OPERA-LG: Efficient and exact scaffolding of large, repeat-rich eukaryotic genomes with performance guarantees

The assembly of large, repeat-rich eukaryotic genomes continues to represent a significant challenge in genomics. While long-read technologies have made the high-quality assembly of small, microbial genomes increasingly feasible, data generation can be prohibitively expensive for larger genomes. Fundamental advances in assembly algorithms are thus essential to exploit the characteristics of short and long-read sequencing technologies to consistently and reliably provide high-qualities assemblies in a cost-efficient manner. Here we present a scalable, exact algorithm (OPERA-LG) for the scaffold assembly of large, repeat-rich genomes that exhibits almost an order of magnitude improvement over the state-of-the-art programs in both correctness (>5X on average) and contiguity (>10X). This provides a systematic approach for combining data from different sequencing technologies, as well as a rigorous framework for scaffolding of repetitive sequences. OPERA-LG represents the first in a new class of algorithms that can efficiently assemble large genomes while providing formal guarantees about assembly quality, providing an avenue for systematic augmentation and improvement of 1000s of existing draft eukaryotic genome assemblies.

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Expanding an expanded genome: long-read sequencing ofTrypanosoma cruzi

10.1101/279174 ◽

2018 ◽

Cited By ~ 3

Author(s):

Luisa Berná ◽

Matías Rodríguez ◽

María Laura Chiribao ◽

Adriana Parodi-Talice ◽

Sebastián Pita ◽

...

Keyword(s):

Repetitive Sequences ◽

Gc Content ◽

Gene Copy ◽

Accurate Estimation ◽

Sequencing Technologies ◽

Copy Numbers ◽

Long Reads ◽

Long Read ◽

Large Clusters ◽

Gene Copy Numbers

Although the genome ofTrypanosoma cruzi, the causative agent of Chagas disease, was first made available in 2005, with additional strains reported later, the intrinsic genome complexity of this parasite (abundance of repetitive sequences and genes organized in tandem) has traditionally hindered high-quality genome assembly and annotation. This also limits diverse types of analyses that require high degree of precision. Long reads generated by third-generation sequencing technologies are particularly suitable to address the challenges associated withT. cruzi´sgenome since they permit directly determining the full sequence of large clusters of repetitive sequences without collapsing them. This, in turn, allows not only accurate estimation of gene copy numbers but also circumvents assembly fragmentation. Here, we present the analysis of the genome sequences of twoT. cruziclones: the hybrid TCC (DTU TcVI) and the non-hybrid Dm28c (DTU TcI), determined by PacBio SMRT technology. The improved assemblies herein obtained permitted us to accurately estimate gene copy numbers, abundance and distribution of repetitive sequences (including satellites and retroelements). We found that the genome ofT. cruziis composed of a "core compartment" and a "disruptive compartment" which exhibit opposite gene and GC content composition. New tandem and disperse repetitive sequences were identified, including some located inside coding sequences. Additionally, homologous chromosomes were separately assembled, allowing us to retrieve haplotypes as separate contigs instead of a unique mosaic sequence. Finally, manual annotation of surface multigene families MUC and trans-sialidases allows now a better overview of these complex groups of genes.

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Picopore: A tool for reducing the storage size of Oxford Nanopore Technologies datasets without loss of functionality

F1000Research ◽

10.12688/f1000research.11022.3 ◽

2017 ◽

Vol 6 ◽

pp. 227 ◽

Cited By ~ 1

Author(s):

Scott Gigante

Keyword(s):

Data Storage ◽

Data Generation ◽

Biologically Relevant ◽

Sequencing Technologies ◽

Long Term Storage ◽

Oxford Nanopore ◽

Long Read ◽

Oxford Nanopore Technologies ◽

Term Storage

Oxford Nanopore Technologies' (ONT's) MinION and PromethION long-read sequencing technologies are emerging as genuine alternatives to established Next-Generation Sequencing technologies. A combination of the highly redundant file format and a rapid increase in data generation have created a significant problem both for immediate data storage on MinION-capable laptops, and for long-term storage on lab data servers. We developed Picopore, a software suite offering three methods of compression. Picopore's lossless and deep lossless methods provide a 25% and 44% average reduction in size, respectively, without removing any data from the files. Picopore's raw method provides an 88% average reduction in size, while retaining biologically relevant data for the end-user. All methods have the capacity to run in real-time in parallel to a sequencing run, reducing demand for both immediate and long-term storage space.

