Advances in metagenomic assembly have led to the discovery of genomes belonging to uncultured microorganisms. Metagenome-assembled genomes (MAGs) often suffer from fragmentation and chimerism. Recently, 20 complete MAGs (cMAGs) have been assembled from Oxford Nanopore long-read sequencing of 13 human fecal samples, but with low nucleotide accuracy. Here, we report 102 cMAGs obtained by Pacific Biosciences (PacBio) high-accuracy long-read (HiFi) metagenomic sequencing of five human fecal samples, whose initial circular contigs were selected for complete prokaryotic genomes using our bioinformatics workflow. Nucleotide accuracy of the final cMAGs was as high as that of Illumina sequencing. The cMAGs could exceed 6 Mbp and included complete genomes of diverse taxa, including entirely uncultured RF39 and TANB77 orders. Moreover, cMAGs revealed that regions hard to assemble by short-read sequencing comprised mostly genomic islands and rRNAs. HiFi metagenomic sequencing will facilitate cataloging accurate and complete genomes from complex microbial communities, including uncultured species.

Advances in metagenomic assembly have led to the discovery of genomes belonging to unculturable microorganisms. Metagenome-assembled genomes (MAGs) often suffer from discontinuity and chimerism. Recently, nanopore metagenomic sequencing assembled 20 complete MAGs (cMAGs) from 13 human fecal samples, but with low nucleotide accuracy. Here, we report 102 cMAGs obtained by high-accuracy long-read (HiFi) metagenomic sequencing of five human fecal samples, whose initial circular contigs were filtered for authentic prokaryotic genomes using our bioinformatics workflow. Nucleotide accuracy of the final cMAGs was similar to that of Illumina sequencing. The cMAGs could exceed 6 Mbp and included complete genomes of diverse taxa, including entirely unculturable RF39 and TANB77 orders, whose genomes have not been characterized yet. Moreover, cMAGs revealed that regions hard to assemble by short-read sequencing comprised mostly genomic islands and rRNAs. HiFi metagenomic sequencing will facilitate cataloging accurate and complete genomes of human gut microbiota, including unculturable species.