Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing.

Clinical-grade preparations of adeno-associated virus (AAV) vectors used for gene therapy typically undergo a series of diagnostics to determine titer, purity, homogeneity, and the presence of DNA contaminants. One type of contaminant that remains poorly investigated is replication-competent (rc)AAVs. rcAAVs form through recombination of DNA originating from production materials, yielding intact, replicative, and potentially infectious virus-like virions. They can be detected through the serial passaging of lysates from cells transduced by AAV vectors in the presence of wildtype adenovirus. Cellular lysates from the last passage are subjected to qPCR to detect the presence of the rep gene. Unfortunately, the method cannot be used to query the diversity of recombination events, nor can qPCR provide insights into how rcAAVs arise. Thus, the formation of rcAAVs through errant recombination events between ITR-flanked gene of interest (GOI) constructs and expression constructs carrying the rep-cap genes is poorly described. We have used single molecule, real-time sequencing (SMRT) to analyze virus-like genomes expanded from rcAAV-positive vector preparations. We present evidence that sequence-independent and non-homologous recombination between the ITR-bearing transgene and the rep/cap plasmid occurs under several events and rcAAVs spawn from diverse clones.

The Development of an Infrastructure to Facilitate the Use of Whole Genome Sequencing for Population Health.

The clinical use of genomic analysis has expanded rapidly resulting in an increased availability and utility of genomic information in clinical care. We have developed an infrastructure utilizing informatics tools and clinical processes to facilitate the use of whole genome sequencing data for population health management across the healthcare system. Our resulting framework scaled well to multiple clinical domains in both pediatric and adult care, although there were domain specific challenges that arose. Our infrastructure was complementary to existing clinical processes and well-received by care providers and patients. Informatics solutions were critical to the successful deployment and scaling of this program. Implementation of genomics at the scale of population health utilizes complicated technologies and processes that for many health systems are not supported by current information systems or in existing clinical workflows. To scale such a system requires a substantial clinical framework backed by informatics tools to facilitate the flow and management of data. Our work represents an early model that has been successful in scaling to 29 different genes with associated genetic conditions in four clinical domains. Work is ongoing to optimize informatics tools; and to identify best practices for translation to smaller healthcare systems.

Towards Next-Generation Sequencing for HIV-1 Drug Resistance Testing in a Clinical Setting.

The HIV genotypic resistance test (GRT) is a standard of care for the clinical management of HIV/AIDS patients. In recent decades, population or Sanger sequencing has been the foundation for drug resistance monitoring in clinical settings. However, the advent of high-throughput or next-generation sequencing has caused a paradigm shift towards the detection and characterization of low-abundance covert mutations that would otherwise be missed by population sequencing. This is clinically significant, as these mutations can potentially compromise the efficacy of antiretroviral therapy, causing poor virologic suppression. Therefore, it is important to develop a more sensitive method so as to reliably detect clinically actionable drug-resistant mutations (DRMs). Here, we evaluated the diagnostic performance of a laboratory-developed, high-throughput, sequencing-based GRT using 103 archived clinical samples that were previously tested for drug resistance using population sequencing. As expected, high-throughput sequencing found all the DRMs that were detectable by population sequencing. Significantly, 78 additional DRMs were identified only by high-throughput sequencing, which is statistically significant based on McNemar's test. Overall, our results complement previous studies, supporting the notion that the two methods are well correlated, and the high-throughput sequencing method appears to be an excellent alternative for drug resistance testing in a clinical setting.

Polymyxin dose tunes the evolutionary dynamics of resistance in multidrug-resistant Acinetobacter baumannii.

OBJECTIVES: Evolutionary principles have informed the design of strategies that slow or prevent antibiotic resistance. However, how antibiotic treatment regimens shape the evolutionary dynamics of resistance mutations remains an open question. Here, we investigate varying concentrations of the last-resort polymyxins on the evolution of resistance in Acinetobacter baumannii. METHODS: Polymyxin resistance was measured in 18 multidrug-resistant A. baumannii AB5075 populations treated over 14 days with concentrations of polymyxin B informed by human pharmacokinetics. Time-resolved whole-population sequencing was conducted to track the genetics and population dynamics of susceptible and resistant subpopulations. RESULTS: A critical threshold concentration of polymyxin B (1 mg/L; i.e. 4 × MIC) was identified. Below this threshold concentration, low levels of resistance repeatedly evolved, but no mutations were fixed, and this resistance was reversed upon removal of the antibiotic. This contrasted with evolution at super-MIC levels (≥4 × MIC) of polymyxin B, which drove the evolution of irreversible resistance, with higher levels of antibiotic correlating with greater rates of molecular evolution. Polymyxin-resistant subpopulations carried mutations in a variety of genes, most commonly pmrB, ompA, glmU/glmS, and wecB/wecC, which contributed to membrane remodelling and virulence in A. baumannii. CONCLUSIONS: Our results show that the strength of the selective pressure applied by polymyxin tunes the dynamics of genetic variants within the population, leading to different evolutionary outcomes for the degree, cost and reversibility of resistance. Our study highlights the critical role of integrating evolutionary findings into pharmacokinetics/pharmacodynamics to optimise antibiotic use in patients.

Interplay of population size and environmental fluctuations: A new explanation for fitness cost rarity in asexuals.

Theoretical models of ecological specialisation commonly assume that adaptation to one environment leads to fitness reductions (costs) in others. However, experiments often fail to detect such costs. We addressed this conundrum using experimental evolution with Escherichia coli in several constant and fluctuating environments at multiple population sizes. We found that in fluctuating environments, smaller populations paid significant costs, but larger ones avoided them altogether. Contrastingly, in constant environments, larger populations paid more costs than the smaller ones. Overall, large population sizes and fluctuating environments led to cost avoidance only when present together. Mutational frequency distributions obtained from whole-genome whole-population sequencing revealed that the primary mechanism of cost avoidance was the enrichment of multiple beneficial mutations within the same lineage. Since the conditions revealed by our study for avoiding costs are widespread, it provides a novel explanation of the conundrum of why the costs expected in theory are rarely detected in experiments.

AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity.

The gene therapy field has been galvanized by two technologies that have revolutionized treating genetic diseases: vectors based on adeno-associated viruses (AAVs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene-editing tools. When combined into one platform, these safe and broadly tropic biotherapies can be engineered to target any region in the human genome to correct genetic flaws. Unfortunately, few investigations into the design compatibility of CRISPR components in AAV vectors exist. Using AAV-genome population sequencing (AAV-GPseq), we previously found that self-complementary AAV vector designs with strong DNA secondary structures can cause a high degree of truncation events, impacting production and vector efficacy. We hypothesized that the single-guide RNA (sgRNA) scaffold, which contains several loop regions, may also compromise vector integrity. We have therefore advanced the AAV-GPseq method to also interrogate single-strand AAV vectors to investigate whether vector genomes carrying Cas9-sgRNA cassettes can cause truncation events. We found that on their own, sgRNA sequences do not produce a high degree of truncation events. However, we demonstrate that vector genome designs that carry dual sgRNA expression cassettes in tail-to-tail configurations lead to truncations. In addition, we revealed that heterogeneity in inverted terminal repeat sequences in the form of regional deletions inherent to certain AAV vector plasmids can be interrogated.

Genome and population sequencing of a chromosome-level genome assembly of the Chinese tapertail anchovy (Coilia nasus) provides novel insights into migratory adaptation.

BACKGROUND: Seasonal migration is one of the most spectacular events in nature; however, the molecular mechanisms related to this phenomenon have not been investigated in detail. The Chinese tapertail, or Japanese grenadier anchovy, Coilia nasus, is a valuable migratory fish of high economic importance and special migratory dimorphism (with certain individuals as non-migratory residents). RESULTS: In this study, an 870.0-Mb high-quality genome was assembled by the combination of Illumina and Pacific Biosciences sequencing. Approximately 812.1 Mb of scaffolds were linked to 24 chromosomes using a high-density genetic map from a family of 104 full siblings and their parents. In addition, population sequencing of 96 representative individuals from diverse areas along the putative migration path identified 150 candidate genes, which are mainly enriched in 3 Ca2+-related pathways. Based on integrative genomic and transcriptomic analyses, we determined that the 3 Ca2+-related pathways are critical for promotion of migratory adaption. A large number of molecular markers were also identified, which distinguished migratory individuals and non-migratory freshwater residents. CONCLUSIONS: We assembled a chromosome-level genome for the Chinese tapertail anchovy. The genome provided a valuable genetic resource for understanding of migratory adaption and population genetics and will benefit the aquaculture and management of this economically important fish.

