Comparison of long-read methods for sequencing and assembly of a plant genome

Abstract Background Sequencing technologies have advanced to the point where it is possible to generate high-accuracy, haplotype-resolved, chromosome-scale assemblies. Several long-read sequencing technologies are available, and a growing number of algorithms have been developed to assemble the reads generated by those technologies. When starting a new genome project, it is therefore challenging to select the most cost-effective sequencing technology, as well as the most appropriate software for assembly and polishing. It is thus important to benchmark different approaches applied to the same sample. Results Here, we report a comparison of 3 long-read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION), and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of Pacific Biosciences and Nanopore reads. Results obtained from combining long-read technologies or short-read and long-read technologies are also presented. The assemblies were compared for contiguity, base accuracy, and completeness, as well as sequencing costs and DNA material requirements. Conclusions The 3 long-read technologies produced highly contiguous and complete genome assemblies of M. jansenii. At the time of sequencing, the cost associated with each method was significantly different, but continuous improvements in technologies have resulted in greater accuracy, increased throughput, and reduced costs. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.

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Comparison of long read methods for sequencing and assembly of a plant genome

10.1101/2020.03.16.992933 ◽

2020 ◽

Cited By ~ 1

Author(s):

Valentine Murigneux ◽

Subash Kumar Rai ◽

Agnelo Furtado ◽

Timothy J.C. Bruxner ◽

Wei Tian ◽

...

Keyword(s):

De Novo ◽

Cost Effective ◽

Genome Project ◽

Plant Genome ◽

Sequencing Data ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Long Read ◽

The Cost ◽

Genome Assemblies

AbstractSequencing technologies have advanced to the point where it is possible to generate high accuracy, haplotype resolved, chromosome scale assemblies. Several long read sequencing technologies are available on the market and a growing number of algorithms have been developed over the last years to assemble the reads generated by those technologies. When starting a new genome project, it is therefore challenging to select the most cost-effective sequencing technology as well as the most appropriate software for assembly and polishing. For this reason, it is important to benchmark different approaches applied to the same sample. Here, we report a comparison of three long read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION) and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of PacBio and Nanopore reads. Results obtained from combining long read technologies or short read and long read technologies are also presented. The assemblies were compared for contiguity, accuracy and completeness as well as sequencing costs and DNA material requirements. Overall, the three long read technologies produced highly contiguous and complete genome assemblies of Macadamia jansenii. At the time of sequencing, the cost associated with each method was significantly different but continuous improvements in technologies have resulted in greater accuracy, increased throughput and reduced costs. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.

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

10.1101/2021.01.25.428044 ◽

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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Genome sequences of horticultural plants: past, present, and future

Horticulture Research ◽

10.1038/s41438-019-0195-6 ◽

2019 ◽

Vol 6 (1) ◽

Cited By ~ 14

Author(s):

Fei Chen ◽

Yunfeng Song ◽

Xiaojiang Li ◽

Junhao Chen ◽

Lan Mo ◽

...

Keyword(s):

Data Storage ◽

Genome Sequencing ◽

Herbal Medicines ◽

Genome Project ◽

Plant Genome ◽

Quality Data ◽

Sequencing Data ◽

Sequencing Technologies ◽

Horticultural Plant ◽

Horticultural Plants

Abstract Horticultural plants play various and critical roles for humans by providing fruits, vegetables, materials for beverages, and herbal medicines and by acting as ornamentals. They have also shaped human art, culture, and environments and thereby have influenced the lifestyles of humans. With the advent of sequencing technologies, there has been a dramatic increase in the number of sequenced genomes of horticultural plant species in the past decade. The genomes of horticultural plants are highly diverse and complex, often with a high degree of heterozygosity and a high ploidy due to their long and complex history of evolution and domestication. Here we summarize the advances in the genome sequencing of horticultural plants, the reconstruction of pan-genomes, and the development of horticultural genome databases. We also discuss past, present, and future studies related to genome sequencing, data storage, data quality, data sharing, and data visualization to provide practical guidance for genomic studies of horticultural plants. Finally, we propose a horticultural plant genome project as well as the roadmap and technical details toward three goals of the project.

