De novo Assembly of the Brugia malayi Genome Using Long Reads from a Single MinION Flowcell

AbstractFilarial nematode infections cause a substantial global disease burden. Genomic studies of filarial worms can improve our understanding of their biology and epidemiology. However, genomic information from field isolates is limited and available reference genomes are often discontinuous. Single molecule sequencing technologies can reduce the cost of genome sequencing and long reads produced from these devices can improve the contiguity and completeness of genome assemblies. In addition, these new technologies can make generation and analysis of large numbers of field isolates feasible. In this study, we assessed the performance of the Oxford Nanopore Technologies MinION for sequencing and assembling the genome of Brugia malayi, a human parasite widely used in filariasis research. Using data from a single MinION flowcell, a 90.3 Mb nuclear genome was assembled into 202 contigs with an N50 of 2.4 Mb. This assembly covered 96.9% of the well-defined B. malayi reference genome with 99.2% identity. The complete mitochondrial genome was obtained with individual reads and the nearly complete genome of the endosymbiotic bacteria Wolbachia was assembled alongside the nuclear genome. Long-read data from the MinION produced an assembly that approached the quality of a well-established reference genome using comparably fewer resources.

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Illumina TruSeq synthetic long-reads empowerde novoassembly and resolve complex, highly repetitive transposable elements

10.1101/001834 ◽

2014 ◽

Cited By ~ 1

Author(s):

Rajiv C McCoy ◽

Ryan W Taylor ◽

Timothy A Blauwkamp ◽

Joanna L Kelley ◽

Michael Kertesz ◽

...

Keyword(s):

Transposable Elements ◽

Reference Genome ◽

De Novo ◽

Model Organism ◽

Genomic Analysis ◽

High Sequence Identity ◽

Current Reference ◽

Sequencing Technologies ◽

Long Reads ◽

Whole Genomes

High-throughput DNA sequencing technologies have revolutionized genomic analysis, including thede novoassembly of whole genomes. Nevertheless, assembly of complex genomes remains challenging, in part due to the presence of dispersed repeats which introduce ambiguity during genome reconstruction. Transposable elements (TEs) can be particularly problematic, especially for TE families exhibiting high sequence identity, high copy number, or present in complex genomic arrangements. While TEs strongly affect genome function and evolution, most currentde novoassembly approaches cannot resolve long, identical, and abundant families of TEs. Here, we applied a novel Illumina technology called TruSeq synthetic long-reads, which are generated through highly parallel library preparation and local assembly of short read data and achieve lengths of 1.5-18.5 Kbp with an extremely low error rate (∼0.03% per base). To test the utility of this technology, we sequenced and assembled the genome of the model organismDrosophila melanogaster(reference genome strainy;cn,bw,sp) achieving an N50 contig size of 69.7 Kbp and covering 96.9% of the euchromatic chromosome arms of the current reference genome. TruSeq synthetic long-read technology enables placement of individual TE copies in their proper genomic locations as well as accurate reconstruction of TE sequences. We entirely recovered and accurately placed 4,229 (77.8%) of the 5,434 of annotated transposable elements with perfect identity to the current reference genome. As TEs are ubiquitous features of genomes of many species, TruSeq synthetic long- reads, and likely other methods that generate long reads, offer a powerful approach to improvede novoassemblies of whole genomes.

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An improved de novo assembly and annotation of the tomato reference genome using single-molecule sequencing, Hi-C proximity ligation and optical maps

10.1101/767764 ◽

2019 ◽

Cited By ~ 16

Author(s):

Prashant S. Hosmani ◽

Mirella Flores-Gonzalez ◽

Henri van de Geest ◽

Florian Maumus ◽

Linda V. Bakker ◽

...

Keyword(s):

Single Molecule ◽

Reference Genome ◽

De Novo ◽

Proximity Ligation ◽

Contact Maps ◽

Long Reads ◽

Blast Database ◽

Optical Maps ◽

Almost All ◽

454 Sequences

AbstractThe original Heinz 1706 reference genome was produced by a large team of scientists from across the globe from a variety of input sources that included 454 sequences in addition to full-length BACs, BAC and fosmid ends sequenced with Sanger technology. We present here the latest tomato reference genome (SL4.0) assembled de novo from PacBio long reads and scaffolded using Hi-C contact maps. The assembly was validated using Bionano optical maps and 10X linked-read sequences. This assembly is highly contiguous with fewer gaps compared to previous genome builds and almost all scaffolds have been anchored and oriented to the 12 tomato chromosomes. We have found more repeats compared to the previous versions and one of the largest repeat classes identified are the LTR retrotransposons. We also describe updates to the reference genome and annotation since the last publication. The corresponding ITAG4.0 annotation has 4,794 novel genes along with 29,281 genes preserved from ITAG2.4. Most of the updated genes have extensions in the 5’ and 3’ UTRs resulting in doubling of annotated UTRs per gene. The genome and annotation can be accessed using SGN through BLAST database, Pathway database (SolCyc), Apollo, JBrowse genome browser and FTP available at https://solgenomics.net.

