scholarly journals Biodiversity genomics of small metazoans: high quality de novo genomes from single specimens of field-collected and ethanol-preserved springtails

2020 ◽  
Author(s):  
Clément Schneider ◽  
Christian Woehle ◽  
Carola Greve ◽  
Cyrille A. D’Haese ◽  
Magnus Wolf ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
De Novo ◽  
Genetic Model ◽  
Nuclear Genome ◽  
High Quality ◽  
Sequencing Technologies ◽  
Natural Product Research ◽  
Genome Wide ◽  
Long Read ◽  
Hmw Dna

ABSTRACTGenome sequencing of all known eukaryotes on Earth promises unprecedented advances in evolutionary sciences, ecology, systematics and in biodiversity-related applied fields such as environmental management and natural product research. Advances in DNA sequencing technologies make genome sequencing feasible for many non-genetic model species. However, genome sequencing today relies on large quantities of high quality, high molecular weight (HMW) DNA which is mostly obtained from fresh tissues. This is problematic for biodiversity genomics of Metazoa as most species are small and yield minute amounts of DNA. Furthermore, briging living specimens to the lab bench not realistic for the majority of species.Here we overcome those difficulties by sequencing two species of springtails (Collembola) from single specimens preserved in ethanol. We used a newly developed, genome-wide amplification-based protocol to generate PacBio libraries for HiFi long-read sequencing.The assembled genomes were highly continuous. They can be considered complete as we recovered over 95% of BUSCOs. Genome-wide amplification does not seem to bias genome recovery. Presence of almost complete copies of the mitochondrial genome in the nuclear genome were pitfalls for automatic assemblers. The genomes fit well into an existing phylogeny of springtails. A neotype is designated for one of the species, blending genome sequencing and creation of taxonomic references.Our study shows that it is possible to obtain high quality genomes from small, field-preserved sub-millimeter metazoans, thus making their vast diversity accessible to the fields of genomics.

scholarly journals Two high-quality de novo genomes from single ethanol-preserved specimens of tiny metazoans (Collembola)

GigaScience ◽  
2021 ◽  
Vol 10 (5) ◽  
Author(s):  
Clément Schneider ◽  
Christian Woehle ◽  
Carola Greve ◽  
Cyrille A D'Haese ◽  
Magnus Wolf ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
De Novo ◽  
Genetic Model ◽  
High Quality ◽  
High Molecular Weight Dna ◽  
Mobile Species ◽  
Natural Product Research ◽  
A Genome ◽  
Long Read

Abstract Background Genome sequencing of all known eukaryotes on Earth promises unprecedented advances in biological sciences and in biodiversity-related applied fields such as environmental management and natural product research. Advances in long-read DNA sequencing make it feasible to generate high-quality genomes for many non–genetic model species. However, long-read sequencing today relies on sizable quantities of high-quality, high molecular weight DNA, which is mostly obtained from fresh tissues. This is a challenge for biodiversity genomics of most metazoan species, which are tiny and need to be preserved immediately after collection. Here we present de novo genomes of 2 species of submillimeter Collembola. For each, we prepared the sequencing library from high molecular weight DNA extracted from a single specimen and using a novel ultra-low input protocol from Pacific Biosciences. This protocol requires a DNA input of only 5 ng, permitted by a whole-genome amplification step. Results The 2 assembled genomes have N50 values >5.5 and 8.5 Mb, respectively, and both contain ∼96% of BUSCO genes. Thus, they are highly contiguous and complete. The genomes are supported by an integrative taxonomy approach including placement in a genome-based phylogeny of Collembola and designation of a neotype for 1 of the species. Higher heterozygosity values are recorded in the more mobile species. Both species are devoid of the biosynthetic pathway for β-lactam antibiotics known in several Collembola, confirming the tight correlation of antibiotic synthesis with the species way of life. Conclusions It is now possible to generate high-quality genomes from single specimens of minute, field-preserved metazoans, exceeding the minimum contig N50 (1 Mb) required by the Earth BioGenome Project.

