Comparative metagenomic assessment of Illumina-compatible library preparation methods, short-read lengths, and PacBio HiFi sequencing reveals differences in microbial and functional diversity recovery from 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-compatible short-read library preparation conditions in triplicate at 2 × 150 bp and 2 × 250 bp read lengths alongside PacBio HiFi long-read sequencing using a composite environmental sample of marine mangrove sediment and terrestrial palm tree soil. Longer short reads (2 × 250 bp) combined with optimal library preparation approaches improved assembly quality, protein detection, and metagenome-assembled genome (MAG) recovery, achieving results approaching those of long-read sequencing. TruSeq libraries at 2 × 250 bp recovered more than sevenfold more unique proteins than the same kit at 2 × 150 bp (811,701 vs 110,108) using the same number of sequencing reads, while recovering a comparable number of high-quality MAGs to PacBio HiFi long-read sequencing (11 vs 18) and surpassing it in protein discovery by almost 10-fold (811,701 vs 87,745) at less than half of the sequencing cost. Furthermore, biosynthetic gene cluster analysis identified 46 biosynthetic gene clusters in TruSeq-250PE assemblies compared to 38 in PacBio HiFi, with several showing no close match in the MIBiG database. Although long reads yield more contiguity 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 that strategic selection of library preparation chemistry and sequencing parameters can reveal more unknown microbial information in complex biomes without requiring additional sequencing depth. IMPORTANCE: Metagenomic 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 2 × 150 bp and 2 × 250 bp across multiple library preparation kits, alongside PacBio HiFi long-read sequencing. We show that sequencing read length and library preparation critically shape assembly quality, protein recovery, and metagenome-assembled genome (MAG) reconstruction. These findings demonstrate that short-read sequencing at 2 × 250 bp, with appropriate library preparation, can match long-read technologies in MAG recovery while substantially surpassing them in protein discovery. With less than half of the sequencing price and a 3.5-fold reduction in cost per gigabase of usable data, this method facilitates more accessible large-scale metagenomic analysis within complex environmental systems.