scholarly journals High-resolution comparative analysis of great ape genomes

Science ◽  
2018 ◽  
Vol 360 (6393) ◽  
pp. eaar6343 ◽  
Author(s):  
Zev N. Kronenberg ◽  
Ian T. Fiddes ◽  
David Gordon ◽  
Shwetha Murali ◽  
Stuart Cantsilieris ◽  
...  
Keyword(s):  
De Novo ◽  
Great Ape ◽  
Structural Variants ◽  
Genetic Studies ◽  
Regulatory Changes ◽  
Long Read ◽  
Orangutan Genome ◽  
Radial Glial ◽  
Genome Assemblies ◽  
Human Specific

Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single– to mega–base pair–sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these variants are enriched near genes that are down-regulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors.

scholarly journals Genotyping structural variants in pangenome graphs using the vg toolkit

2019 ◽  
Author(s):  
Glenn Hickey ◽  
David Heller ◽  
Jean Monlong ◽  
Jonas A. Sibbesen ◽  
Jouni Sirén ◽  
...  
Keyword(s):  
De Novo ◽  
State Of The Art ◽  
Effective Means ◽  
Point Mutations ◽  
Structural Variants ◽  
Short Read ◽  
Yeast Strains ◽  
Sequencing Studies ◽  
Long Read

AbstractStructural variants (SVs) remain challenging to represent and study relative to point mutations despite their demonstrated importance. We show that variation graphs, as implemented in the vg toolkit, provide an effective means for leveraging SV catalogs for short-read SV genotyping experiments. We benchmarked vg against state-of-the-art SV genotypers using three sequence-resolved SV catalogs generated by recent long-read sequencing studies. In addition, we use assemblies from 12 yeast strains to show that graphs constructed directly from aligned de novo assemblies improve genotyping compared to graphs built from intermediate SV catalogs in the VCF format.

scholarly journals Contiguity: Contig adjacency graph construction and visualisation

2015 ◽  
Author(s):  
Mitchell J Sullivan ◽  
Nouri L Ben Zakour ◽  
Brian M Forde ◽  
Mitchell Stanton-Cook ◽  
Scott A Beatson
Keyword(s):  
De Novo ◽  
Reference Sequence ◽  
De Bruijn Graph ◽  
Interactive Software ◽  
Graph Exploration ◽  
Adjacency Graph ◽  
Highly Sensitive ◽  
Long Read ◽  
Genome Assemblies ◽  
Adjacency Graphs

Contiguity is an interactive software for the visualization and manipulation of de novo genome assemblies. Contiguity creates and displays information on contig adjacency which is contextualized by the simultaneous display of a comparison between assembled contigs and reference sequence. Where scaffolders allow unambiguous connections between contigs to be resolved into a single scaffold, Contiguity allows the user to create all potential scaffolds in ambiguous regions of the genome. This enables the resolution of novel sequence or structural variants from the assembly. In addition, Contiguity provides a sequencing and assembly agnostic approach for the creation of contig adjacency graphs. To maximize the number of contig adjacencies determined, Contiguity combines information from read pair mappings, sequence overlap and De Bruijn graph exploration. We demonstrate how highly sensitive graphs can be achieved using this method. Contig adjacency graphs allow the user to visualize potential arrangements of contigs in unresolvable areas of the genome. By combining adjacency information with comparative genomics, Contiguity provides an intuitive approach for exploring and improving sequence assemblies. It is also useful in guiding manual closure of long read sequence assemblies. Contiguity is an open source application, implemented using Python and the Tkinter GUI package that can run on any Unix, OSX and Windows operating system. It has been designed and optimized for bacterial assemblies. Contiguity is available at http://mjsull.github.io/Contiguity .

scholarly journals Mapping and phasing of structural variation in patient genomes using nanopore sequencing

2017 ◽  
Author(s):  
Mircea Cretu Stancu ◽  
Markus J. van Roosmalen ◽  
Ivo Renkens ◽  
Marleen Nieboer ◽  
Sjors Middelkamp ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Structural Variants ◽  
Human Genetic Disease ◽  
Structural Genomic ◽  
Short Read ◽  
Sequencing Technologies ◽  
Genome Wide ◽  
Long Read ◽  
Complex Structural

