Improvements in the sequencing and assembly of plant genomes

Advances in DNA sequencing have made it easier to sequence and assemble plant genomes. Here, we extend an earlier study, and compare recent methods for long read sequencing and assembly. Updated Oxford Nanopore Technology software improved assemblies. Using more accurate sequences produced by repeated sequencing of the same molecule (Pacific Biosciences HiFi) resulted in less fragmented assembly of sequencing reads. Using data for increased genome coverage resulted in longer contigs, but reduced total assembly length and improved genome completeness. The original model species, Macadamia jansenii, was also compared with three other Macadamia species, as well as avocado (Persea americana) and jojoba (Simmondsia chinensis). In these angiosperms, increasing sequence data volumes caused a linear increase in contig size, decreased assembly length and further improved already high completeness. Differences in genome size and sequence complexity influenced the success of assembly. Advances in long read sequencing technology continue to improve plant genome sequencing and assembly. However, results were improved by greater genome coverage, with the amount needed to achieve a particular level of assembly being species dependent.

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Improvements in the Sequencing and Assembly of Plant Genomes

10.1101/2021.01.22.427724 ◽

2021 ◽

Author(s):

Priyanka Sharma ◽

Othman Aldossary ◽

Bader Alsubaie ◽

Ibrahim Al-Mssallem ◽

Onka Nath ◽

...

Keyword(s):

Sequence Data ◽

Linear Increase ◽

Persea Americana ◽

Sequencing Technology ◽

Model Species ◽

Genome Coverage ◽

Plant Genomes ◽

Sequence Complexity ◽

Oxford Nanopore ◽

Long Read

AbstractBackgroundAdvances in DNA sequencing have reduced the difficulty of sequencing and assembling plant genomes. A range of methods for long read sequencing and assembly have been recently compared and we now extend the earlier study and report a comparison with more recent methods.ResultsUpdated Oxford Nanopore Technology software supported improved assemblies. The use of more accurate sequences produced by repeated sequencing of the same molecule (PacBio HiFi) resulted in much less fragmented assembly of sequencing reads. The use of more data to give increased genome coverage resulted in longer contigs (higher N50) but reduced the total length of the assemblies and improved genome completeness (BUSCO). The original model species, Macadamia jansenii, a basal eudicot, was also compared with the 3 other Macadamia species and with avocado (Persea americana), a magnoliid, and jojoba (Simmondsia chinensis) a core eudicot. In these phylogenetically diverse angiosperms, increasing sequence data volumes also caused a highly linear increase in contig size, decreased assembly length and further improved already high completeness. Differences in genome size and sequence complexity apparently influenced the success of assembly from these different species.ConclusionsAdvances in long read sequencing technology have continued to significantly improve the results of sequencing and assembly of plant genomes. However, results were consistently improved by greater genome coverage (using an increased number of reads) with the amount needed to achieve a particular level of assembly being species dependant.

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LeafGo: Leaf to Genome, a quick workflow to produce high-quality de novo plant genomes using long-read sequencing technology

Genome Biology ◽

10.1186/s13059-021-02475-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Patrick Driguez ◽

Salim Bougouffa ◽

Karen Carty ◽

Alexander Putra ◽

Kamel Jabbari ◽

...

Keyword(s):

De Novo ◽

Plant Genome ◽

Time Cost ◽

Sequencing Technology ◽

High Quality ◽

Plant Genomes ◽

Long Read ◽

Generate Plant ◽

Sequencing Platforms ◽

Chromosome Level

AbstractCurrently, different sequencing platforms are used to generate plant genomes and no workflow has been properly developed to optimize time, cost, and assembly quality. We present LeafGo, a complete de novo plant genome workflow, that starts from tissue and produces genomes with modest laboratory and bioinformatic resources in approximately 7 days and using one long-read sequencing technology. LeafGo is optimized with ten different plant species, three of which are used to generate high-quality chromosome-level assemblies without any scaffolding technologies. Finally, we report the diploid genomes of Eucalyptus rudis and E. camaldulensis and the allotetraploid genome of Arachis hypogaea.

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Oxford Nanopore sequencing: new opportunities for plant genomics?

