After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end ...to end, and hundreds of unresolved gaps persist
. Here we present a human genome assembly that surpasses the continuity of GRCh38
, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome
, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.
Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and ...diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of disease.
The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at ...targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that "enhanced specificity" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.
New genome assemblies have been arriving at a rapidly increasing pace, thanks to decreases in sequencing costs and improvements in third-generation sequencing technologies
. For example, the number ...of vertebrate genome assemblies currently in the NCBI (National Center for Biotechnology Information) database
increased by more than 50% to 1,485 assemblies in the year from July 2018 to July 2019. In addition to this influx of assemblies from different species, new human de novo assemblies
are being produced, which enable the analysis of not only small polymorphisms, but also complex, large-scale structural differences between human individuals and haplotypes. This coming era and its unprecedented amount of data offer the opportunity to uncover many insights into genome evolution but also present challenges in how to adapt current analysis methods to meet the increased scale. Cactus
, a reference-free multiple genome alignment program, has been shown to be highly accurate, but the existing implementation scales poorly with increasing numbers of genomes, and struggles in regions of highly duplicated sequences. Here we describe progressive extensions to Cactus to create Progressive Cactus, which enables the reference-free alignment of tens to thousands of large vertebrate genomes while maintaining high alignment quality. We describe results from an alignment of more than 600 amniote genomes, which is to our knowledge the largest multiple vertebrate genome alignment created so far.
Most genetic variants that contribute to disease
are challenging to correct efficiently and without excess byproducts
. Here we describe prime editing, a versatile and precise genome editing method ...that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.
The Trans-Omics for Precision Medicine (TOPMed) programme seeks to elucidate the genetic architecture and biology of heart, lung, blood and sleep disorders, with the ultimate goal of improving ...diagnosis, treatment and prevention of these diseases. The initial phases of the programme focused on whole-genome sequencing of individuals with rich phenotypic data and diverse backgrounds. Here we describe the TOPMed goals and design as well as the available resources and early insights obtained from the sequence data. The resources include a variant browser, a genotype imputation server, and genomic and phenotypic data that are available through dbGaP (Database of Genotypes and Phenotypes)
. In the first 53,831 TOPMed samples, we detected more than 400 million single-nucleotide and insertion or deletion variants after alignment with the reference genome. Additional previously undescribed variants were detected through assembly of unmapped reads and customized analysis in highly variable loci. Among the more than 400 million detected variants, 97% have frequencies of less than 1% and 46% are singletons that are present in only one individual (53% among unrelated individuals). These rare variants provide insights into mutational processes and recent human evolutionary history. The extensive catalogue of genetic variation in TOPMed studies provides unique opportunities for exploring the contributions of rare and noncoding sequence variants to phenotypic variation. Furthermore, combining TOPMed haplotypes with modern imputation methods improves the power and reach of genome-wide association studies to include variants down to a frequency of approximately 0.01%.
Inorganic-organic hybrid materials such as organically templated metal oxides, metal-organic frameworks (MOFs) and organohalide perovskites have been studied for decades, and hydrothermal and ...(non-aqueous) solvothermal syntheses have produced thousands of new materials that collectively contain nearly all the metals in the periodic table. Nevertheless, the formation of these compounds is not fully understood, and development of new compounds relies primarily on exploratory syntheses. Simulation- and data-driven approaches (promoted by efforts such as the Materials Genome Initiative) provide an alternative to experimental trial-and-error. Three major strategies are: simulation-based predictions of physical properties (for example, charge mobility, photovoltaic properties, gas adsorption capacity or lithium-ion intercalation) to identify promising target candidates for synthetic efforts; determination of the structure-property relationship from large bodies of experimental data, enabled by integration with high-throughput synthesis and measurement tools; and clustering on the basis of similar crystallographic structure (for example, zeolite structure classification or gas adsorption properties). Here we demonstrate an alternative approach that uses machine-learning algorithms trained on reaction data to predict reaction outcomes for the crystallization of templated vanadium selenites. We used information on 'dark' reactions--failed or unsuccessful hydrothermal syntheses--collected from archived laboratory notebooks from our laboratory, and added physicochemical property descriptions to the raw notebook information using cheminformatics techniques. We used the resulting data to train a machine-learning model to predict reaction success. When carrying out hydrothermal synthesis experiments using previously untested, commercially available organic building blocks, our machine-learning model outperformed traditional human strategies, and successfully predicted conditions for new organically templated inorganic product formation with a success rate of 89 per cent. Inverting the machine-learning model reveals new hypotheses regarding the conditions for successful product formation.