AbstractStructural genomic variants form a common type of genetic alteration underlying human genetic disease and phenotypic variation. Despite major improvements in genome sequencing technology and data analysis, the detection of structural variants still poses challenges, particularly when variants are of high complexity. Emerging long-read single-molecule sequencing technologies provide new opportunities for detection of structural variants. Here, we demonstrate sequencing of the genomes of two patients with congenital abnormalities using the ONT MinION at 11x and 16x mean coverage, respectively. We developed a bioinformatic pipeline - NanoSV - to efficiently map genomic structural variants (SVs) from the long-read data. We demonstrate that the nanopore data are superior to corresponding short-read data with regard to detection of de novo rearrangements originating from complex chromothripsis events in the patients. Additionally, genome-wide surveillance of SVs, revealed 3,253 (33%) novel variants that were missed in short-read data of the same sample, the majority of which are duplications < 200bp in size. Long sequencing reads enabled efficient phasing of genetic variations, allowing the construction of genome-wide maps of phased SVs and SNVs. We employed read-based phasing to show that all de novo chromothripsis breakpoints occurred on paternal chromosomes and we resolved the long-range structure of the chromothripsis. This work demonstrates the value of long-read sequencing for screening whole genomes of patients for complex structural variants.

scholarly journals De novo assembly of a Tibetan genome and identification of novel structural variants associated with high-altitude adaptation

2019 ◽  
Vol 7 (2) ◽  
pp. 391-402 ◽  
Author(s):  
Yaoxi He ◽  
Haiyi Lou ◽  
Chaoying Cui ◽  
Lian Deng ◽  
Yang Gao ◽  
...  
Keyword(s):  
High Altitude ◽  
De Novo Assembly ◽  
De Novo ◽  
Population Analysis ◽  
Extreme Environments ◽  
Structural Variants ◽  
Base Pairs ◽  
High Quality ◽  
Long Read ◽  
Altitude Adaptation

Abstract Structural variants (SVs) may play important roles in human adaptation to extreme environments such as high altitude but have been under-investigated. Here, combining long-read sequencing with multiple scaffolding techniques, we assembled a high-quality Tibetan genome (ZF1), with a contig N50 length of 24.57 mega-base pairs (Mb) and a scaffold N50 length of 58.80 Mb. The ZF1 assembly filled 80 remaining N-gaps (0.25 Mb in total length) in the reference human genome (GRCh38). Markedly, we detected 17 900 SVs, among which the ZF1-specific SVs are enriched in GTPase activity that is required for activation of the hypoxic pathway. Further population analysis uncovered a 163-bp intronic deletion in the MKL1 gene showing large divergence between highland Tibetans and lowland Han Chinese. This deletion is significantly associated with lower systolic pulmonary arterial pressure, one of the key adaptive physiological traits in Tibetans. Moreover, with the use of the high-quality de novo assembly, we observed a much higher rate of genome-wide archaic hominid (Altai Neanderthal and Denisovan) shared non-reference sequences in ZF1 (1.32%–1.53%) compared to other East Asian genomes (0.70%–0.98%), reflecting a unique genomic composition of Tibetans. One such archaic hominid shared sequence—a 662-bp intronic insertion in the SCUBE2 gene—is enriched and associated with better lung function (the FEV1/FVC ratio) in Tibetans. Collectively, we generated the first high-resolution Tibetan reference genome, and the identified SVs may serve as valuable resources for future evolutionary and medical studies.

scholarly journals Trio deep-sequencing does not reveal unexpected off-target and on-target mutations in Cas9-edited rhesus monkeys

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Xin Luo ◽  
Yaoxi He ◽  
Chao Zhang ◽  
Xiechao He ◽  
Lanzhen Yan ◽  
...  
Keyword(s):  
Rhesus Monkeys ◽  
De Novo ◽  
Preclinical Model ◽  
Structural Variants ◽  
Sequencing Data ◽  
De Novo Mutations ◽  
Target Region ◽  
Long Read ◽  
Target Effect

AbstractCRISPR-Cas9 is a widely-used genome editing tool, but its off-target effect and on-target complex mutations remain a concern, especially in view of future clinical applications. Non-human primates (NHPs) share close genetic and physiological similarities with humans, making them an ideal preclinical model for developing Cas9-based therapies. However, to our knowledge no comprehensive in vivo off-target and on-target assessment has been conducted in NHPs. Here, we perform whole genome trio sequencing of Cas9-treated rhesus monkeys. We only find a small number of de novo mutations that can be explained by expected spontaneous mutations, and no unexpected off-target mutations (OTMs) were detected. Furthermore, the long-read sequencing data does not detect large structural variants in the target region.

