scholarly journals Long-reads assembly of the Brassica napus reference genome, Darmor-bzh

2020 ◽  
Author(s):  
Mathieu Rousseau-Gueutin ◽  
Caroline Belser ◽  
Corinne Da Silva ◽  
Gautier Richard ◽  
Benjamin Istace ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Reference Genome ◽  
Crop Improvement ◽  
Genetic Maps ◽  
Sequencing Data ◽  
Long Reads ◽  
Gene Catalogue ◽  
Genome Assemblies ◽  
Agronomically Important Traits ◽  
Selection Of

AbstractBackgroundThe combination of long-reads and long-range information to produce genome assemblies is now accepted as a common standard. This strategy not only allow to access the gene catalogue of a given species but also reveals the architecture and organisation of chromosomes, including complex regions like telomeres and centromeres. The Brassica genus is not exempt and many assemblies based on long reads are now available. The reference genome for Brassica napus, Darmor-bzh, which was published in 2014, has been produced using short-reads and its contiguity was extremely low if compared to current assemblies of the Brassica genus.FindingsHere, we report the new long-reads assembly of Darmor-bzh genome (Brassica napus) generated by combining long-reads sequencing data, optical and genetic maps. Using the PromethION device and six flowcells, we generated about 16M long-reads representing 93X coverage and more importantly 6X with reads longer than 100Kb. This ultralong-reads dataset allows us to generate one of the most contiguous and complete assembly of a Brassica genome to date (contigs N50 > 10Mb). In addition, we exploited all the advantages of the nanopore technology to detect modified bases and sequence transcriptomic data using direct RNA to annotate the genome and focus on resistance genes.ConclusionUsing these cutting edge technologies, and in particular by relying on all the advantages of the nanopore technology, we provide the most contiguous Brassica napus assembly, a resource that will be valuable for the Brassica community for crop improvement and will facilitate the rapid selection of agronomically important traits.

scholarly journals Long-read assembly of the Brassica napus reference genome Darmor-bzh

GigaScience ◽  
2020 ◽  
Vol 9 (12) ◽  
Author(s):  
Mathieu Rousseau-Gueutin ◽  
Caroline Belser ◽  
Corinne Da Silva ◽  
Gautier Richard ◽  
Benjamin Istace ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Reference Genome ◽  
Crop Improvement ◽  
Genetic Maps ◽  
Sequencing Data ◽  
Long Reads ◽  
Gene Catalogue ◽  
Long Read ◽  
Genome Assemblies ◽  
Selection Of

Abstract Background The combination of long reads and long-range information to produce genome assemblies is now accepted as a common standard. This strategy not only allows access to the gene catalogue of a given species but also reveals the architecture and organization of chromosomes, including complex regions such as telomeres and centromeres. The Brassica genus is not exempt, and many assemblies based on long reads are now available. The reference genome for Brassica napus, Darmor-bzh, which was published in 2014, was produced using short reads and its contiguity was extremely low compared with current assemblies of the Brassica genus. Findings Herein, we report the new long-read assembly of Darmor-bzh genome (Brassica napus) generated by combining long-read sequencing data and optical and genetic maps. Using the PromethION device and 6 flowcells, we generated ∼16 million long reads representing 93× coverage and, more importantly, 6× with reads longer than 100 kb. This ultralong-read dataset allows us to generate one of the most contiguous and complete assemblies of a Brassica genome to date (contig N50 > 10 Mb). In addition, we exploited all the advantages of the nanopore technology to detect modified bases and sequence transcriptomic data using direct RNA to annotate the genome and focus on resistance genes. Conclusion Using these cutting-edge technologies, and in particular by relying on all the advantages of the nanopore technology, we provide the most contiguous Brassica napus assembly, a resource that will be valuable to the Brassica community for crop improvement and will facilitate the rapid selection of agronomically important traits.

scholarly journals GALA: gap-free chromosome-scale assembly with long reads

2020 ◽  
Author(s):  
Mohamed Awad ◽  
Xiangchao Gan
Keyword(s):  
Genome Assembly ◽  
Reference Genome ◽  
De Novo ◽  
Genetic Maps ◽  
Sequencing Data ◽  
C Elegans ◽  
Assembly Method ◽  
Long Reads ◽  
Long Read ◽  
Assembly Technology