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 A comprehensive toolkit to enable MinION sequencing in any laboratory

2018 ◽  
Author(s):  
Miriam Schalamun ◽  
David Kainer ◽  
Eleanor Beavan ◽  
Ramawatar Nagar ◽  
David Eccles ◽  
...  
Keyword(s):  
Read Length ◽  
Full Potential ◽  
Sequencing Data ◽  
Special Equipment ◽  
High Quality ◽  
Sequencing Technologies ◽  
Entry Cost ◽  
Long Read ◽  
Hmw Dna

AbstractLong-read sequencing technologies are transforming our ability to assemble highly complex genomes. Realising their full potential relies crucially on extracting high quality, high molecular weight (HMW) DNA from the organisms of interest. This is especially the case for the portable MinION sequencer which potentiates all laboratories to undertake their own genome sequencing projects, due to its low entry cost and minimal spatial footprint. One challenge of the MinION is that each group has to independently establish effective protocols for using the instrument, which can be time consuming and costly. Here we present a workflow and protocols that enabled us to establish MinION sequencing in our own laboratories, based on optimising DNA extractions from a challenging plant tissue as a case study. Following the workflow illustrated we were able to reliably and repeatedly obtain > 8.5 Gb of long read sequencing data with a mean read length of 13 kb and an N50 of 26 kb. Our protocols are open-source and can be performed in any laboratory without special equipment. We also illustrate some more elaborate workflows which can increase mean and average read lengths if this is desired. We envision that our workflow for establishing MinION sequencing, including the illustration of potential pitfalls, will be useful to others who plan to establish long-read sequencing in their own laboratories.

scholarly journals Whole-genome sequencing and de novo assembly of a 2019 novel coronavirus (SARS-CoV-2) strain isolated in Vietnam

2020 ◽  
Vol 18 (2) ◽  
pp. 197-208
Author(s):  
Le Tung Lam ◽  
Nguyen Trung Hieu ◽  
Nguyen Hong Trang ◽  
Ho Thi Thuong ◽  
Tran Huyen Linh ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Sanger Sequencing ◽  
De Novo ◽  
Whole Genome ◽  
Smrt Sequencing ◽  
Sequencing Technology ◽  
High Quality ◽  
Long Read ◽  
Novel Coronavirus

The pandemic COVID-19 caused by the virus SARS-CoV-2 has devastated countries worldwide, infecting more than 4.5 million people and leading to more than 300,000 deaths as of May 16th, 2020. Whole-genome sequencing (WGS) is an effective tool to monitor emerging strains and provide information for intervention, thus help to inform outbreak control decisions. Here, we reported the first effort to sequence and de novo assemble the whole genome of SARS-CoV-2 using PacBio’s SMRT sequencing technology in Vietnam. We also presented the annotation results and a brief analysis of the variants found in our SARS-CoV-2 strain, which was isolated from a Vietnamese patient. The sequencing was successfully completed and de novo assembled in less than 30 hours, resulting in one contig with no gap and a length of 29,766 bp. All detected variants as compared to the NCBI reference were highly accurate, as confirmed by Sanger sequencing. The results have shown the potential of long read sequencing to provide high quality WGS data to support public health responses and advance understanding of this and future pandemics.

scholarly journals A high-quality de novo genome assembly based on nanopore sequencing of a wild-caught coconut rhinoceros beetle (Oryctes rhinoceros)

2021 ◽  
Author(s):  
Igor Filipović ◽  
Gordana Rašić ◽  
James Hereward ◽  
Maria Gharuka ◽  
Gregor J Devine ◽  
...  
Keyword(s):  
Genome Assembly ◽  
De Novo ◽  
Nuclear Genome ◽  
Assembly Process ◽  
Structural Annotation ◽  
High Quality ◽  
Oryctes Rhinoceros ◽  
Rhinoceros Beetle ◽  
Long Read ◽  
Genome Assemblies