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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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Benchmarking metagenomic classification tools for long-read sequencing data

10.1101/2020.11.25.397729 ◽

2020 ◽

Author(s):

Josip Marić ◽

Krešimir Križanović ◽

Sylvain Riondet ◽

Niranjan Nagarajan ◽

Mile Šikić

Keyword(s):

De Novo ◽

Real Life ◽

Metagenomic Analysis ◽

Sequencing Data ◽

De Novo Genome Assembly ◽

Sequencing Technologies ◽

Long Reads ◽

Species Abundances ◽

Long Read ◽

Eukaryotic Genomes

ABSTRACTIn recent years, both long-read sequencing and metagenomic analysis have been significantly advanced. Although long-read sequencing technologies have been primarily used for de novo genome assembly, they are rapidly maturing for widespread use in other applications. In particular, long reads could potentially lead to more precise taxonomic identification, which has sparked an interest in using them for metagenomic analysis.Here we present a benchmark of several state-of-the-art tools for metagenomic taxonomic classification, tested on in-silico datasets constructed using real long reads from isolate sequencing. We compare tools that were either newly developed or modified to work with long reads, including k-mer based tools Kraken2, Centrifuge and CLARK, and mapping-based tools MetaMaps and MEGAN-LR. The test datasets were constructed with varying numbers of bacterial and eukaryotic genomes to simulate different real-life metagenomic applications. The tools were tested to detect species accurately and precisely estimate species abundances in the samples.Our analysis shows that all tested classifiers provide useful results, and the composition of the used database strongly influences the performance. Using the same database, tested tools achieve comparable results except for MetaMaps, which slightly outperform others in most metrics, but it is significantly slower than k-mer based tools.We deem there is significant room for improvement for all tested tools, especially in lowering the number of false-positive detections.

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Picopore: A tool for reducing the storage size of Oxford Nanopore Technologies datasets without loss of functionality

F1000Research ◽

10.12688/f1000research.11022.1 ◽

2017 ◽

Vol 6 ◽

pp. 227 ◽

Cited By ~ 2

Author(s):

Scott Gigante

Keyword(s):

Data Storage ◽

Data Generation ◽

Biologically Relevant ◽

Sequencing Technologies ◽

Long Term Storage ◽

Oxford Nanopore ◽

Long Read ◽

Oxford Nanopore Technologies ◽

Term Storage

Oxford Nanopore Technologies' (ONT) MinION and PromethION long-read sequencing technologies are emerging as genuine alternatives to established Next-Generation Sequencing technologies. A combination of the highly redundant file format and a rapid increase in data generation have created a significant problem both for immediate data storage on MinION-capable laptops, and for long-term storage on lab data servers. We developed Picopore, a software suite offering three methods of compression. Picopore's lossless and deep lossless methods provide a 25% and 44% average reduction in size, respectively, without removing any data from the files. Picopore's raw method provides an 88% average reduction in size, while retaining biologically relevant data for the end-user. All methods have the capacity to run in real-time in parallel to a sequencing run, reducing demand for both immediate and long-term storage space.

