HiFi long-read sequencing enables more complete microbial genome reconstruction than short-read sequencing, but its cost and low coverage can limit binning. The authors propose a novel hybrid approach: using high-depth short-read data to improve the binning of HiFi contigs rather than for assembly. Tested on tunnel-cultivated soil, this method substantially increases the number and quality of recovered genomes, showing that combining short- and long-read data remains valuable for complex microbiomes and maximizes the use of existing short-read datasets.

Background Advances in high-fidelity long-read (HiFi-LR) sequencing technologies offer unprecedented opportunities to uncover the microbial genomic diversity of complex environments, such as soil. While short-read (SR) sequencing has enabled broad insights at gene-level diversity, its inherently limited read length constrains the reconstruction of complete genomes. Conversely, HiFi-LR sequencing enhances the quality and completeness of metagenome-assembled genomes (MAGs), enabling higher-resolution taxonomic and functional annotation. However, the high cost and relatively low throughput of HiFi-LR sequencing can limit genome recovery, particularly at the binning stage, where coverage depth is critical. Results Here, we present a novel hybrid strategy that differs from classical hybrid assemblies, where SR and LR reads are jointly used at the assembly step. Instead, we use high-depth SR data to guide and improve the binning of HiFi-LR contigs. Using both SR and HiFi-LR metagenomic datasets generated from a tunnel-cultivated soil sample, we demonstrate that SR-derived coverage profiles significantly improve the binning of HiFi-LR assemblies. This results in a substantial increase in the number and quality of recovered MAGs compared to using HiFi-LR data alone. Conclusion Our findings highlight that, even in the context of HiFi reads, combining SR and LR remains beneficial in highly diverse environments, such as soil, not for hybrid assembly per se, but to enhance the downstream binning process. Our findings demonstrate that, even with the advent of high-quality HiFi long reads, integrating SR data remains highly valuable in complex and diverse environments such as soil. The combination of SR and LR data substantially improves the downstream binning process and overall genome recovery. Importantly, this approach underscores the potential of leveraging the vast amount of publicly available Illumina metagenomic datasets. By completing existing SR resources with additional PacBio HiFi sequencing can maximise assembly contiguity and binning accuracy using data already generated. This highlights a practical and forward-looking strategy for microbiome research, where new LR technologies amplify rather than replace the value of previous short-read efforts.