Single-molecule real-time sequencing combined with optical mapping yields completely finished fungal genome

Next-generation sequencing (NGS) technologies have increased the scalability, speed, and resolution of genomic sequencing and, thus, have revolutionized genomic studies. However, eukaryotic genome sequencing initiatives typically yield considerably fragmented genome assemblies. Here, we assessed various state-of-the-art sequencing and assembly strategies in order to produce a contiguous and complete eukaryotic genome assembly, focusing on the filamentous fungus Verticillium dahliae. Compared with Illumina-based assemblies of the V. dahliae genome, hybrid assemblies that also include PacBio- generated long reads establish superior contiguity. Intriguingly, provided that sufficient sequence depth is reached, assemblies solely based on PacBio reads outperform hybrid assemblies and even result in fully assembled chromosomes. Furthermore, the addition of optical map data allowed us to produce a gapless and complete V. dahliae genome assembly of the expected eight chromosomes from telomere to telomere. Consequently, we can now study genomic regions that were previously not assembled or poorly assembled, including regions that are populated by repetitive sequences, such as transposons, allowing us to fully appreciate an organism’s biological complexity. Our data show that a combination of PacBio-generated long reads and optical mapping can be used to generate complete and gapless assemblies of fungal genomes. IMPORTANCE Studying whole-genome sequences has become an important aspect of biological research. The advent of nextgeneration sequencing (NGS) technologies has nowadays brought genomic science within reach of most research laboratories, including those that study nonmodel organisms. However, most genome sequencing initiatives typically yield (highly) fragmented genome assemblies. Nevertheless, considerable relevant information related to genome structure and evolution is likely hidden in those nonassembled regions. Here, we investigated a diverse set of strategies to obtain gapless genome assemblies, using the genome of a typical ascomycete fungus as the template. Eventually, we were able to show that a combination of PacBiogenerated long reads and optical mapping yields a gapless telomere-to-telomere genome assembly, allowing in-depth genome sanalyses to facilitate functional studies into an organism’s biology

Sequencing the banana genome (W069)

Bananas (Musa) are the fourth most important crop in developing countries. They are important as an export crop but also play a major role in local food security. Crops of Musa are susceptible to an ever increasing range of diseases requiring massive use of pesticides that have dramatic environmental and health impacts and threaten the sustainability of the crop. There is thus an urgent need for a wider diversity of genetically improved banana cultivars with more robust disease resistance, increased productivity and better adaptability to a large range of growing conditions. The production of export dessert bananas relies on very few related genotypes of the Cavendish subgroup with an AAA genome constitution. However, numerous dessert and cooking types with an AAA, AAB (including plantain) or ABB genome constitution are grown for local consumption. The Musa accession selected for sequencing is a doubled haploid of the accession 'Pahang' (DH Pahang). This accession belongs to the Musa acuminata species (AA genome) malaccensis subspecies. We generated 20x coverage using paired and single 454 reads, complemented by Sanger BESs and ~50 x coverage of Illumina shotgun data. The assembly was performed with Newbler, and the scaffolds were anchored to a genetic map. Genes were predicted using a reconciliation approach taking ESTs, protein sequences and ab initio data as input. A genetic map of the parent accession 'Pahang' was developed with SSR and DArT markers to assemble the scaffolds in pseudo-molecules. (Texte intégral

Are we there yet? : reliably estimating the completeness of plant genome sequences

Genome sequencing is becoming cheaper and faster thanks to the introduction of next-generation sequencing techniques. Dozens of new plant genome sequences have been released in recent years, ranging from small to gigantic repeat-rich or polyploid genomes. Most genome projects have a dual purpose: delivering a contiguous, complete genome assembly and creating a full catalog of correctly predicted genes. Frequently, the completeness of a species' gene catalog is measured using a set of marker genes that are expected to be present. This expectation can be defined along an evolutionary gradient, ranging from highly conserved genes to species-specific genes. Large-scale population resequencing studies have revealed that gene space is fairly variable even between closely related individuals, which limits the definition of the expected gene space, and, consequently, the accuracy of estimates used to assess genome and gene space completeness. We argue that, based on the desired applications of a genome sequencing project, different completeness scores for the genome assembly and/or gene space should be determined. Using examples from several dicot and monocot genomes, we outline some pitfalls and recommendations regarding methods to estimate completeness during different steps of genome assembly and annotation

Pyrosequencing/Sanger Plant Genome Assembly (Limitations, Problems And Solutions) - On The Way To Cucumber (Cucumis sativus L. cv. Borszczagowski) Draft Genome Sequence Publishing

Genome sequencing give us an opportunity to broaden the range and improve the quality of omics studies. New sequencing technologies make it possible to achieve the sequencing reads fast and cheap. Since the assembly step of such next generation reads is still not well standardized it is the most cumbersome part of sequencing projects.
We focus here on the wide range of approaches undertaken to assemble the draft cucumber (Cucumis sativus L. cv. Borszczagowski) genome done by using 12x coverage of pyrosequencing Titanium chemistry shotgun paired and unpaired reads combined with Sanger reads

Toward 959 nematode genomes

The sequencing of the complete genome of the nematode Caenorhabditis elegans was a landmark achievement and ushered in a new era of whole-organism, systems analyses of the biology of this powerful model organism. The success of the C. elegans genome sequencing project also inspired communities working on other organisms to approach genome sequencing of their species. The phylum Nematoda is rich and diverse and of interest to a wide range of research fields from basic biology through ecology and parasitic disease. For all these communities, it is now clear that access to genome scale data will be key to advancing understanding, and in the case of parasites, developing new ways to control or cure diseases. The advent of second-generation sequencing technologies, improvements in computing algorithms and infrastructure and growth in bioinformatics and genomics literacy is making the addition of genome sequencing to the research goals of any nematode research program a less daunting prospect. To inspire, promote and coordinate genomic sequencing across the diversity of the phylum, we have launched a community wiki and the 959 Nematode Genomes initiative (www.nematodegenomes.org/). Just as the deciphering of the developmental lineage of the 959 cells of the adult hermaphrodite C. elegans was the gateway to broad advances in biomedical science, we hope that a nematode phylogeny with (at least) 959 sequenced species will underpin further advances in understanding the origins of parasitism, the dynamics of genomic change and the adaptations that have made Nematoda one of the most successful animal phyla

A Novel Genome-Wide Association Study Approach Using Genotyping by Exome Sequencing Leads to the Identification of a Primary Open Angle Glaucoma Associated Inversion Disrupting ADAMTS17

Closed breeding populations in the dog in conjunction with advances in gene mapping and sequencing techniques facilitate mapping of autosomal recessive diseases and identification of novel disease-causing variants, often using unorthodox experimental designs. In our investigation we demonstrate successful mapping of the locus for primary open angle glaucoma in the Petit Basset Griffon Vendéen dog breed with 12 cases and 12 controls, using a novel genotyping by exome sequencing approach. The resulting genome-wide association signal was followed up by genome sequencing of an individual case, leading to the identification of an inversion with a breakpoint disrupting the ADAMTS17 gene. Genotyping of additional controls and expression analysis provide strong evidence that the inversion is disease causing. Evidence of cryptic splicing resulting in novel exon transcription as a consequence of the inversion in ADAMTS17 is identified through RNAseq experiments. This investigation demonstrates how a novel genotyping by exome sequencing approach can be used to map an autosomal recessive disorder in the dog, with the use of genome sequencing to facilitate identification of a disease-associated variant

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing >50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations