enhanced resolution of native epigenetic modifications and full-length transcriptomes. Despite these advancements, widespread implementation remains constrained by multidimensional challenges, including stringent high-molecular-weight DNA requirements, depth deficits, and computational overhead. Nevertheless, LRS is increasingly becoming the method of choice for isolate genomics and metagenomics. As detection technologies and algorithms progress, LRS will further improve our ability to decipher the structural and functional diversity of microbial ecosystems.