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Unlike hybrid approaches that use multiple libraries for de novo assembly, the hierarchical genome-assembly process uses data from only a single long-read SMRT sequencing library to produce high-quality finished microbial genome or BAC assemblies in an automated workflow. We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph–based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.

Evolutionary forecasting models inform seasonal influenza vaccine design by predicting which current genetic variants will dominate in the influenza season 12 months later. Forecasting models depend on hemagglutinin sequences from global public health networks to identify current genetic variants (clades) and estimate clade fitnesses. The lag between collection of a clinical sample and public availability of its sequence averages ∼3 months, complicating the 12-month forecasting problem by reducing our understanding of current clade frequencies. Despite continued methodological improvements to forecasting models, these constraints of a 12-month forecast horizon and 3-month submission lags impose an upper bound on any model’s accuracy. The SARS-CoV-2 pandemic revealed that modern vaccine technology reduces forecast horizons to 6 months and expanded sequencing support reduces submission lags to 1 month on average. We quantified the potential effects of these public health policy changes on forecast accuracy for A/H3N2 populations. Reducing forecast horizons to 6 months reduced average absolute forecasting errors to 25% of the 12-month average, while reducing submission lags decreased uncertainty in current clade frequencies by 50%. These results show the potential to improve the accuracy of existing forecasting models through realistic changes to public health policy.

Deciphering the genetic basis of human disease requires a comprehensive knowledge of genetic variants irrespective of their class or frequency. Although an impressive number of human genetic variants have been catalogued, a large fraction of the genetic difference that distinguishes two human genomes is still not understood at the base-pair level. This is because the emphasis has been on single-nucleotide variation as opposed to less tractable and more complex genetic variants, including indels and structural variants. The latter, we propose, will have a large impact on human phenotypes but require a more systematic assessment of genomes at deeper coverage and alternate sequencing and mapping technologies.

Human influenza virus rapidly accumulates mutations in its major surface protein hemagglutinin (HA). The evolutionary success of influenza virus lineages depends on how these mutations affect HA’s functionality and antigenicity. Here we experimentally measure the effects on viral growth in cell culture of all single amino acid mutations to the HA from a recent human H3N2 influenza virus strain. We show that mutations that are measured to be more favorable for viral growth are enriched in evolutionarily successful H3N2 viral lineages relative to mutations that are measured to be less favorable for viral growth. Therefore, despite the well-known caveats about cell-culture measurements of viral fitness, such measurements can still be informative for understanding evolution in nature. We also compare our measurements for H3 HA to similar data previously generated for a distantly related H1 HA and find substantial differences in which amino acids are preferred at many sites. For instance, the H3 HA has less disparity in mutational tolerance between the head and stalk domains than the H1 HA. Overall, our work suggests that experimental measurements of mutational effects can be leveraged to help understand the evolutionary fates of viral lineages in nature—but only when the measurements are made on a viral strain similar to the ones being studied in nature.

Single-molecule, real-time DNA sequencing is used to analyse a haploid human genome (CHM1), thus closing or extending more than half of the remaining 164 euchromatic gaps in the human genome; the complete sequences of euchromatic structural variants (including inversions, complex insertions and tandem repeats) are resolved at the base-pair level, suggesting that a greater complexity of the human genome can now be accessed. Deep-sequencing the human genome The human genome is considered sequenced, yet more than 160 euchromatic gaps remain and many aspects of its structural variation are poorly understood. Evan Eichler and colleagues sequenced and analysed a haploid human genome (CHM1) using single-molecule, real-time (SMRT) DNA sequencing and by doing so closed — or in some cases extended — more than half of the remaining gaps. They also resolved the complete sequence of numerous euchromatic structural variants at the base-pair level, revealing inversions, complex insertions and long tracts of tandem repeats, some of them previously unknown. Thanks to the longer-read sequencing technology applied here, the complexity of the human genome that stems from variation of longer and more complex repetitive DNA can now be largely resolved. The human genome is arguably the most complete mammalian reference assembly 1 , 2 , 3 , yet more than 160 euchromatic gaps remain 4 , 5 , 6 and aspects of its structural variation remain poorly understood ten years after its completion 7 , 8 , 9 . To identify missing sequence and genetic variation, here we sequence and analyse a haploid human genome (CHM1) using single-molecule, real-time DNA sequencing 10 . We close or extend 55% of the remaining interstitial gaps in the human GRCh37 reference genome—78% of which carried long runs of degenerate short tandem repeats, often several kilobases in length, embedded within (G+C)-rich genomic regions. We resolve the complete sequence of 26,079 euchromatic structural variants at the base-pair level, including inversions, complex insertions and long tracts of tandem repeats. Most have not been previously reported, with the greatest increases in sensitivity occurring for events less than 5 kilobases in size. Compared to the human reference, we find a significant insertional bias (3:1) in regions corresponding to complex insertions and long short tandem repeats. Our results suggest a greater complexity of the human genome in the form of variation of longer and more complex repetitive DNA that can now be largely resolved with the application of this longer-read sequencing technology.