AbstractHigh-quality genome assembly has wide applications in genetics and medical studies. However, it is still very challenging to achieve gap-free chromosome-scale assemblies using current workflows for long-read platforms. Here we propose GALA (Gap-free long-read assembler), a chromosome-by-chromosome assembly method implemented through a multi-layer computer graph that identifies mis-assemblies within preliminary assemblies or chimeric raw reads and partitions the data into chromosome-scale linkage groups. The subsequent independent assembly of each linkage group generates a gap-free assembly free from the mis-assembly errors which usually hamper existing workflows. This flexible framework also allows us to integrate data from various technologies, such as Hi-C, genetic maps, a reference genome and even motif analyses, to generate gap-free chromosome-scale assemblies. We de novo assembled the C. elegans and A. thaliana genomes using combined Pacbio and Nanopore sequencing data from publicly available datasets. We also demonstrated the new method’s applicability with a gap-free assembly of a human genome with the help a reference genome. In addition, GALA showed promising performance for Pacbio high-fidelity long reads. Thus, our method enables straightforward assembly of genomes with multiple data sources and overcomes barriers that at present restrict the application of de novo genome assembly technology.

scholarly journals New gains with a slimmer genome – An automated approach to improve reference genome assemblies

2020 ◽  
Author(s):  
Mikko Kivikoski ◽  
Pasi Rastas ◽  
Ari Löytynoja ◽  
Juha Merilä
Keyword(s):  
Gasterosteus Aculeatus ◽  
Reference Genome ◽  
De Novo ◽  
Biological Research ◽  
Integrative Approach ◽  
Sequencing Data ◽  
Pungitius Pungitius ◽  
Model Study ◽  
Long Reads ◽  
Genome Assemblies

AbstractThe utility of genome-wide sequencing data in biological research depends heavily on the quality of the reference genome. Although the reference genomes have improved, it is evident that the assemblies could still be refined, especially in non-model study organisms. Here, we describe an integrative approach to improve contiguity and haploidy of a reference genome assembly. With two novel features of Lep-Anchor software and a combination of dense linkage maps, overlap detection and bridging long reads we generated an improved assembly of the nine-spined stickleback (Pungitius pungitius) reference genome. We were able to remove a significant number of haplotypic contigs, detect more genetic variation and improve the contiguity of the genome, especially that of X chromosome. However, improved scaffolding cannot correct for mosaicism of erroneously assembled contigs, demonstrated by a de novo assembly of a 1.7 Mbp inversion. Qualitatively similar gains were obtained with the genome of three-spined stickleback (Gasterosteus aculeatus).

scholarly journals Genome Assembly of the Popular Korean Soybean Cultivar Hwangkeum

2021 ◽  
Author(s):  
Myung-Shin Kim ◽  
Taeyoung Lee ◽  
Jeonghun Baek ◽  
Ji Hong Kim ◽  
Changhoon Kim ◽  
...  
Keyword(s):  
Reference Genome ◽  
De Novo ◽  
Anthocyanin Biosynthesis ◽  
Genetic Maps ◽  
Soybean Cultivar ◽  
Gene Arrangement ◽  
Nucleotide Polymorphisms ◽  
Sequencing Data ◽  
Crop Species ◽  
Satellite Repeats

AbstractMassive resequencing efforts have been undertaken to catalog allelic variants in major crop species including soybean, but the scope of the information for genetic variation often depends on short sequence reads mapped to the extant reference genome. Additional de novo assembled genome sequences provide a unique opportunity to explore a dispensable genome fraction in the pan-genome of a species. Here, we report the de novo assembly and annotation of Hwangkeum, a popular soybean cultivar in Korea. The assembly was constructed using PromethION nanopore sequencing data and two genetic maps, and was then error-corrected using Illumina short-reads and PacBio SMRT reads. The 933.12 Mb assembly was annotated 79,870 transcripts for 58,550 genes using RNA-Seq data and the public soybean annotation set. Comparison of the Hwangkeum assembly with the Williams 82 soybean reference genome sequence revealed 1.8 million single-nucleotide polymorphisms, 0.5 million indels, and 25 thousand putative structural variants. However, there was no natural megabase-scale chromosomal rearrangement. Incidentally, by adding two novel groups, we found that soybean contains four clearly separated groups of centromeric satellite repeats. Analyses of satellite repeats and gene content suggested that the Hwangkeum assembly is a high-quality assembly. This was further supported by comparison of the marker arrangement of anthocyanin biosynthesis genes and of gene arrangement at the Rsv3 locus. Therefore, the results indicate that the de novo assembly of Hwangkeum is a valuable additional reference genome resource for characterizing traits for the improvement of this important crop species.

scholarly journals AFLAP: Assembly-Free Linkage Analysis Pipeline using k-mers from whole genome sequencing data

2020 ◽  
Author(s):  
Kyle Fletcher ◽  
Lin Zhang ◽  
Juliana Gil ◽  
Rongkui Han ◽  
Keri Cavanaugh ◽  
...  
Keyword(s):  
Linkage Analysis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genetic Map ◽  
Genotyping By Sequencing ◽  
Genetic Maps ◽  
Whole Genome ◽  
Sequencing Data ◽  
Analysis Pipeline ◽  
Genome Assemblies