Background: An optimal starting point for relating genome function to organismal biology is a high-quality nuclear genome assembly, and long-read sequencing is revolutionizing the production of this genomic resource in insects. Despite this, nuclear genome assemblies have been under-represented for agricultural insect pests, particularly from the order Coleoptera. Here we present a de novo genome assembly and structural annotation for the coconut rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae), based on Oxford Nanopore Technologies (ONT) long-read data generated from a wild-caught female, as well as the assembly process that also led to the recovery of the complete circular genome assemblies of the beetle's mitochondrial genome and that of the biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). As an invasive pest of palm trees, O. rhinoceros is undergoing an expansion in its range across the Pacific Islands, requiring new approaches to management that may include strategies facilitated by genome assembly and annotation. Results: High-quality DNA isolated from an adult female was used to create four ONT libraries that were sequenced using four MinION flow cells, producing a total of 27.2 Gb of high-quality long-read sequences. We employed an iterative assembly process and polishing with one lane of high-accuracy Illumina reads, obtaining a final size of the assembly of 377.36 Mb that had high contiguity (fragment N50 length = 12 Mb) and accuracy, as evidenced by the exceptionally high completeness of the benchmarked set of conserved single-copy orthologous genes (BUSCO completeness = 99.11%). These quality metrics place our assembly as the most complete of the published Coleopteran genomes. The structural annotation of the nuclear genome assembly contained a highly-accurate set of 16,371 protein-coding genes showing BUSCO completeness of 92.09%, as well as the expected number of non-coding RNAs and the number and structure of paralogous genes in a gene family like Sigma GST. Conclusions: The genomic resources produced in this study form a foundation for further functional genetic research and management programs that may inform the control and surveillance of O. rhinoceros populations, and we demonstrate the efficacy of de novo genome assembly using long-read ONT data from a single field-caught insect.

scholarly journals WENGAN: Efficient and high quality hybrid de novo assembly of human genomes

2019 ◽  
Author(s):  
Alex Di Genova ◽  
Elena Buena-Atienza ◽  
Stephan Ossowski ◽  
Marie-France Sagot
Keyword(s):  
De Novo ◽  
Computational Cost ◽  
Sequence Information ◽  
Sequencing Data ◽  
High Quality ◽  
Sequencing Technologies ◽  
Human Genomes ◽  
Long Reads ◽  
Long Read ◽  
Genome Assemblies

The continuous improvement of long-read sequencing technologies along with the development of ad-doc algorithms has launched a new de novo assembly era that promises high-quality genomes. However, it has proven difficult to use only long reads to generate accurate genome assemblies of large, repeat-rich human genomes. To date, most of the human genomes assembled from long error-prone reads add accurate short reads to further polish the consensus quality. Here, we report the development of a novel algorithm for hybrid assembly, WENGAN, and the de novo assembly of four human genomes using a combination of sequencing data generated on ONT PromethION, PacBio Sequel, Illumina and MGI technology. WENGAN implements efficient algorithms that exploit the sequence information of short and long reads to tackle assembly contiguity as well as consensus quality. The resulting genome assemblies have high contiguity (contig NG50:16.67-62.06 Mb), few assembly errors (contig NGA50:10.9-45.91 Mb), good consensus quality (QV:27.79-33.61), and high gene completeness (BUSCO complete: 94.6-95.1%), while consuming low computational resources (CPU hours:153-1027). In particular, the WENGAN assembly of the haploid CHM13 sample achieved a contig NG50 of 62.06 Mb (NGA50:45.91 Mb), which surpasses the contiguity of the current human reference genome (GRCh38 contig NG50:57.88 Mb). Providing highest quality at low computational cost, WENGAN is an important step towards the democratization of the de novo assembly of human genomes. The WENGAN assembler is available at https://github.com/adigenova/wengan

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

2019 ◽  
Author(s):  
Ouzhuluobu ◽  
Yaoxi He ◽  
Haiyi Lou ◽  
Chaoying Cui ◽  
Lian Deng ◽  
...  
Keyword(s):  
High Altitude ◽  
De Novo ◽  
Population Analysis ◽  
Extreme Environments ◽  
Structural Variants ◽  
Base Pairs ◽  
High Quality ◽  
Genome Wide ◽  
Long Read ◽  
Altitude Adaptation

AbstractStructural variants (SVs) may play important roles in human adaption 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 Hapo-G, haplotype-aware polishing of genome assemblies with accurate reads

2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Jean-Marc Aury ◽  
Benjamin Istace
Keyword(s):  
Single Molecule ◽  
Direct Consequence ◽  
High Quality ◽  
Sequencing Errors ◽  
Coding Regions ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genome Assemblies