Chromosomal level assembly and population sequencing of the Chinese tree shrew genome.

Chinese tree shrews (Tupaia belangeri chinensis) have become an increasingly important experimental animal in biomedical research due to their close relationship to primates. An accurately sequenced and assembled genome is essential for understanding the genetic features and biology of this animal. In this study, we used long-read single-molecule sequencing and high-throughput chromosome conformation capture (Hi-C) technology to obtain a high-qualitychromosome-scale scaffolding of the Chinese tree shrew genome. The new reference genome (KIZ version 2: TS_2.0) resolved problems in presently available tree shrew genomes and enabled accurate identification of large and complex repeat regions, gene structures, and species-specific genomic structural variants. In addition, by sequencing the genomes of six Chinese tree shrew individuals, we produced a comprehensive map of 12.8 M single nucleotide polymorphisms and confirmed that the major histocompatibility complex (MHC) loci and immunoglobulin gene family exhibited high nucleotide diversity in the tree shrew genome. We updated the tree shrew genome database (TreeshrewDB v2.0: http://www.treeshrewdb.org) to include the genome annotation information and genetic variations. The new high-quality reference genome of the Chinese tree shrew and the updated TreeshrewDB will facilitate the use of this animal in many different fields of research.

Population Selection and Sequencing of Caenorhabditis elegans Wild Isolates Identifies a Region on Chromosome III Affecting Starvation Resistance.

To understand the genetic basis of complex traits, it is important to be able to efficiently phenotype many genetically distinct individuals. In the nematode Caenorhabditis elegans, individuals have been isolated from diverse populations around the globe and whole-genome sequenced. As a result, hundreds of wild strains with known genome sequences can be used for genome-wide association studies (GWAS). However, phenotypic analysis of these strains can be laborious, particularly for quantitative traits requiring multiple measurements per strain. Starvation resistance is likely a fitness-proximal trait for nematodes, and it is related to metabolic disease risk in humans. However, natural variation in C. elegans starvation resistance has not been systematically characterized, and precise measurement of the trait is time-intensive. Here, we developed a population-selection-and-sequencing-based approach to phenotype starvation resistance in a pool of 96 wild strains. We used restriction site-associated DNA sequencing (RAD-seq) to infer the frequency of each strain among survivors in a mixed culture over time during starvation. We used manual starvation survival assays to validate the trait data, confirming that strains that increased in frequency over time are starvation-resistant relative to strains that decreased in frequency. Further, we found that variation in starvation resistance is significantly associated with variation at a region on chromosome III. Using a near-isogenic line (NIL), we showed the importance of this genomic interval for starvation resistance. This study demonstrates the feasibility of using population selection and sequencing in an animal model for phenotypic analysis of quantitative traits, documents natural variation of starvation resistance in C. elegans, and identifies a genomic region that contributes to such variation.

Clinical and virological properties of hepatitis C virus genotype 4 infection in patients treated with different direct-acting antiviral agents.

BACKGROUND: The efficacy of direct-acting antivirals (DAAs) depends on the hepatitis C virus (HCV) genotype 4 (GT4) subtype which are used in the treatment of HCV. We aimed to retrospectively investigate the baseline prevalence of HCV NS5A and NS5B polymorphisms and their impact on virological outcome in GT4-infected patients treated with various DAA regimens. PATIENTS AND METHODS: Available plasma samples from HCV GT4-infected patients treated with different DAA regimens were analyzed at baseline and after treatment failure, where applicable. Sanger sequencing of patient-derived NS5A and NS5B regions was performed on all available samples, while ultradeep pyrosequencing (UDPS) of NS5A and NS5B regions was performed only on samples from treatment failures at different time points. RESULTS: Sustained virological response (SVR) was achieved by 96% (48/50) of patients. Of 16 patients with baseline NS5A sequence, polymorphisms at amino acid positions associated with drug resistance were detected only at position 58: P58 (69.2%) and T58 (30.8%). Of 21 patients with baseline NS5B sequence, N142S was detected only in the two treatment failures, both with GT4d were treated with sofosbuvir (SOF)-based regimens, suggesting a potential involvement in SOF efficacy. Two patients (patient 1 [Pt1] and patient 2 [Pt2]) relapsed. In Pt1, NS5A-T56I and NS5A-Y93H/S emerged. In Pt2, NS5A-L28F emerged and a novel NS5B resistance-associated substitution (RAS), L204F, representing 1.5% of the viral population at baseline, enriched to 71% and 91.6% during and after treatment failure, respectively. UDPS of NS5B from Pt2 indicated a mixed infection of approximately 1:5, GT1a:GT4d, at baseline and GT4d during failure. Phylogenetic analysis of NS5A sequences indicated no clustering of HCV strains from patients achieving SVR vs patients who relapsed. The mean genetic distance in NS5A sequences was 5.8%, while a lower genetic distance (3.1%) was observed in NS5B sequences. CONCLUSION: Results from these analyses confirm the importance of UDPS in the analysis of viral quasispecies variability and the identification of novel RASs potentially associated with DAA treatment failure in HCV GT4-infected patients.