Abstract Seasonal influenza viruses repeatedly infect humans in part because they rapidly change their antigenic properties and evade host immune responses, necessitating frequent updates of the vaccine composition. Accurate predictions of strains circulating in the future could therefore improve the vaccine match. Here, we studied the predictability of frequency dynamics and fixation of amino acid substitutions. Current frequency was the strongest predictor of eventual fixation, as expected in neutral evolution. Other properties, such as occurrence in previously characterized epitopes or high Local Branching Index (LBI) had little predictive power. Parallel evolution was found to be moderately predictive of fixation. Although the LBI had little power to predict frequency dynamics, it was still successful at picking strains representative of future populations. The latter is due to a tendency of the LBI to be high for consensus-like sequences that are closer to the future than the average sequence. Simulations of models of adapting populations, in contrast, show clear signals of predictability. This indicates that the evolution of influenza HA and NA, while driven by strong selection pressure to change, is poorly described by common models of directional selection such as traveling fitness waves.

In many repeat diseases, such as Huntington’s disease (HD), ongoing repeat expansions in affected tissues contribute to disease onset, progression and severity. Inducing contractions of expanded repeats by exogenous agents is not yet possible. Traditional approaches would target proteins driving repeat mutations. Here we report a compound, naphthyridine-azaquinolone (NA), that specifically binds slipped-CAG DNA intermediates of expansion mutations, a previously unsuspected target. NA efficiently induces repeat contractions in HD patient cells as well as en masse contractions in medium spiny neurons of HD mouse striatum. Contractions are specific for the expanded allele, independently of DNA replication, require transcription across the coding CTG strand and arise by blocking repair of CAG slip-outs. NA-induced contractions depend on active expansions driven by MutSβ. NA injections in HD mouse striatum reduce mutant HTT protein aggregates, a biomarker of HD pathogenesis and severity. Repeat-structure-specific DNA ligands are a novel avenue to contract expanded repeats. Naphthyridine-azaquinolone specifically binds slipped-CAG DNA intermediates, induces contractions of expanded repeats and reduces mutant HTT protein aggregates in cell and animal models of Huntington’s disease.

Access to complete, high-quality genomes of nonhuman primates will also help us understand human biology. Gordon et al. used long-read sequencing technology to improve genome data on our close relative the gorilla. Sequencing from a single individual decreased assembly fragmentation and recovered previously missed genes and noncoding loci. Mapping short-read sequences from additional gorillas helped reconstruct a “pan” gorilla sequence documenting genetic variation. Comparison with human genomes revealed species-specific differences ranging in size from one to thousands of bases in length, including some that are likely to affect gene regulation. Science , this issue p. 10.1126/science.aae0344 A new approach to looking at the gorilla genome improves estimates of the differences between humans and gorillas. Accurate sequence and assembly of genomes is a critical first step for studies of genetic variation. We generated a high-quality assembly of the gorilla genome using single-molecule, real-time sequence technology and a string graph de novo assembly algorithm. The new assembly improves contiguity by two to three orders of magnitude with respect to previously released assemblies, recovering 87% of missing reference exons and incomplete gene models. Although regions of large, high-identity segmental duplications remain largely unresolved, this comprehensive assembly provides new biological insight into genetic diversity, structural variation, gene loss, and representation of repeat structures within the gorilla genome. The approach provides a path forward for the routine assembly of mammalian genomes at a level approaching that of the current quality of the human genome.

Duplications and deletions can lead to variation in copy number for genes and genomic loci among humans. Such variants can reveal evolutionary patterns and have implications for human health. Sudmant et al. examined copy-number variation across 236 individual genomes from 125 human populations. Deletions were under more selection, whereas duplications showed more population-specific structure. Interestingly, Oceanic populations retain large duplications postulated to have originated in an ancient Denisovan lineage. Science , this issue 10.1126/science.aab3761 Copy-number variation reveals how selection affects the human genome across the globe. In order to explore the diversity and selective signatures of duplication and deletion human copy-number variants (CNVs), we sequenced 236 individuals from 125 distinct human populations. We observed that duplications exhibit fundamentally different population genetic and selective signatures than deletions and are more likely to be stratified between human populations. Through reconstruction of the ancestral human genome, we identify megabases of DNA lost in different human lineages and pinpoint large duplications that introgressed from the extinct Denisova lineage now found at high frequency exclusively in Oceanic populations. We find that the proportion of CNV base pairs to single-nucleotide–variant base pairs is greater among non-Africans than it is among African populations, but we conclude that this difference is likely due to unique aspects of non-African population history as opposed to differences in CNV load.

Short-read sequencing has enabled the de novo assembly of several individual human genomes, but with inherent limitations in characterizing repeat elements. Here we sequence a Chinese individual HX1 by single-molecule real-time (SMRT) long-read sequencing, construct a physical map by NanoChannel arrays and generate a de novo assembly of 2.93 Gb (contig N50: 8.3 Mb, scaffold N50: 22.0 Mb, including 39.3 Mb N-bases), together with 206 Mb of alternative haplotypes. The assembly fully or partially fills 274 (28.4%) N-gaps in the reference genome GRCh38. Comparison to GRCh38 reveals 12.8 Mb of HX1-specific sequences, including 4.1 Mb that are not present in previously reported Asian genomes. Furthermore, long-read sequencing of the transcriptome reveals novel spliced genes that are not annotated in GENCODE and are missed by short-read RNA-Seq. Our results imply that improved characterization of genome functional variation may require the use of a range of genomic technologies on diverse human populations. Short-read sequencing has inherent limitations in the characterisation of long repeat elements. Shi and Guo et al. combine single-molecule real-time sequencing and IrysChip to construct a Chinese reference genome that fills many gaps in the reference genome, and identify novel spliced genes.