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Evaluation of Germline Structural Variant Calling Methods for Nanopore Sequencing Data

Frontiers in Genetics ◽

10.3389/fgene.2021.761791 ◽

2021 ◽

Vol 12 ◽

Author(s):

Davide Bolognini ◽

Alberto Magi

Keyword(s):

Variant Calling ◽

Research Report ◽

Nanopore Sequencing ◽

Sequencing Data ◽

Factors Affecting ◽

Sequencing Technologies ◽

Long Reads ◽

Oxford Nanopore ◽

Sequencing Studies ◽

Long Read

Structural variants (SVs) are genomic rearrangements that involve at least 50 nucleotides and are known to have a serious impact on human health. While prior short-read sequencing technologies have often proved inadequate for a comprehensive assessment of structural variation, more recent long reads from Oxford Nanopore Technologies have already been proven invaluable for the discovery of large SVs and hold the potential to facilitate the resolution of the full SV spectrum. With many long-read sequencing studies to follow, it is crucial to assess factors affecting current SV calling pipelines for nanopore sequencing data. In this brief research report, we evaluate and compare the performances of five long-read SV callers across four long-read aligners using both real and synthetic nanopore datasets. In particular, we focus on the effects of read alignment, sequencing coverage, and variant allele depth on the detection and genotyping of SVs of different types and size ranges and provide insights into precision and recall of SV callsets generated by integrating the various long-read aligners and SV callers. The computational pipeline we propose is publicly available at https://github.com/davidebolo1993/EViNCe and can be adjusted to further evaluate future nanopore sequencing datasets.

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A chromosome-scale assembly of the sorghum genome using nanopore sequencing and optical mapping

10.1101/327817 ◽

2018 ◽

Cited By ~ 2

Author(s):

Stáphane Deschamps ◽

Yun Zhang ◽

Victor Llaca ◽

Liang Ye ◽

Gregory May ◽

...

Keyword(s):

Sorghum Bicolor ◽

De Novo ◽

Hybrid Assembly ◽

The Public ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Long Read ◽

Optical Maps ◽

Eukaryotic Genomes ◽

Direct Label

The advent of long-read sequencing technologies has greatly facilitated assemblies of large eukaryotic genomes. In this paper, Oxford Nanopore sequences generated on a MinION sequencer were combined with BioNano Genomics Direct Label and Stain (DLS) optical maps to generate a chromosome-scale de novo assembly of the repeat-rich Sorghum bicolor Tx430 genome. The final hybrid assembly consists of 29 scaffolds, encompassing in most cases entire chromosome arms. It has a scaffold N50 value of 33.28Mbps and covers >90% of Sorghum bicolor expected genome length. A sequence accuracy of 99.67% was obtained in unique regions after aligning contigs against Illumina Tx430 data. Alignments showed that 99.4% of the 34,211 public gene models are present in the assembly, including 94.2% mapping end-to-end. Comparisons of the DLS optical maps against the public Sorghum Bicolor v3.0.1 BTx623 genome assembly suggest the presence of substantial genomic rearrangements whose origin remains to be determined.

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Extensive sequencing of seven human genomes to characterize benchmark reference materials

10.1101/026468 ◽

2015 ◽

Cited By ~ 9

Author(s):

Justin M Zook ◽

David Catoe ◽

Jennifer McDaniel ◽

Lindsay Vang ◽

Noah Spies ◽

...

Keyword(s):

Human Genome ◽

Reference Materials ◽

De Novo ◽

Variant Calling ◽

Genome Project ◽

Genome Comparison ◽

Personal Genome ◽

Sequencing Data ◽

Sequencing Technologies ◽

Human Genomes

The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCodeTM WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly.

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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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Draft genome assemblies using sequencing reads from Oxford Nanopore Technology and Illumina platforms for four species of North American Fundulus killifish

GigaScience ◽

10.1093/gigascience/giaa067 ◽

2020 ◽

Vol 9 (6) ◽

Cited By ~ 3

Author(s):

Lisa K Johnson ◽

Ruta Sahasrabudhe ◽

James Anthony Gill ◽

Jennifer L Roach ◽

Lutz Froenicke ◽

...