De Novo Assembly of Two Swedish Genomes Reveals Missing Segments from the Human GRCh38 Reference and Improves Variant Calling of Population-Scale Sequencing Data.

The current human reference sequence (GRCh38) is a foundation for large-scale sequencing projects. However, recent studies have suggested that GRCh38 may be incomplete and give a suboptimal representation of specific population groups. Here, we performed a de novo assembly of two Swedish genomes that revealed over 10 Mb of sequences absent from the human GRCh38 reference in each individual. Around 6 Mb of these novel sequences (NS) are shared with a Chinese personal genome. The NS are highly repetitive, have an elevated GC-content, and are primarily located in centromeric or telomeric regions. Up to 1 Mb of NS can be assigned to chromosome Y, and large segments are also missing from GRCh38 at chromosomes 14, 17, and 21. Inclusion of NS into the GRCh38 reference radically improves the alignment and variant calling from short-read whole-genome sequencing data at several genomic loci. A re-analysis of a Swedish population-scale sequencing project yields > 75,000 putative novel single nucleotide variants (SNVs) and removes > 10,000 false positive SNV calls per individual, some of which are located in protein coding regions. Our results highlight that the GRCh38 reference is not yet complete and demonstrate that personal genome assemblies from local populations can improve the analysis of short-read whole-genome sequencing data.

Parallel genetic adaptation across environments differing in mode of growth or resource availability.

Evolution experiments have demonstrated high levels of genetic parallelism between populations evolving in identical environments. However, natural populations evolve in complex environments that can vary in many ways, likely sharing some characteristics but not others. Here, we ask whether shared selection pressures drive parallel evolution across distinct environments. We addressed this question in experimentally evolved populations founded from a clone of the bacterium Burkholderia cenocepacia. These populations evolved for 90 days (approximately 600 generations) under all combinations of high or low carbon availability and selection for either planktonic or biofilm modes of growth. Populations that evolved in environments with shared selection pressures (either level of carbon availability or mode of growth) were more genetically similar to each other than populations from environments that shared neither characteristic. However, not all shared selection pressures led to parallel evolution. Genetic parallelism between low-carbon biofilm and low-carbon planktonic populations was very low despite shared selection for growth under low-carbon conditions, suggesting that evolution in low-carbon environments may generate stronger trade-offs between biofilm and planktonic modes of growth. For all environments, a population's fitness in a particular environment was positively correlated with the genetic similarity between that population and the populations that evolved in that particular environment. Although genetic similarity was low between low-carbon environments, overall, evolution in similar environments led to higher levels of genetic parallelism and that genetic parallelism, in turn, was correlated with fitness in a particular environment.

Patterns of emergent resistance-associated mutations after initiation of non-nucleoside reverse-transcriptase inhibitor-containing antiretroviral regimens in Taiwan: a multicenter cohort study.

