Improved Assembly of Metagenome-Assembled Genomes and Viruses in Tibetan Saline Lake Sediment by HiFi Metagenomic Sequencing.

With the development and reduced costs of high-throughput sequencing technology, environmental dark matter, such as novel metagenome-assembled genomes (MAGs) and viruses, is now being discovered easily. However, due to read length limitations, MAGs and viromes often suffer from genome discontinuity and deficiencies in key functional elements. Here, by applying long-read sequencing technology to sediment samples from a Tibetan saline lake, we comprehensively analyzed the performance of high-fidelity (HiFi) reads and the possibility of integration with short-read next-generation sequencing (NGS) data. In total, 207 full-length nonredundant 16S rRNA gene sequences and 19 full-length nonredundant 18S rRNA genes were directly obtained from HiFi reads, which greatly surpassed the retrieval performance of NGS technology. We carried out a cross-sectional comparison among multiple assembly strategies, referred to as 'NGS', 'Hybrid (NGS+HiFi)', and 'HiFi'. Two MAGs and 29 viruses with circular genomes were reconstructed using HiFi reads alone, indicating the great power of the 'HiFi' approach to assemble high-quality microbial genomes. Among the 3 strategies, the 'Hybrid' approach produced the highest number of medium/high-quality MAGs and viral genomes, while the ratio of MAGs containing 16S rRNA genes was significantly improved in the 'HiFi' assembly results. Overall, our study provides a practical metagenomic resolution for analyzing complex environmental samples by taking advantage of both the short-read and HiFi long-read sequencing methods to extract the maximum amount of information, including data on prokaryotes, eukaryotes, and viruses, via the 'Hybrid' approach.