AbstractBackgroundGenetic maps are an important resource for validation of genome assemblies, trait discovery, and breeding. Next generation sequencing has enabled production of high-density genetic maps constructed with 10,000s of markers. Most current approaches require a genome assembly to identify markers. Our Assembly Free Linkage Analysis Pipeline (AFLAP) removes this requirement by using uniquely segregating k-mers as markers to rapidly construct a genotype table and perform subsequent linkage analysis. This avoids potential biases including preferential read alignment and variant calling.ResultsThe performance of AFLAP was determined in simulations and contrasted to a conventional workflow. We tested AFLAP using 100 F2 individuals of Arabidopsis thaliana, sequenced to low coverage. Genetic maps generated using k-mers contained over 130,000 markers that were concordant with the genomic assembly. The utility of AFLAP was then demonstrated by generating an accurate genetic map using genotyping-by-sequencing data of 235 recombinant inbred lines of Lactuca spp. AFLAP was then applied to 83 F1 individuals of the oomycete Bremia lactucae, sequenced to >5x coverage. The genetic map contained over 90,000 markers ordered in 19 large linkage groups. This genetic map was used to fragment, order, orient, and scaffold the genome, resulting in a much-improved reference assembly.ConclusionsAFLAP can be used to generate high density linkage maps and improve genome assemblies of any organism when a mapping population is available using whole genome sequencing or genotyping-by-sequencing data. Genetic maps produced for B. lactucae were accurately aligned to the genome and guided significant improvements of the reference assembly.

scholarly journals Assembly of chromosome-scale contigs by efficiently resolving repetitive sequences with long reads

2018 ◽  
Author(s):  
Huilong Du ◽  
Chengzhi Liang
Keyword(s):  
Single Molecule ◽  
High Efficiency ◽  
Reference Genome ◽  
Repetitive Sequences ◽  
Sequencing Data ◽  
High Quality ◽  
Single Molecule Sequencing ◽  
Genome Maps ◽  
Long Reads ◽  
Novel Method

AbstractDue to the large number of repetitive sequences in complex eukaryotic genomes, fragmented and incompletely assembled genomes lose value as reference sequences, often due to short contigs that cannot be anchored or mispositioned onto chromosomes. Here we report a novel method Highly Efficient Repeat Assembly (HERA), which includes a new concept called a connection graph as well as algorithms for constructing the graph. HERA resolves repeats at high efficiency with single-molecule sequencing data, and enables the assembly of chromosome-scale contigs by further integrating genome maps and Hi-C data. We tested HERA with the genomes of rice R498, maize B73, human HX1 and Tartary buckwheat Pinku1. HERA can correctly assemble most of the tandemly repetitive sequences in rice using single-molecule sequencing data only. Using the same maize and human sequencing data published by Jiao et al. (2017) and Shi et al. (2016), respectively, we dramatically improved on the sequence contiguity compared with the published assemblies, increasing the contig N50 from 1.3 Mb to 61.2 Mb in maize B73 assembly and from 8.3 Mb to 54.4 Mb in human HX1 assembly with HERA. We provided a high-quality maize reference genome with 96.9% of the gaps filled (only 76 gaps left) and several incorrectly positioned sequences fixed compared with the B73 RefGen_v4 assembly. Comparisons between the HERA assembly of HX1 and the human GRCh38 reference genome showed that many gaps in GRCh38 could be filled, and that GRCh38 contained some potential errors that could be fixed. We assembled the Pinku1 genome into 12 scaffolds with a contig N50 size of 27.85 Mb. HERA serves as a new genome assembly/phasing method to generate high quality sequences for complex genomes and as a curation tool to improve the contiguity and completeness of existing reference genomes, including the correction of assembly errors in repetitive regions.

scholarly journals Telomere-to-telomere gapless chromosomes of banana using nanopore sequencing

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Caroline Belser ◽  
Franc-Christophe Baurens ◽  
Benjamin Noel ◽  
Guillaume Martin ◽  
Corinne Cruaud ◽  
...  
Keyword(s):  
Musa Acuminata ◽  
Genetic Maps ◽  
Nanopore Sequencing ◽  
Genome Coverage ◽  
Long Reads ◽  
Oxford Nanopore ◽  
A Genome ◽  
Long Read ◽  
Genome Assemblies ◽  
First Time

AbstractLong-read technologies hold the promise to obtain more complete genome assemblies and to make them easier. Coupled with long-range technologies, they can reveal the architecture of complex regions, like centromeres or rDNA clusters. These technologies also make it possible to know the complete organization of chromosomes, which remained complicated before even when using genetic maps. However, generating a gapless and telomere-to-telomere assembly is still not trivial, and requires a combination of several technologies and the choice of suitable software. Here, we report a chromosome-scale assembly of a banana genome (Musa acuminata) generated using Oxford Nanopore long-reads. We generated a genome coverage of 177X from a single PromethION flowcell with near 17X with reads longer than 75 kbp. From the 11 chromosomes, 5 were entirely reconstructed in a single contig from telomere to telomere, revealing for the first time the content of complex regions like centromeres or clusters of paralogous genes.