BACKGROUND: Increasing trends of resistance-associated mutations (RAMs) to non-nucleoside reverse-transcriptase inhibitors (nNRTIs) have raised concerns about the effectiveness of the regimens in the national HIV treatment programs in resource-limited countries. We aimed to retrospectively investigate the incidence and patterns of emergent RAMs of HIV-1 in HIV-positive adults experiencing virological failure to first-line nNRTI-containing combination antiretroviral therapy (cART) in Taiwan. PATIENTS AND METHODS: Between June 2012 and March 2016, 1138 antiretroviral-naïve HIV-positive adults without baseline RAMs who initiated nNRTI-containing regimens were included for analysis. Virological failure was defined as plasma viral load (PVL) ≥200 copies/mL after 6 months of cART or confirmed PVL ≥200 copies/mL after achieving PVL <50 copies/mL. Population sequencing was retrospectively performed to detect baseline and emergent RAMs. RAMs were interpreted using the International AIDS Society-USA 2016 mutations list. RESULTS: Seventy-one patients (6.2%) developed virological failure, which occurred in 14.8% (43/291), 3.9% (26/675), and 1.2% (2/172) of patients receiving 2 nucleoside reverse-transcriptase inhibitors (NRTIs) plus nevirapine, efavirenz, and rilpivirine, respectively. Among those, 53 (74.6%) had emergent RAMs identified, which included 43 (81.1%), 53 (100.0%), and 1 (1.9%) with RAMs to NRTIs, nNRTIs, and protease inhibitors, respectively; and 43 (81.1%) had multi-drug resistance. The most common emergent RAMs to NRTIs were M184V/I (42.3%) and K65R (28.2%), and those to nNRTIs were Y181C (42.3%), K103N (15.5%), G190A/E/Q (12.7%), V179D/E (12.7%), and V108I (9.9%). CONCLUSION: While the rates of virological failure varied with the nNRTI used, the rate of emergent RAMs of HIV-1 to NRTIs and nNRTIs among the antiretroviral-naïve patients who failed nNRTI-containing cART remained low.

Error rates, PCR recombination, and sampling depth in HIV-1 whole genome deep sequencing.

Deep sequencing is a powerful and cost-effective tool to characterize the genetic diversity and evolution of virus populations. While modern sequencing instruments readily cover viral genomes many thousand fold and very rare variants can in principle be detected, sequencing errors, amplification biases, and other artifacts can limit sensitivity and complicate data interpretation. For this reason, the number of studies using whole genome deep sequencing to characterize viral quasi-species in clinical samples is still limited. We have previously undertaken a large scale whole genome deep sequencing study of HIV-1 populations. Here we discuss the challenges, error profiles, control experiments, and computational test we developed to quantify the accuracy of variant frequency estimation.

Whole Genome Analysis of 132 Clinical Saccharomyces cerevisiae Strains Reveals Extensive Ploidy Variation.

Budding yeast has undergone several independent transitions from commercial to clinical lifestyles. The frequency of such transitions suggests that clinical yeast strains are derived from environmentally available yeast populations, including commercial sources. However, despite their important role in adaptive evolution, the prevalence of polyploidy and aneuploidy has not been extensively analyzed in clinical strains. In this study, we have looked for patterns governing the transition to clinical invasion in the largest screen of clinical yeast isolates to date. In particular, we have focused on the hypothesis that ploidy changes have influenced adaptive processes. We sequenced 144 yeast strains, 132 of which are clinical isolates. We found pervasive large-scale genomic variation in both overall ploidy (34% of strains identified as 3n/4n) and individual chromosomal copy numbers (36% of strains identified as aneuploid). We also found evidence for the highly dynamic nature of yeast genomes, with 35 strains showing partial chromosomal copy number changes and eight strains showing multiple independent chromosomal events. Intriguingly, a lineage identified to be baker's/commercial derived with a unique damaging mutation in NDC80 was particularly prone to polyploidy, with 83% of its members being triploid or tetraploid. Polyploidy was in turn associated with a >2× increase in aneuploidy rates as compared to other lineages. This dataset provides a rich source of information on the genomics of clinical yeast strains and highlights the potential importance of large-scale genomic copy variation in yeast adaptation.

Diversity and population-genetic properties of copy number variations and multicopy genes in cattle.