Keyword(s):

North American ◽

De Novo ◽

Draft Genome ◽

Whole Genome Sequencing Data ◽

Sequencing Data ◽

Sequence Coverage ◽

Short Read ◽

Oxford Nanopore ◽

Long Read ◽

Genome Assemblies

Abstract Background Whole-genome sequencing data from wild-caught individuals of closely related North American killifish species (Fundulus xenicus, Fundulus catenatus, Fundulus nottii, and Fundulus olivaceus) were obtained using long-read Oxford Nanopore Technology (ONT) PromethION and short-read Illumina platforms. Findings Draft de novo reference genome assemblies were generated using a combination of long and short sequencing reads. For each species, the PromethION platform was used to generate 30–45× sequence coverage, and the Illumina platform was used to generate 50–160× sequence coverage. Illumina-only assemblies were fragmented with high numbers of contigs, while ONT-only assemblies were error prone with low BUSCO scores. The highest N50 values, ranging from 0.4 to 2.7 Mb, were from assemblies generated using a combination of short- and long-read data. BUSCO scores were consistently >90% complete using the Eukaryota database. Conclusions High-quality genomes can be obtained from a combination of using short-read Illumina data to polish assemblies generated with long-read ONT data. Draft assemblies and raw sequencing data are available for public use. We encourage use and reuse of these data for assembly benchmarking and other analyses.

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SVJedi: Genotyping structural variations with long reads

10.1101/849208 ◽

2019 ◽

Author(s):

Lolita Lecompte ◽

Pierre Peterlongo ◽

Dominique Lavenier ◽

Claire Lemaitre

Keyword(s):

Sequencing Data ◽

Structural Variations ◽

Short Read ◽

Sequencing Technologies ◽

Long Reads ◽

Oxford Nanopore ◽

Clinical Diagnoses ◽

Long Read ◽

Reference Sequences ◽

The One

AbstractMotivationStudies on structural variants (SV) are expanding rapidly. As a result, and thanks to third generation sequencing technologies, the number of discovered SVs is increasing, especially in the human genome. At the same time, for several applications such as clinical diagnoses, it is important to genotype newly sequenced individuals on well defined and characterized SVs. Whereas several SV genotypers have been developed for short read data, there is a lack of such dedicated tool to assess whether known SVs are present or not in a new long read sequenced sample, such as the one produced by Pacific Biosciences or Oxford Nanopore Technologies.ResultsWe present a novel method to genotype known SVs from long read sequencing data. The method is based on the generation of a set of reference sequences that represent the two alleles of each structural variant. Long reads are aligned to these reference sequences. Alignments are then analyzed and filtered out to keep only informative ones, to quantify and estimate the presence of each SV allele and the allele frequencies. We provide an implementation of the method, SVJedi, to genotype insertions and deletions with long reads. The tool has been applied to both simulated and real human datasets and achieves high genotyping accuracy. We also demonstrate that SV genotyping is considerably improved with SVJedi compared to other approaches, namely SV discovery and short read SV genotyping approaches.Availabilityhttps://github.com/llecompte/[email protected]

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Clustering de Novo by Gene of Long Reads from Transcriptomics Data

10.1101/170035 ◽

2017 ◽

Cited By ~ 3

Author(s):

Camille Marchet ◽

Lolita Lecompte ◽

Corinne Da Silva ◽

Corinne Cruaud ◽

Jean-Marc Aury ◽

...

Keyword(s):

De Novo ◽

Free Access ◽

Sequencing Data ◽

Base Pairs ◽

Long Reads ◽

Oxford Nanopore ◽

Processing Step ◽

Whole Transcriptome Sequencing ◽

Long Read ◽

Transcriptomics Data

AbstractLong-read sequencing currently provides sequences of several thousand base pairs. This allows to obtain complete transcripts, which offers an un-precedented vision of the cellular transcriptome.However the literature is lacking tools to cluster such data de novo, in particular for Oxford Nanopore Technologies reads, because of the inherent high error rate compared to short reads.Our goal is to process reads from whole transcriptome sequencing data accurately and without a reference genome in order to reliably group reads coming from the same gene. This de novo approach is therefore particularly suitable for non-model species, but can also serve as a useful pre-processing step to improve read mapping. Our contribution is both to propose a new algorithm adapted to clustering of reads by gene and a practical and free access tool that permits to scale the complete processing of eukaryotic transcriptomes.We sequenced a mouse RNA sample using the MinION device, this dataset is used to compare our solution to other algorithms used in the context of biological clustering. We demonstrate its is better-suited for transcriptomics long reads. When a reference is available thus mapping possible, we show that it stands as an alternative method that predicts complementary clusters.

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