From short to long: The impact of read length on metagenome assembly and binning

Abstract Metagenome sequencing not only plays a pivotal role in unravelling the genetic diversity and functional potential of microbial communities but also facilitates the discovery of genome context for microbial dark matter. This study presents a comparative analysis of metagenome sequencing strategies, focusing on the impact of read length on the assembly quality of metagenome binning. We employed metaSPAdes assembly with varying k ‐mer lists and the read lengths on 19 Illumina datasets, revealing that longer reads significantly improve the number of contigs and their length, despite a trade‐off in N50. Specially, longer reads also contribute to better performance of gene fragment reconstruction from contigs. Next, the substantial potential of Nanopore sequencing was further evaluated by comparing the short‐read assembly by Illumina, long‐read assembly by Nanopore and hybrid assembly strategies on samples from extreme environments, including both cold seep and hot spring. The binning of assembled contigs and subsequent metagenome‐assembled genome quality assessment highlighted the superiority of long‐read data in reconstructing medium‐ and high‐quality drafted genomes, specifically, increasing medium‐quality species‐level representative genomes by 1.32‐fold. These findings advocate for the integration of extended read lengths and Nanopore sequencing in metagenome analysis, which can lead to a more nuanced comprehension of the environmental microbiome.