Long-Read Sequencing Improves Recovery of Picoeukaryotic Genomes and Zooplankton Marker Genes from Marine Metagenomes

Ocean microbes provide critical ecosystem services, but most remain uncultivated. Their communities can be studied through shotgun metagenomic sequencing and bioinformatic analyses, including binning draft microbial genomes. Long-read sequencing offers the potential to improve metagenome assemblies and provide more robust assessments of microbial community composition and function than short-read sequencing. We applied Pacific Biosciences (PacBio) CCS (circular consensus sequencing) HiFi shotgun sequencing to 14 marine water column samples and compared the results with those for short-read metagenomes from the corresponding environmental DNA samples. We found that long-read metagenomes varied widely in quality and biological information. The community compositions of the corresponding long- and short-read metagenomes were frequently dissimilar, suggesting higher stochasticity and/or bias associated with PacBio sequencing. Long reads provided few improvements to the assembly qualities, gene annotations, and prokaryotic metagenome-assembled genome (MAG) binning results. However, only long reads produced high-quality eukaryotic MAGs and contigs containing complete zooplankton marker gene sequences. These results suggest that high-quality long-read metagenomes can improve marine community composition analyses and provide important insight into eukaryotic phyto- and zooplankton genetics, but the benefits may be outweighed by the inconsistent data quality.IMPORTANCE Ocean microbes provide critical ecosystem services, but most remain uncultivated. Their communities can be studied through shotgun metagenomic sequencing and bioinformatic analyses, including binning draft microbial genomes. However, most sequencing to date has been done using short-read technology, which rarely yields genome sequences of key microbes like SAR11. Long-read sequencing can improve metagenome assemblies but is hampered by technological shortcomings and high costs. In this study, we compared long- and short-read sequencing of marine metagenomes. We found a wide range of long-read metagenome qualities and minimal improvements to microbiome analyses. However, long reads generated draft genomes of eukaryotic algal species and provided full-length marker gene sequences of zooplankton species, including krill and copepods. These results suggest that long-read sequencing can provide greater genetic insight into the wide diversity of eukaryotic phyto- and zooplankton that interact as part of and with the marine microbiome.

Automated summary

The study compared long- and short-read sequencing of marine metagenomes, finding a wide range of long-read metagenome qualities and minimal improvements to microbiome analyses. However, long reads were able to generate draft genomes of eukaryotic algal species and provide full-length marker gene sequences of zooplankton species. This suggests that long-read sequencing can provide greater genetic insight into the wide diversity of eukaryotic phyto- and zooplankton present in marine environments.