Accurate MAG reconstruction from a complex soil microbiome through combined short- and HiFi long-reads metagenomics

Abstract

Abstract Background Advances in high-fidelity long-read (HiFi-LR) sequencing technologies have opened new opportunities to explore the microbial genomic diversity of complex environments, such as soils. While short-read (SR) sequencing has enabled broad insights at the gene level, the limited read length constrains the reconstruction of complete genomes. HiFi-LRs, in contrast, improve assembly continuity and completeness, supporting higher-resolution taxonomic and functional annotation. However, the cost and relatively low throughput of HiFi-LR sequencing can limit genome recovery—particularly at the binning stage, where coverage depth is critical. In this study, we assess the benefit of combining HiFi-LR and SR sequencing for genome-resolved characterization of a soil microbiome. Results We generated metagenomic data for a tunnel-cultivated soil sample using high coverage Illumina SRs as well as a combination of two HiFi-LR sequencing platforms (PacBio Sequel II and PacBio Revio). We found that assemblies generated from pooled HiFi-LR data alone exhibited higher completeness compared to those from ultra-deep SR data. Incorporating SR-derived coverage information for the binning of HiFi-LR contigs further increased both the number and quality of recovered metagenome-assembled genomes (MAGs), with a 24% increase in MAG recovery (313 vs. 252) and lower contamination levels (116 vs. 132 contaminated bins; mean 7.09 vs. 8.07), compared to using HiFi-LR data alone. This approach enabled the recovery of 61 additional MAGs, including 67% of low-abundance and taxonomically diverse lineages such as Archaea, representing 36 novel lineages. Conclusion Our results demonstrate that integrating HiFi-LR and SR sequencing markedly enhances genome recovery and binning accuracy in a highly diverse environment such as soil. The hybrid approach employed leverages the strengths of both technologies, leading to more contiguous assemblies and enabling the recovery of a broader range of genomes, including low-abundance and taxonomically diverse taxa. While factors such as sequencing depth, cost, and DNA quality remain important considerations, our study provides practical guidance for designing future soil metagenomics projects and underscores the value of adopting long-read technologies for more comprehensive characterization of complex microbial communities.