SGTK: a toolkit for visualization and assessment of scaffold graphs

2018 ◽  
Vol 35 (13) ◽  
pp. 2303-2305 ◽  
Author(s):  
Olga Kunyavskaya ◽  
Andrey D Prjibelski
Keyword(s):  
Software Package ◽  
Supplementary Information ◽  
Sequencing Data ◽  
Software Developers ◽  
Long Reads ◽  
Mate Pair ◽  
Linkage Information ◽  
Assembly Pipeline ◽  
Genome Assemblies ◽  
Assembly Software

Abstract Summary Scaffolding is an important step in every genome assembly pipeline, which allows to order contigs into longer sequences using various types of linkage information, such as mate-pair libraries and long reads. In this work, we operate with a notion of a scaffold graph—a graph, vertices of which correspond to the assembled contigs and edges represent connections between them. We present a software package called Scaffold Graph ToolKit that allows to construct and visualize scaffold graphs using different kinds of sequencing data. We show that the scaffold graph appears to be useful for analyzing and assessing genome assemblies, and demonstrate several use cases that can be helpful for both assembly software developers and their users. Availability and implementation SGTK is implemented in C++, Python and JavaScript and is freely available at https://github.com/olga24912/SGTK. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals AFLAP: assembly-free linkage analysis pipeline using k-mers from genome sequencing data

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Kyle Fletcher ◽  
Lin Zhang ◽  
Juliana Gil ◽  
Rongkui Han ◽  
Keri Cavanaugh ◽  
...  
Keyword(s):  
Linkage Analysis ◽  
Genome Sequencing ◽  
Genome Assembly ◽  
Simulated Data ◽  
Genetic Maps ◽  
Sequencing Data ◽  
Analysis Pipeline ◽  
A Genome ◽  
Genotype By Sequencing ◽  
Genome Assemblies

AbstractOur assembly-free linkage analysis pipeline (AFLAP) identifies segregating markers as k-mers in the raw reads without using a reference genome assembly for calling variants and provides genotype tables for the construction of unbiased, high-density genetic maps without a genome assembly. AFLAP is validated and contrasted to a conventional workflow using simulated data. AFLAP is applied to whole genome sequencing and genotype-by-sequencing data of F1, F2, and recombinant inbred populations of two different plant species, producing genetic maps that are concordant with genome assemblies. The AFLAP-based genetic map for Bremia lactucae enables the production of a chromosome-scale genome assembly.

scholarly journals Chromosome-scale reference genome assembly of a diploid potato clone derived from an elite variety

2021 ◽  
Author(s):  
Ruth Freire ◽  
Marius Weisweiler ◽  
Ricardo Guerreiro ◽  
Nadia Baig ◽  
Bruno Hüttel ◽  
...  
Keyword(s):  
Reference Genome ◽  
Solanum Tuberosum L ◽  
Consensus Sequence ◽  
Sequence Divergence ◽  
Potato Genome ◽  
Proximity Ligation ◽  
Final Assembly ◽  
Diploid Clone ◽  
Long Reads ◽  
Genome Assemblies

Abstract Potato (Solanum tuberosum L.) is one of the most important crops with a world-wide production of 370 million metric tons. The objectives of this study were (i) to create a high quality consensus sequence across the two haplotypes of a diploid clone derived from a tetraploid elite variety and assess the sequence divergence from the available potato genome assemblies, as well as among the two haplotypes; (ii) to evaluate the new assembly’s usefulness for various genomic methods and (iii) to assess the performance of phasing in diploid and tetraploid clones, using linked read sequencing technology. We used PacBio long reads coupled with 10x Genomics reads and proximity-ligation scaffolding to create the dAg1_v1.0 reference genome sequence. With a final assembly size of 812 Mb, where 750 Mb are anchored to 12 chromosomes, our assembly is larger than other available potato reference sequences and high proportions of properly paired reads were observed for clones unrelated by pedigree to dAg1. Comparisons of the new dAg1_v1.0 sequence to other potato genome sequences point out the high divergence between the different potato varieties and illustrate the potential of using dAg1_v1.0 sequence in breeding applications.

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