The diversity and population genetics of copy number variation (CNV) in domesticated animals are not well understood. In this study, we analysed 75 genomes of major taurine and indicine cattle breeds (including Angus, Brahman, Gir, Holstein, Jersey, Limousin, Nelore, and Romagnola), sequenced to 11-fold coverage to identify 1,853 non-redundant CNV regions. Supported by high validation rates in array comparative genomic hybridization (CGH) and qPCR experiments, these CNV regions accounted for 3.1% (87.5 Mb) of the cattle reference genome, representing a significant increase over previous estimates of the area of the genome that is copy number variable (∼2%). Further population genetics and evolutionary genomics analyses based on these CNVs revealed the population structures of the cattle taurine and indicine breeds and uncovered potential diversely selected CNVs near important functional genes, including AOX1, ASZ1, GAT, GLYAT, and KRTAP9-1 Additionally, 121 CNV gene regions were found to be either breed specific or differentially variable across breeds, such as RICTOR in dairy breeds and PNPLA3 in beef breeds. In contrast, clusters of the PRP and PAG genes were found to be duplicated in all sequenced animals, suggesting that subfunctionalization, neofunctionalization, or overdominance play roles in diversifying those fertility-related genes. These CNV results provide a new glimpse into the diverse selection histories of cattle breeds and a basis for correlating structural variation with complex traits in the future.

How Archiving by Freezing Affects the Genome-Scale Diversity of Escherichia coli Populations.

In the experimental evolution of microbes such as Escherichia coli, many replicate populations are evolved from a common ancestor. Freezing a population sample supplemented with the cryoprotectant glycerol permits later analysis or restarting of an evolution experiment. Typically, each evolving population, and thus each sample archived in this way, consists of many unique genotypes and phenotypes. The effect of archiving on such a heterogeneous population is unknown. Here, we identified optimal archiving conditions for E. coli. We also used genome sequencing of archived samples to study the effects that archiving has on genomic population diversity. We observed no allele substitutions and mostly small changes in allele frequency. Nevertheless, principal component analysis of genome-scale allelic diversity shows that archiving affects diversity across many loci. We showed that this change in diversity is due to selection rather than drift. In addition, ∼1% of rare alleles that occurred at low frequencies were lost after treatment. Our observations imply that archived populations may be used to conduct fitness or other phenotypic assays of populations, in which the loss of a rare allele may have negligible effects. However, caution is appropriate when sequencing populations restarted from glycerol stocks, as well as when using glycerol stocks to restart or replay evolution. This is because the loss of rare alleles can alter the future evolutionary trajectory of a population if the lost alleles were strongly beneficial.

Deep-sequencing analysis of the gene encoding the hepatitis C virus nonstructural 3-4A protease confirms a low prevalence of telaprevir-resistant variants at baseline and the end of the REALIZE study.

BACKGROUND: Population sequencing (PS) has shown that telaprevir-resistant variants are not typically detectable at baseline (prevalence, ≤5% of patients), and most variants present at the time of treatment failure are no longer detectable at the end of the study. METHODS: To gain insight into the evolution of telaprevir-resistant variants, their baseline prevalence and persistence after treatment was investigated using a more sensitive, deep-sequencing (DS) technique in a large number of treatment-experienced patients from the REALIZE study who were infected with hepatitis C virus genotype 1. RESULTS: Before treatment initiation, telaprevir-resistant variants (T54A, T54S, or R155K in 1%-2% of the viral population) were detected by DS in a fraction (2%) of patients for whom PS failed to detect resistance; these variants were not necessarily detected at the time of treatment failure. Of 49 patients in whom telaprevir-resistant variants were detected by PS at the time of treatment failure but not at the end of the study, DS revealed the presence of variants (V36A/L/M, T54S, or R155K in 1%-36% of the viral population) in 16 patients (33%) at the end of the study. CONCLUSIONS: Similar to PS findings, DS analysis revealed that the frequency of telaprevir-resistant variants before treatment was also low, and variants detected at the time of treatment failure were no longer detectable in the majority of patients during follow-up.

Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ).

Next-generation whole-genome shotgun assemblies of complex genomes are highly useful, but fail to link nearby sequence contigs with each other or provide a linear order of contigs along individual chromosomes. Here, we introduce a strategy based on sequencing progeny of a segregating population that allows de novo production of a genetically anchored linear assembly of the gene space of an organism. We demonstrate the power of the approach by reconstructing the chromosomal organization of the gene space of barley, a large, complex and highly repetitive 5.1 Gb genome. We evaluate the robustness of the new assembly by comparison to a recently released physical and genetic framework of the barley genome, and to various genetically ordered sequence-based genotypic datasets. The method is independent of the need for any prior sequence resources, and will enable rapid and cost-efficient establishment of powerful genomic information for many species.