The DNA sequencing technologies in use today produce either highly accurate short reads or lessaccurate long reads. We report the optimization of circular consensus sequencing (CCS) to improve the accuracy of single-molecule real-time (SMRT) sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5 kilobases (kb). We applied our approach to sequence the well-characterized human HG002/NA24385 genome and obtained precision and recall rates of at least 99.91% for single-nucleotide variants (SNVs), 95.98% for insertions and deletions <50 bp (indels) and 95.99% for structural variants. Our CCS method matches or exceeds the ability of short-read sequencing to detect small variants and structural variants. We estimate that 2,434 discordances are correctable mistakes in the 'genome in a bottle' (GIAB) benchmark set. Nearly all (99.64%) variants can be phased into haplotypes, further improving variant detection. De novo genome assembly using CCS reads alone produced a contiguous and accurate genome with a contig N50 of >15 megabases (Mb) and concordance of 99.997%, substantially outperforming assembly with less-accurate long reads.

Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion–base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.

Highlights d Long-read sequencing of 100 tomato genomes uncovered 238,490 structural variants d Transposons underlie many SVs, and SV hotspots revealed large introgressions d SVs associated with genes are predictive of population-scale changes in expression d New genome assemblies resolved complex breeding QTLs caused by SVs

We present RaGOO, a reference-guided contig ordering and orienting tool that leverages the speed and sensitivity of Minimap2 to accurately achieve chromosome-scale assemblies in minutes. After the pseudomolecules are constructed, RaGOO identifies structural variants, including those spanning sequencing gaps. We show that RaGOO accurately orders and orients 3 de novo tomato genome assemblies, including the widely used M82 reference cultivar. We then demonstrate the scalability and utility of RaGOO with a pan-genome analysis of 103 Arabidopsis thaliana accessions by examining the structural variants detected in the newly assembled pseudomolecules. RaGOO is available open source at https://github.com/malonge/RaGOO.

A closer look at centromeres Centromeres are key for anchoring chromosomes to the mitotic spindle, but they have been difficult to sequence because they can contain many repeating DNA elements. These repeats, however, carry regularly spaced, distinctive sequence markers because of sequence heterogeneity between the mostly, but not completely, identical DNA sequence repeats. Such differences aid sequence assembly. Naish et al . used ultra-long-read DNA sequencing to establish a reference assembly that resolves all five centromeres in the small mustard plant Arabidopsis . Their view into the subtly homogenized world of centromeres reveals retrotransposons that interrupt centromere organization and repressive DNA methylation that excludes centromeres from meiotic crossover repair. Thus, Arabidopsis centromeres evolve under the opposing forces of sequence homogenization and retrotransposon disruption. —PJH