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Nanopore metagenomic sequencing enables rapid annotating microbiological ecosystems, and the previous glacier-related sequencing applications (e.g., targeted ice sheets, ice lake, and cryoconite holes) inspire us to explore high-altitude glacier meltwater at Qilian Mountain, China (3000 to 4000 m above sea level, MASL). Our findings suggest that (1) despite only several hundred meters apart, the microbial communities and functionalities are quite different among vertical alpine distributions; (2) the high-altitude Qilian meltwater microbiome serve several main metabolic functions, including sulfur oxidation, selenite decomposing, photosynthesis, energy production, enzymic, and UV tolerant activities. Meanwhile, our Nanopore metagenomic results indicate that the microbial classifications and functionalities (e.g., chaperones, cold-shock, specific tRNA species, oxidative stress, and resistance to toxic compounds) of Qilian meltwater are highly consistent with the other glacial microbiome, emphasizing that only certain microbial species can survive in the cold environment and the molecular adaptions and lifestyles remain stable all over the world. Besides, we have shown Nanopore metagenomic sequencing can provide reliable prokaryotic classifications within or among studies, which therefore can encourage more applications in the field given faster turnaround time. However, we recommend accumulating at least 400 ng nucleic acids (after extraction) and maximizing Nanopore library preparation efficiency before on-site sequencing to obtain better resolutions., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Rare species are vital members of a microbial community, but retrieving their genomes is difficult because of their low abundance. The ReadUntil (RU) approach allows nanopore devices to sequence specific DNA molecules selectively in real time, which provides an opportunity for enriching rare species. Despite the robustness of enriching rare species by reducing the sequencing depth of known host sequences, such as the human genome, there is still a gap in RU-based enriching of rare species in environmental samples whose community composition is unclear, and many rare species have poor or incomplete reference genomes in public databases. Therefore, here we present metaRUpore to overcome this challenge. When we applied metaRUpore to a thermophilic anaerobic digester (TAD) community and human gut microbial community, it reduced coverage of the high-abundance populations and modestly increased (∼2×) the genome coverage of the rare taxa, facilitating successful recovery of near-finished metagenome-assembled genomes (nf-MAGs) of rare species. The simplicity and robustness of the approach make it accessible for laboratories with moderate computational resources, and hold the potential to become the standard practice in future metagenomic sequencing of complicated microbiomes., (© 2023 Sun et al.; Published by Cold Spring Harbor Laboratory Press.)