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Highly-accurate long-read sequencing improves variant detection and assembly of a human genome

10.1101/519025 ◽

2019 ◽

Cited By ~ 27

Author(s):

Aaron M. Wenger ◽

Paul Peluso ◽

William J. Rowell ◽

Pi-Chuan Chang ◽

Richard J. Hall ◽

...

Keyword(s):

Single Molecule ◽

De Novo ◽

Structural Variants ◽

Short Reads ◽

Sequencing Technologies ◽

Long Reads ◽

Long Read ◽

Variant Detection ◽

High Quality Genome ◽

Circular Consensus Sequencing

AbstractThe major DNA sequencing technologies in use today produce either highly-accurate short reads or noisy long reads. We developed a protocol based on single-molecule, circular consensus sequencing (CCS) to generate highly-accurate (99.8%) long reads averaging 13.5 kb and applied it to sequence the well-characterized human HG002/NA24385. We optimized existing tools to comprehensively detect variants, achieving precision and recall above 99.91% for SNVs, 95.98% for indels, and 95.99% for structural variants. We estimate that 2,434 discordances are correctable mistakes in the high-quality Genome in a Bottle benchmark. Nearly all (99.64%) variants are phased into haplotypes, which further improves variant detection. De novo assembly produces a highly contiguous and accurate genome with contig N50 above 15 Mb and concordance of 99.998%. CCS reads match short reads for small variant detection, while enabling structural variant detection and de novo assembly at similar contiguity and markedly higher concordance than noisy long reads.

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Reference-free reconstruction and quantification of transcriptomes from Nanopore long-read sequencing

10.1101/2020.02.08.939942 ◽

2020 ◽

Author(s):

Ivan de la Rubia ◽

Joel A. Indi ◽

Silvia Carbonell-Sala ◽

Julien Lagarde ◽

M Mar Albà ◽

...

Keyword(s):

Single Molecule ◽

Reference Genome ◽

Simulated Data ◽

Cost Effective ◽

Dna Assembly ◽

Sequencing Data ◽

Consensus Sequences ◽

Sequencing Technologies ◽

Long Reads ◽

Long Read

AbstractSingle-molecule long-read sequencing with Nanopore provides an unprecedented opportunity to measure transcriptomes from any sample1–3. However, current analysis methods rely on the comparison with a reference genome or transcriptome2,4,5, or the use of multiple sequencing technologies6,7, thereby precluding cost-effective studies in species with no genome assembly available, in individuals underrepresented in the existing reference, and for the discovery of disease-specific transcripts not directly identifiable from a reference genome. Methods for DNA assembly8–10 cannot be directly transferred to transcriptomes since their consensus sequences lack the required interpretability for genes with multiple transcript isoforms. To address these challenges, we have developed RATTLE, the first tool to perform reference-free reconstruction and quantification of transcripts from Nanopore long reads. Using simulated data, isoform spike-ins, and sequencing data from tissues and cell lines, we demonstrate that RATTLE accurately determines transcript sequence and abundance, is comparable to reference-based methods, and shows saturation in the number of predicted transcripts with increasing number of input reads.

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Hapo-G, haplotype-aware polishing of genome assemblies with accurate reads

NAR Genomics and Bioinformatics ◽

10.1093/nargab/lqab034 ◽

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.

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AStrap: identification of alternative splicing from transcript sequences without a reference genome

Bioinformatics ◽

10.1093/bioinformatics/bty1008 ◽

2018 ◽

Vol 35 (15) ◽

pp. 2654-2656 ◽

Cited By ~ 5

Author(s):

Guoli Ji ◽

Wenbin Ye ◽

Yaru Su ◽

Moliang Chen ◽

Guangzao Huang ◽

...

Keyword(s):

Machine Learning ◽

Alternative Splicing ◽

Single Molecule ◽

Reference Genome ◽

De Novo ◽

Supplementary Information ◽

Model Organisms ◽

Sequencing Data ◽

Extensive Evaluation ◽

Reference Genomes

Abstract Summary Alternative splicing (AS) is a well-established mechanism for increasing transcriptome and proteome diversity, however, detecting AS events and distinguishing among AS types in organisms without available reference genomes remains challenging. We developed a de novo approach called AStrap for AS analysis without using a reference genome. AStrap identifies AS events by extensive pair-wise alignments of transcript sequences and predicts AS types by a machine-learning model integrating more than 500 assembled features. We evaluated AStrap using collected AS events from reference genomes of rice and human as well as single-molecule real-time sequencing data from Amborella trichopoda. Results show that AStrap can identify much more AS events with comparable or higher accuracy than the competing method. AStrap also possesses a unique feature of predicting AS types, which achieves an overall accuracy of ∼0.87 for different species. Extensive evaluation of AStrap using different parameters, sample sizes and machine-learning models on different species also demonstrates the robustness and flexibility of AStrap. AStrap could be a valuable addition to the community for the study of AS in non-model organisms with limited genetic resources. Availability and implementation AStrap is available for download at https://github.com/BMILAB/AStrap. Supplementary information Supplementary data are available at Bioinformatics online.