The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase ...III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE
and Roadmap Epigenomics
data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.
•Bacillus group are well known as producers of a wide array of antimicrobials.•Most important molecules are non-ribosomally synthesized peptides and lipopeptides.•Very strong antimicrobial activity ...was confirmed through numerous studies.•Bacillus lipopeptides have strong impact through direct antibiosis on plant pathogens.
Bacteria from the Bacillus group are microorganisms that inhabit a large number of different habitats. They are well known as producers of a wide array of antagonistic compounds of different structures, having between 5 to 8% of the total genome devoted to biosynthesis of secondary metabolites. Most important bioactive molecules from the genus Bacillus are non-ribosomally synthesized peptides and lipopeptides, polyketide compounds, bacteriocins and siderophores. Lipopeptides from Bacillus have very complex mechanisms of biosynthesis catalyzed by non-ribosomal peptide synthetases (NRPSs), large enzyme complexes with modular structure, with each module being in charge for the incorporation of a particular amino acid. In general, they have a broad spectrum of antagonistic activity against plant pathogenic bacteria, fungi and viruses. Most important molecules from this group, circular lipopeptides from surfactin, iturin and fengycin families affect the target cells on the membrane level. Bacillus strains exhibit their biocontrol capacity predominantly through inhibitory activity on the growth of plant pathogens, as well as inducing systemic resistance in plants and competing for ecological niches with plant pathogens. Our previous studies showed the presence of multiple biosynthetic operons for synthesis of non-ribosomal lipopeptides in the collection of natural isolates of Bacillus, with many strains having more than one of them. Several strains of Bacillus sp. that we have recently characterized showed very strong antibacterial and antifungal activity against phytopathogens. The PCR analysis showed the presence of biosynthetic operons for iturin, bacillomycin, fengycin and surfactin in tested strains. Measurement of the kinetics of production of antimicrobial substances showed that, in most cases, synthesis started at the beginning of exponential phase of growth, reaching the maximum of antimicrobial activity at the beginning of the stationary growth phase and stayed at this level for the whole duration of observed period. Preparations of cell-free supernatants of tested strains were active against many fungal and bacterial pathogens, in vitro and in vivo. Mass spectrometry and HPTLC bioautography analysis of purified compounds confirmed the presence of lipopeptides of mentioned families, hence confirming the biocontrol capacity of Bacillus isolates.
A chemical genetic roadmap to improved tomato flavor Tieman, Denise; Zhu, Guangtao; Resende, Marcio F. R. ...
Science (American Association for the Advancement of Science),
01/2017, Letnik:
355, Številka:
6323
Journal Article
Recenzirano
Modern commercial tomato varieties are substantially less flavorful than heirloom varieties. To understand and ultimately correct this deficiency, we quantified flavor-associated chemicals in 398 ...modern, heirloom, and wild accessions. A subset of these accessions was evaluated in consumer panels, identifying the chemicals that made the most important contributions to flavor and consumer liking. We found that modern commercial varieties contain significantly lower amounts of many of these important flavor chemicals than older varieties. Whole-genome sequencing and a genome-wide association study permitted identification of genetic loci that affect most of the target flavor chemicals, including sugars, acids, and volatiles. Together, these results provide an understanding of the flavor deficiencies in modern commercial varieties and the information necessary for the recovery of good flavor through molecular breeding.
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