Comparative Metagenomic Assessment Of Short- And Long-Read Sequencing Technologies Reveals Unknown Microbial Information In A Complex Environmental Sample

Metagenomics enables comprehensive exploration of microbial communities but is influenced by library preparation and sequencing technologies, affecting recovery of microbial genomes and proteins. Here, we benchmarked six Illumina short-read library kits at 2x150 bp and 2x250 bp read lengths alongside PacBio HiFi long-read sequencing using a complex environmental sample. Longer short reads (2x250 bp) combined with optimal library preparation approaches notably improved assembly quality, protein detection, and metagenome-assembled genome (MAG) recovery, achieving results similar to those of long-read sequencing. Although long reads yield more contiguous and complete genomes, longer short reads offer a cost-effective, scalable alternative for uncovering microbial and functional diversity. These findings provide critical guidance for metagenomic experimental design, demonstrating the importance of strategic selection of library preparation chemistry and sequencing parameters in revealing unknown microbial information in complex biomes without requiring additional sequencing depth. IMPORTANCEMetagenomic outcomes are strongly influenced by library preparation and sequencing strategies, yet their combined effects in complex environmental samples remain poorly defined. Here, we provide the first direct comparison of Illumina NovaSeq short-read metagenomic sequencing at 2x150 bp and 2x250 bp across multiple library preparation kits, alongside PacBio long-read sequencing. We show that sequencing read length and library preparation critically shape assembly quality, protein recovery, and metagenome-assembled genome (MAG) reconstruction. Optimized short-read sequencing at 2x250 bp recovered high-quality MAGs approaching those obtained with long-read technologies while substantially improving protein discovery compared to 2x150 bp at the same sequencing depth. Together, these results provide actionable guidance for experimental design and reveal how sequencing read length can be leveraged to improve the recovery of microbial and functional diversity in complex biomes.