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RECORD: Reference-Assisted Genome Assembly for Closely Related Genomes

International Journal of Genomics ◽

10.1155/2015/563482 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Krisztian Buza ◽

Bartek Wilczynski ◽

Norbert Dojer

Keyword(s):

Reference Genome ◽

De Novo ◽

Real Data ◽

Reference Sequence ◽

Individual Genome ◽

Single Experiment ◽

Sequencing Technologies ◽

Sequencing Cost ◽

The Individual ◽

Assembly Software

Background. Next-generation sequencing technologies are now producing multiple times the genome size in total reads from a single experiment. This is enough information to reconstruct at least some of the differences between the individual genome studied in the experiment and the reference genome of the species. However, in most typical protocols, this information is disregarded and the reference genome is used.Results. We provide a new approach that allows researchers to reconstruct genomes very closely related to the reference genome (e.g., mutants of the same species) directly from the reads used in the experiment. Our approach applies de novo assembly software to experimental reads and so-called pseudoreads and uses the resulting contigs to generate a modified reference sequence. In this way, it can very quickly, and at no additional sequencing cost, generate new, modified reference sequence that is closer to the actual sequenced genome and has a full coverage. In this paper, we describe our approach and test its implementation called RECORD. We evaluate RECORD on both simulated and real data. We made our software publicly available on sourceforge.Conclusion. Our tests show that on closely related sequences RECORD outperforms more general assisted-assembly software.

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Mapping and phasing of structural variation in patient genomes using nanopore sequencing

10.1101/129379 ◽

2017 ◽

Cited By ~ 4

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.

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The sequencing and de novo assembly of the Larimichthys crocea genome using PacBio and Hi-C technologies

Scientific Data ◽

10.1038/s41597-019-0194-3 ◽

2019 ◽

Vol 6 (1) ◽

Cited By ~ 8

Author(s):

Baohua Chen ◽

Zhixiong Zhou ◽

Qiaozhen Ke ◽

Yidi Wu ◽

Huaqiang Bai ◽

...

Keyword(s):

Marine Fish ◽

Single Molecule ◽

Large Scale ◽

Reference Genome ◽

De Novo ◽

Larimichthys Crocea ◽

Chromosome Conformation ◽

Protein Coding ◽

Total Length ◽

Chromosome Level

Abstract Larimichthys crocea is an endemic marine fish in East Asia that belongs to Sciaenidae in Perciformes. L. crocea has now been recognized as an “iconic” marine fish species in China because not only is it a popular food fish in China, it is a representative victim of overfishing and still provides high value fish products supported by the modern large-scale mariculture industry. Here, we report a chromosome-level reference genome of L. crocea generated by employing the PacBio single molecule sequencing technique (SMRT) and high-throughput chromosome conformation capture (Hi-C) technologies. The genome sequences were assembled into 1,591 contigs with a total length of 723.86 Mb and a contig N50 length of 2.83 Mb. After chromosome-level scaffolding, 24 scaffolds were constructed with a total length of 668.67 Mb (92.48% of the total length). Genome annotation identified 23,657 protein-coding genes and 7262 ncRNAs. This highly accurate, chromosome-level reference genome of L. crocea provides an essential genome resource to support the development of genome-scale selective breeding and restocking strategies of L. crocea.

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Computational Approaches for Transcriptome Assembly Based on Sequencing Technologies

Current Bioinformatics ◽

10.2174/1574893614666190410155603 ◽

2020 ◽

Vol 15 (1) ◽

pp. 2-16

Author(s):

Yuwen Luo ◽

Xingyu Liao ◽

Fang-Xiang Wu ◽

Jianxin Wang

Keyword(s):

De Novo ◽

Transcriptome Assembly ◽

Critical Role ◽

High Sensitivity ◽

Biological Properties ◽

Sequencing Data ◽

Sequencing Technologies ◽

Long Reads ◽

Massive Sequencing ◽

Generation Sequencing

Transcriptome assembly plays a critical role in studying biological properties and examining the expression levels of genomes in specific cells. It is also the basis of many downstream analyses. With the increase of speed and the decrease in cost, massive sequencing data continues to accumulate. A large number of assembly strategies based on different computational methods and experiments have been developed. How to efficiently perform transcriptome assembly with high sensitivity and accuracy becomes a key issue. In this work, the issues with transcriptome assembly are explored based on different sequencing technologies. Specifically, transcriptome assemblies with next-generation sequencing reads are divided into reference-based assemblies and de novo assemblies. The examples of different species are used to illustrate that long reads produced by the third-generation sequencing technologies can cover fulllength transcripts without assemblies. In addition, different transcriptome assemblies using the Hybrid-seq methods and other tools are also summarized. Finally, we discuss the future directions of transcriptome assemblies.

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