Nanopore and Illumina Sequencing Reveal Different Viral Populations from Human Gut Samples

Abstract

The advent of viral metagenomics, or viromics, has improved our knowledge and understanding of global viral diversity. High-throughput sequencing technologies enable explorations of the ecological roles, contributions to host metabolism, and the influence of viruses in various environments including the human gut microbiome. However, the bacterial metagenomic studies frequently have the advantage. The adoption of advanced technologies like long-read sequencing has the potential to be transformative in refining viromics and metagenomics. Here, we examined the effectiveness of long-read and hybrid sequencing by comparing Illumina short-read and Oxford Nanopore Technology (ONT) long-read sequencing technologies and different assembly strategies on recovering viral genomes from human faecal samples. Our findings showed that if a single sequencing technology is to be chosen for virome analysis, Illumina was preferable due to its superior ability to recover fully resolved viral genomes and minimise erroneous genomes. While ONT assemblies were effective in recovering viral diversity, the challenges related to input requirements and the necessity for amplification made it less ideal as a standalone solution. However, using a combined, hybrid approach enabled a more authentic representation of viral diversity to be obtained within samples. Impact StatementViral metagenomics, or viromics, has revolutionised our understanding of global viral diversity however long-read and hybrid approaches are not yet widespread in this field. Here, we compared the performance of Illumina short-read and Nanopore long-read assembly approaches for recovering fully resolved viral genomes from human faecal samples. We highlight Illuminas short-read sequencing for recovering fully resolved viral genomes, while acknowledging Oxford Nanopore Technologys long-read sequencing for capturing broader viral diversity. However, a hybrid approach, utilising both technologies, may mitigate the limitations of one technology alone. Data SummaryAll reads used in this study are available on European Nucleotide Archive (ENA) within the project PRJEB47625.