Journal of Experimental Botany ◽

10.1093/jxb/eraa263 ◽

2020 ◽

Vol 71 (18) ◽

pp. 5313-5322 ◽

Cited By ~ 2

Author(s):

Kathryn Dumschott ◽

Maximilian H-W Schmidt ◽

Harmeet Singh Chawla ◽

Rod Snowdon ◽

Björn Usadel

Keyword(s):

Plant Genome ◽

Third Generation ◽

Plant Genomics ◽

High Coverage ◽

Plant Genomes ◽

Sequencing Technologies ◽

Third Generation Sequencing ◽

Oxford Nanopore ◽

Long Read ◽

Generation Sequencing

Abstract DNA sequencing was dominated by Sanger’s chain termination method until the mid-2000s, when it was progressively supplanted by new sequencing technologies that can generate much larger quantities of data in a shorter time. At the forefront of these developments, long-read sequencing technologies (third-generation sequencing) can produce reads that are several kilobases in length. This greatly improves the accuracy of genome assemblies by spanning the highly repetitive segments that cause difficulty for second-generation short-read technologies. Third-generation sequencing is especially appealing for plant genomes, which can be extremely large with long stretches of highly repetitive DNA. Until recently, the low basecalling accuracy of third-generation technologies meant that accurate genome assembly required expensive, high-coverage sequencing followed by computational analysis to correct for errors. However, today’s long-read technologies are more accurate and less expensive, making them the method of choice for the assembly of complex genomes. Oxford Nanopore Technologies (ONT), a third-generation platform for the sequencing of native DNA strands, is particularly suitable for the generation of high-quality assemblies of highly repetitive plant genomes. Here we discuss the benefits of ONT, especially for the plant science community, and describe the issues that remain to be addressed when using ONT for plant genome sequencing.

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Constructing a Reference Genome in a Single Lab: The Possibility to Use Oxford Nanopore Technology

Plants ◽

10.3390/plants8080270 ◽

2019 ◽

Vol 8 (8) ◽

pp. 270 ◽

Cited By ~ 4

Author(s):

Yun Gyeong Lee ◽

Sang Chul Choi ◽

Yuna Kang ◽

Kyeong Min Kim ◽

Chon-Sik Kang ◽

...

Keyword(s):

Plant Species ◽

Genome Sequencing ◽

Reference Genome ◽

Genome Structure ◽

Plant Genome ◽

Sequence Information ◽

Sequencing Analysis ◽

Oxford Nanopore ◽

A Genome ◽

Long Read

The whole genome sequencing (WGS) has become a crucial tool in understanding genome structure and genetic variation. The MinION sequencing of Oxford Nanopore Technologies (ONT) is an excellent approach for performing WGS and it has advantages in comparison with other Next-Generation Sequencing (NGS): It is relatively inexpensive, portable, has simple library preparation, can be monitored in real-time, and has no theoretical limits on reading length. Sorghum bicolor (L.) Moench is diploid (2n = 2x = 20) with a genome size of about 730 Mb, and its genome sequence information is released in the Phytozome database. Therefore, sorghum can be used as a good reference. However, plant species have complex and large genomes when compared to animals or microorganisms. As a result, complete genome sequencing is difficult for plant species. MinION sequencing that produces long-reads can be an excellent tool for overcoming the weak assembly of short-reads generated from NGS by minimizing the generation of gaps or covering the repetitive sequence that appears on the plant genome. Here, we conducted the genome sequencing for S. bicolor cv. BTx623 while using the MinION platform and obtained 895,678 reads and 17.9 gigabytes (Gb) (ca. 25× coverage of reference) from long-read sequence data. A total of 6124 contigs (covering 45.9%) were generated from Canu, and a total of 2661 contigs (covering 50%) were generated from Minimap and Miniasm with a Racon through a de novo assembly using two different tools and mapped assembled contigs against the sorghum reference genome. Our results provide an optimal series of long-read sequencing analysis for plant species while using the MinION platform and a clue to determine the total sequencing scale for optimal coverage that is based on various genome sizes.