Abstract Single-molecule sequencing technologies have recently been commercialized by Pacific Biosciences and Oxford Nanopore with the promise of sequencing long DNA fragments (kilobases to megabases order) and then, using efficient algorithms, provide high quality assemblies in terms of contiguity and completeness of repetitive regions. However, the error rate of long-read technologies is higher than that of short-read technologies. This has a direct consequence on the base quality of genome assemblies, particularly in coding regions where sequencing errors can disrupt the coding frame of genes. In the case of diploid genomes, the consensus of a given gene can be a mixture between the two haplotypes and can lead to premature stop codons. Several methods have been developed to polish genome assemblies using short reads and generally, they inspect the nucleotide one by one, and provide a correction for each nucleotide of the input assembly. As a result, these algorithms are not able to properly process diploid genomes and they typically switch from one haplotype to another. Herein we proposed Hapo-G (Haplotype-Aware Polishing Of Genomes), a new algorithm capable of incorporating phasing information from high-quality reads (short or long-reads) to polish genome assemblies and in particular assemblies of diploid and heterozygous genomes.

scholarly journals Identifying the causes and consequences of assembly gaps using a multiplatform genome assembly of a bird-of-paradise

2019 ◽  
Author(s):  
Valentina Peona ◽  
Mozes P.K. Blom ◽  
Luohao Xu ◽  
Reto Burri ◽  
Shawn Sullivan ◽  
...  
Keyword(s):  
Dark Matter ◽  
Genome Assembly ◽  
Sex Chromosome ◽  
De Novo ◽  
Model Organism ◽  
Technology Choice ◽  
High Quality ◽  
Sequencing Technologies ◽  
Downstream Analysis ◽  
Genome Assemblies

AbstractGenome assemblies are currently being produced at an impressive rate by consortia and individual laboratories. The low costs and increasing efficiency of sequencing technologies have opened up a whole new world of genomic biodiversity. Although these technologies generate high-quality genome assemblies, there are still genomic regions difficult to assemble, like repetitive elements and GC-rich regions (genomic “dark matter”). In this study, we compare the efficiency of currently used sequencing technologies (short/linked/long reads and proximity ligation maps) and combinations thereof in assembling genomic dark matter starting from the same sample. By adopting different de-novo assembly strategies, we were able to compare each individual draft assembly to a curated multiplatform one and identify the nature of the previously missing dark matter with a particular focus on transposable elements, multi-copy MHC genes, and GC-rich regions. Thanks to this multiplatform approach, we demonstrate the feasibility of producing a high-quality chromosome-level assembly for a non-model organism (paradise crow) for which only suboptimal samples are available. Our approach was able to reconstruct complex chromosomes like the repeat-rich W sex chromosome and several GC-rich microchromosomes. Telomere-to-telomere assemblies are not a reality yet for most organisms, but by leveraging technology choice it is possible to minimize genome assembly gaps for downstream analysis. We provide a roadmap to tailor sequencing projects around the completeness of both the coding and non-coding parts of the genomes.

scholarly journals LeafGo: Leaf to Genome, a quick workflow to produce high-quality De novo genomes with Third Generation Sequencing technology

2021 ◽  
Author(s):  
Patrick Driguez ◽  
Salim Bougouffa ◽  
Karen Carty ◽  
Alexander Putra ◽  
Kamel Jabbari ◽  
...  
Keyword(s):  
De Novo ◽  
Rapid Development ◽  
Plant Genome ◽  
Plant Genomics ◽  
High Quality ◽  
High Molecular Weight Dna ◽  
Tissue Samples ◽  
Sequencing Technologies ◽  
The Cost ◽  
New Generation

AbstractRecent years have witnessed a rapid development of sequencing technologies. Fundamental differences and limitations among various platforms impact the time, the cost and the accuracy for sequencing whole genomes. Here we designed a complete de novo plant genome generation workflow that starts from plant tissue samples and produces high-quality draft genomes with relatively modest laboratory and bioinformatic resources within seven days. To optimize our workflow we selected different species of plants which were used to extract high molecular weight DNA, to make PacBio and ONT libraries for sequencing with the Sequel I, Sequel II and GridION platforms. We assembled high-quality draft genomes of two different Eucalyptus species E. rudis, and E. camaldulensis to chromosome level without using additional scaffolding technologies. For the rapid production of de novo genome assembly of plant species we showed that our DNA extraction protocol followed by PacBio high fidelity sequencing, and assembly with new generation assemblers such as hifiasm produce excellent results. Our findings will be a valuable benchmark for groups planning wet- and dry-lab plant genomics research and for high throughput plant genomics initiatives.

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