scholarly journals Long-read assembly of the Chinese rhesus macaque genome and identification of ape-specific structural variants

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Yaoxi He ◽  
Xin Luo ◽  
Bin Zhou ◽  
Ting Hu ◽  
Xiaoyu Meng ◽  
...  
Keyword(s):  
Rhesus Macaque ◽  
Genome Assembly ◽  
De Novo ◽  
Gene Annotation ◽  
Large Body ◽  
Phenotypic Traits ◽  
Structural Variants ◽  
De Novo Genome Assembly ◽  
Chinese Rhesus Macaque ◽  
Long Read

Abstract We present a high-quality de novo genome assembly (rheMacS) of the Chinese rhesus macaque (Macaca mulatta) using long-read sequencing and multiplatform scaffolding approaches. Compared to the current Indian rhesus macaque reference genome (rheMac8), rheMacS increases sequence contiguity 75-fold, closing 21,940 of the remaining assembly gaps (60.8 Mbp). We improve gene annotation by generating more than two million full-length transcripts from ten different tissues by long-read RNA sequencing. We sequence resolve 53,916 structural variants (96% novel) and identify 17,000 ape-specific structural variants (ASSVs) based on comparison to ape genomes. Many ASSVs map within ChIP-seq predicted enhancer regions where apes and macaque show diverged enhancer activity and gene expression. We further characterize a subset that may contribute to ape- or great-ape-specific phenotypic traits, including taillessness, brain volume expansion, improved manual dexterity, and large body size. The rheMacS genome assembly serves as an ideal reference for future biomedical and evolutionary studies.

scholarly journals A high-quality genome assembly from a single, field-collected spotted lanternfly (Lycorma delicatula) using the PacBio Sequel II system

GigaScience ◽  
2019 ◽  
Vol 8 (10) ◽  
Author(s):  
Sarah B Kingan ◽  
Julie Urban ◽  
Christine C Lambert ◽  
Primo Baybayan ◽  
Anna K Childers ◽  
...  
Keyword(s):  
Invasive Species ◽  
Genome Assembly ◽  
De Novo ◽  
Fragment Size ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Lycorma Delicatula ◽  
Long Read ◽  
Genome Assemblies ◽  
High Quality Genome

ABSTRACT Background A high-quality reference genome is an essential tool for applied and basic research on arthropods. Long-read sequencing technologies may be used to generate more complete and contiguous genome assemblies than alternate technologies; however, long-read methods have historically had greater input DNA requirements and higher costs than next-generation sequencing, which are barriers to their use on many samples. Here, we present a 2.3 Gb de novo genome assembly of a field-collected adult female spotted lanternfly (Lycorma delicatula) using a single Pacific Biosciences SMRT Cell. The spotted lanternfly is an invasive species recently discovered in the northeastern United States that threatens to damage economically important crop plants in the region. Results The DNA from 1 individual was used to make 1 standard, size-selected library with an average DNA fragment size of ∼20 kb. The library was run on 1 Sequel II SMRT Cell 8M, generating a total of 132 Gb of long-read sequences, of which 82 Gb were from unique library molecules, representing ∼36× coverage of the genome. The assembly had high contiguity (contig N50 length = 1.5 Mb), completeness, and sequence level accuracy as estimated by conserved gene set analysis (96.8% of conserved genes both complete and without frame shift errors). Furthermore, it was possible to segregate more than half of the diploid genome into the 2 separate haplotypes. The assembly also recovered 2 microbial symbiont genomes known to be associated with L. delicatula, each microbial genome being assembled into a single contig. Conclusions We demonstrate that field-collected arthropods can be used for the rapid generation of high-quality genome assemblies, an attractive approach for projects on emerging invasive species, disease vectors, or conservation efforts of endangered species.

scholarly journals High contiguity long read assembly of Brassica nigra allows localization of active centromeres and provides insights into the ancestral Brassica genome