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Genomic dynamics of species and mobile genetic elements in a prolonged blaIMP-4-associated carbapenemase outbreak in an Australian hospital

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz526 ◽

2020 ◽

Vol 75 (4) ◽

pp. 873-882 ◽

Cited By ~ 3

Author(s):

A Kizny Gordon ◽

H T T Phan ◽

S I Lipworth ◽

E Cheong ◽

T Gottlieb ◽

...

Keyword(s):

Resistance Genes ◽

Genetic Relatedness ◽

Sequence Data ◽

Environmental Isolates ◽

Class 1 Integron ◽

Oxford Nanopore ◽

Class 1 ◽

Long Read ◽

Cleaning Methods ◽

Burns Unit

Abstract Background Hospital outbreaks of carbapenemase-producing organisms, such as blaIMP-4-containing organisms, are an increasing threat to patient safety. Objectives To investigate the genomic dynamics of a 10 year (2006–15) outbreak of blaIMP-4-containing organisms in a burns unit in a hospital in Sydney, Australia. Methods All carbapenem-non-susceptible or MDR clinical isolates (2006–15) and a random selection of equivalent or ESBL-producing environmental isolates (2012–15) were sequenced [short-read (Illumina), long-read (Oxford Nanopore Technology)]. Sequence data were used to assess genetic relatedness of isolates (Mash; mapping and recombination-adjusted phylogenies), perform in silico typing (MLST, resistance genes and plasmid replicons) and reconstruct a subset of blaIMP plasmids for comparative plasmid genomics. Results A total of 46/58 clinical and 67/96 environmental isolates contained blaIMP-4. All blaIMP-4-positive organisms contained five or more other resistance genes. Enterobacter cloacae was the predominant organism, with 12 other species mainly found in either the environment or patients, some persisting despite several cleaning methods. On phylogenetic analysis there were three genetic clusters of E. cloacae containing both clinical and environmental isolates, and an additional four clusters restricted to either reservoir. blaIMP-4 was mostly found as part of a cassette array (blaIMP-4-qacG2-aacA4-catB3) in a class 1 integron within a previously described IncM2 plasmid (pEl1573), with almost complete conservation of this cassette across the species over the 10 years. Several other plasmids were also implicated, including an IncF plasmid backbone not previously widely described in association with blaIMP-4. Conclusions Genetic backgrounds disseminating blaIMP-4 can persist, diversify and evolve amongst both human and environmental reservoirs during a prolonged outbreak despite intensive prevention efforts.

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BleTIES: Annotation of natural genome editing in ciliates using long read sequencing

10.1101/2021.05.18.444610 ◽

2021 ◽

Author(s):

Brandon K. B. Seah ◽

Estienne C. Swart

Keyword(s):

Dna Sequences ◽

Sequence Data ◽

Low Complexity ◽

Supplementary Information ◽

Neighboring Element ◽

Sequencing Technologies ◽

Long Reads ◽

Oxford Nanopore ◽

Long Read ◽

Element Elimination

Ciliates are single-celled eukaryotes that eliminate specific, interspersed DNA sequences (internally eliminated sequences, IESs) from their genomes during development. These are challenging to annotate and assemble because IES-containing sequences are much less abundant in the cell than those without, and IES sequences themselves often contain repetitive and low-complexity sequences. Long read sequencing technologies from Pacific Biosciences and Oxford Nanopore have the potential to reconstruct longer IESs than has been possible with short reads, and also the ability to detect correlations of neighboring element elimination. Here we present BleTIES, a software toolkit for detecting, assembling, and analyzing IESs using mapped long reads. Availability and implementation: BleTIES is implemented in Python 3. Source code is available at https://github.com/Swart-lab/bleties (MIT license), and also distributed via Bioconda. Contact: [email protected] Supplementary information: Benchmarking of BleTIES with published sequence data.

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

Communications Biology ◽

10.1038/s42003-021-02559-3 ◽

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.

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Review on the Development and Applications of Medicinal Plant Genomes

Frontiers in Plant Science ◽

10.3389/fpls.2021.791219 ◽

2021 ◽

Vol 12 ◽

Author(s):

Qi-Qing Cheng ◽

Yue Ouyang ◽

Zi-Yu Tang ◽

Chi-Chou Lao ◽

Yan-Yu Zhang ◽

...

