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

Abstract

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 metagenome-assembled genomes (MAGs). HiFi-LR, in contrast, improves 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. 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-LRs data alone exhibited higher completeness than those generated from ultra-deep SR data. Incorporating SR-derived coverage information into the binning of HiFi-LR contigs increased MAG recovery by 24% (313 vs. 252) while reducing contamination (116 vs. 132 contaminated bins; mean contamination 7.09% vs. 8.07%). Importantly, 67% of the newly recovered MAGs belonged to low-abundance and taxonomically diverse lineages representing 36 novel lineages, including Archaea. Our results demonstrate that the integration of HiFi-LR and SR sequencing markedly enhances genome recovery and binning accuracy in a highly diverse environment such as soil. The hybrid approach used 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. Although factors such as sequencing depth, cost, and DNA quality remain important considerations, our study provides practical guidance for designing future soil metagenomics projects. It underscores the value of adopting long-read technologies for more comprehensive characterization of complex microbial communities.