2020 ◽  
Author(s):  
Sampath Perumal ◽  
Chu Shin Koh ◽  
Lingling Jin ◽  
Miles Buchwaldt ◽  
Erin Higgins ◽  
...  
Keyword(s):  
De Novo ◽  
Low Complexity ◽  
Error Rates ◽  
Brassica Nigra ◽  
Genome Integrity ◽  
Ancestral Genome ◽  
Genomic Distance ◽  
Long Read ◽  
Genome Assemblies ◽  
Technology Comparison

AbstractHigh-quality nanopore genome assemblies were generated for two Brassica nigra genotypes (Ni100 and CN115125); a member of the agronomically important Brassica species. The N50 contig length for the two assemblies were 17.1 Mb (58 contigs) and 0.29 Mb (963 contigs), respectively, reflecting recent improvements in the technology. Comparison with a de novo short read assembly for Ni100 corroborated genome integrity and quantified sequence related error rates (0.002%). The contiguity and coverage allowed unprecedented access to low complexity regions of the genome. Pericentromeric regions and coincidence of hypo-methylation enabled localization of active centromeres and identified a novel centromere-associated ALE class I element which appears to have proliferated through relatively recent nested transposition events (<1 million years ago). Computational abstraction was used to define a post-triplication Brassica specific ancestral genome and to calculate the extensive rearrangements that define the genomic distance separating B. nigra from its diploid relatives.

scholarly journals Merqury: reference-free quality, completeness, and phasing assessment for genome assemblies

2020 ◽  
Author(s):  
Arang Rhie ◽  
Brian P. Walenz ◽  
Sergey Koren ◽  
Adam M. Phillippy
Keyword(s):  
De Novo ◽  
High Accuracy ◽  
Link Type ◽  
Base Level ◽  
Project Home Page ◽  
Set Operations ◽  
Assembly Evaluation ◽  
Long Read ◽  
Genome Assemblies ◽  
Reference Genomes

AbstractRecent long-read assemblies often exceed the quality and completeness of available reference genomes, making validation challenging. Here we present Merqury, a novel tool for reference-free assembly evaluation based on efficient k-mer set operations. By comparing k-mers in a de novo assembly to those found in unassembled high-accuracy reads, Merqury estimates base-level accuracy and completeness. For trios, Merqury can also evaluate haplotype-specific accuracy, completeness, phase block continuity, and switch errors. Multiple visualizations, such as k-mer spectrum plots, can be generated for evaluation. We demonstrate on both human and plant genomes that Merqury is a fast and robust method for assembly validation.Availability of data and materialProject name: MerquryProject home page: https://github.com/marbl/merqury, https://github.com/marbl/merylArchived version: https://github.com/marbl/merqury/releases/tag/v1.0Operating system(s): Platform independentProgramming language: C++, Java, PerlOther requirements: gcc 4.8 or higher, java 1.6 or higherLicense: Public domain (see https://github.com/marbl/merqury/blob/master/README.license) Any restrictions to use by non-academics: No restrictions applied

scholarly journals Purge Haplotigs: Synteny Reduction for Third-gen Diploid Genome Assemblies

2018 ◽  
Author(s):  
Michael J Roach ◽  
Simon Schmidt ◽  
Anthony R Borneman
Keyword(s):  
De Novo ◽  
Haplotype Reconstruction ◽  
Minimal Impact ◽  
Variant Discovery ◽  
Rapid Release ◽  
Long Read ◽  
Recent Developments ◽  
Reference Quality ◽  
Downstream Analysis ◽  
Genome Assemblies

AbstractRecent developments in third-gen long read sequencing and diploid-aware assemblers have resulted in the rapid release of numerous reference-quality assemblies for diploid genomes. However, assembling highly heterozygous genomes is still facing a major problem where the two haplotypes for a region are highly polymorphic and the synteny is not recognised during assembly. This causes issues with downstream analysis, for example variant discovery using the haploid assembly, or haplotype reconstruction using the diploid assembly. A new pipeline—Purge Haplotigs—was developed specifically for third-gen assemblies to identify and reassign the duplicate contigs. The pipeline takes a draft haplotype-fused assembly or a diploid assembly, and read alignments to produce an improved assembly. The pipeline was tested on a simulated dataset and on four recent diploid (phased) de novo assemblies from third-generation long-read sequencing. All assemblies after processing with Purge Haplotigs were less duplicated with minimal impact on genome completeness. The software is available at https://bitbucket.org/mroachawri/purge_haplotigs under a permissive MIT licence.

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