Keyword(s):

Medicinal Plants ◽

Medicinal Plant ◽

Sequencing Technology ◽

Effective Utilization ◽

Next Generation Sequencing Technology ◽

Plant Genomes ◽

Sequencing Technologies ◽

Long Read ◽

Genetic Level ◽

Sustainable Protection

With the development of sequencing technology, the research on medicinal plants is no longer limited to the aspects of chemistry, pharmacology, and pharmacodynamics, but reveals them from the genetic level. As the price of next-generation sequencing technology becomes affordable, and the long-read sequencing technology is established, the medicinal plant genomes with large sizes have been sequenced and assembled more easily. Although the review of plant genomes has been reported several times, there is no review giving a systematic and comprehensive introduction about the development and application of medicinal plant genomes that have been reported until now. Here, we provide a historical perspective on the current situation of genomes in medicinal plant biology, highlight the use of the rapidly developing sequencing technologies, and conduct a comprehensive summary on how the genomes apply to solve the practical problems in medicinal plants, like genomics-assisted herb breeding, evolution history revelation, herbal synthetic biology study, and geoherbal research, which are important for effective utilization, rational use and sustainable protection of medicinal plants.

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Rapid, raw-read reference and identification (R4IDs): A flexible platform for rapid generic species ID using long-read sequencing technology

10.1101/281048 ◽

2018 ◽

Cited By ~ 2

Author(s):

Joe Parker ◽

Andrew Helmstetter ◽

James Crowe ◽

John Iacona ◽

Dion Devey ◽

...

Keyword(s):

Dna Sequencing ◽

Species Identification ◽

Sequence Data ◽

Vascular Plant ◽

Reference Sequence ◽

Read Length ◽

Reference Database ◽

Sequencing Technology ◽

Long Read ◽

Suitable Reference

AbstractThe versatility of the current DNA sequencing platforms and the development of portable, nanopore sequencers means that it has never been easier to collect genetic data for unknown sample ID. DNA barcoding and meta-barcoding have become increasingly popular and barcode databases continue to grow at an impressive rate. However, the number of canonical genome assemblies (reference or draft) that are publically available is relatively tiny, hindering the more widespread use of genome scale DNA sequencing technology for accurate species identification and discovery. Here, we show that rapid raw-read reference datasets, or R4IDs for short, generated in a matter of hours on the Oxford Nanopore MinION, can bridge this gap and accelerate the generation of useable reference sequence data. By exploiting the long read length of this technology, shotgun genomic sequencing of a small portion of an organism’s genome can act as a suitable reference database despite the low sequencing coverage. These R4IDs can then be used for accurate species identification with minimal amounts of re-sequencing effort (1000s of reads). We demonstrated the capabilities of this approach with six vascular plant species for which we created R4IDs in the laboratory and then re-sequenced, live at the Kew Science Festival 2016. We further validated our method using simulations to determine the broader applicability of the approach. Our data analysis pipeline has been made available as a Dockerised workflow for simple, scalable deployment for a range of uses.

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Draft Genome Sequence of Pandrug-Resistant Pseudomonas aeruginosa SPA03, Isolated from a Patient with Benign Prostatic Hyperplasia

Microbiology Resource Announcements ◽

10.1128/mra.00336-21 ◽

2021 ◽

Vol 10 (22) ◽

Author(s):

Chanakya Pachi Pulusu ◽

Balaram Khamari ◽

Manmath Lama ◽

Arun Sai Kumar Peketi ◽

Prakash Kumar ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Benign Prostatic Hyperplasia ◽

Sequence Data ◽

Draft Genome ◽

Prostatic Hyperplasia ◽

Illumina Hiseq ◽

Content Type ◽

Oxford Nanopore ◽

Long Read ◽

Oxford Nanopore Technologies

The draft genome of pandrug-resistant Pseudomonas aeruginosa strain SPA03, which belongs to global high-risk sequence type 357 (ST357) and was isolated from a patient with benign prostatic hyperplasia, is presented in this report. The genome assembly was generated by combining short-read Illumina HiSeq-X Ten and long-read Oxford Nanopore Technologies MinION sequence data using the Unicycler assembler.

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