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Picopore: A tool for reducing the storage size of Oxford Nanopore Technologies datasets without loss of functionality

F1000Research ◽

10.12688/f1000research.11022.2 ◽

2017 ◽

Vol 6 ◽

pp. 227 ◽

Cited By ~ 1

Author(s):

Scott Gigante

Keyword(s):

Data Storage ◽

Data Generation ◽

Biologically Relevant ◽

Sequencing Technologies ◽

Long Term Storage ◽

Oxford Nanopore ◽

Long Read ◽

Oxford Nanopore Technologies ◽

Term Storage

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Scaffolding and Completing Genome Assemblies in Real-time with Nanopore Sequencing

10.1101/054783 ◽

2016 ◽

Cited By ~ 8

Author(s):

Minh Duc Cao ◽

Son Hoang Nguyen ◽

Devika Ganesamoorthy ◽

Alysha G. Elliott ◽

Matthew Cooper ◽

...

Keyword(s):

Real Time ◽

Sequence Data ◽

Repetitive Sequences ◽

Computational Method ◽

Nanopore Sequencing ◽

Short Read ◽

Sequencing Technologies ◽

Long Read ◽

Computational Resources ◽

Genome Assemblies

AbstractGenome assemblies obtained from short read sequencing technologies are often fragmented into many contigs because of the abundance of repetitive sequences. Long read sequencing technologies allow the generation of reads spanning most repeat sequences, providing the opportunity to complete these genome assemblies. However, substantial amounts of sequence data and computational resources are required to overcome the high per-base error rate inherent to these technologies. Furthermore, most existing methods only assemble the genomes after sequencing has completed which could result in either generation of more sequence data at greater cost than required or a low-quality assembly if insufficient data are generated. Here we present the first computational method which utilises real-time nanopore sequencing to scaffold and complete short-read assemblies while the long read sequence data is being generated. The method reports the progress of completing the assembly in real-time so users can terminate the sequencing once an assembly of sufficient quality and completeness is obtained. We use our method to complete four bacterial genomes and one eukaryotic genome, and show that it is able to construct more complete and more accurate assemblies, and at the same time, requires less sequencing data and computational resources than existing pipelines. We also demonstrate that the method can facilitate real-time analyses of positional information such as identification of bacterial genes encoded in plasmids and pathogenicity islands.

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Nebula: ultra-efficient mapping-free structural variant genotyper

Nucleic Acids Research ◽

10.1093/nar/gkab025 ◽

2021 ◽

Author(s):

Parsoa Khorsand ◽

Fereydoun Hormozdiari

Keyword(s):

Large Scale ◽

Structural Variants ◽

Sequencing Technologies ◽

Generic Framework ◽

Common Genetic Variants ◽

Order Of Magnitude ◽

Complex Events ◽

Comparable Accuracy ◽

Using Data ◽

Computational Resources

Abstract Large scale catalogs of common genetic variants (including indels and structural variants) are being created using data from second and third generation whole-genome sequencing technologies. However, the genotyping of these variants in newly sequenced samples is a nontrivial task that requires extensive computational resources. Furthermore, current approaches are mostly limited to only specific types of variants and are generally prone to various errors and ambiguities when genotyping complex events. We are proposing an ultra-efficient approach for genotyping any type of structural variation that is not limited by the shortcomings and complexities of current mapping-based approaches. Our method Nebula utilizes the changes in the count of k-mers to predict the genotype of structural variants. We have shown that not only Nebula is an order of magnitude faster than mapping based approaches for genotyping structural variants, but also has comparable accuracy to state-of-the-art approaches. Furthermore, Nebula is a generic framework not limited to any specific type of event. Nebula is publicly available at https://github.com/Parsoa/Nebula.

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Comprehensive identification of transposable element insertions using multiple sequencing technologies

Nature Communications ◽

10.1038/s41467-021-24041-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chong Chu ◽

Rebeca Borges-Monroy ◽

Vinayak V. Viswanadham ◽

Soohyun Lee ◽

Heng Li ◽

...

Keyword(s):

Transposable Element ◽

Structure And Function ◽

Endogenous Retroviruses ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data ◽

Short Read ◽

Sequencing Technologies ◽

Long Read ◽

And Function

AbstractTransposable elements (TEs) help shape the structure and function of the human genome. When inserted into some locations, TEs may disrupt gene regulation and cause diseases. Here, we present xTea (x-Transposable element analyzer), a tool for identifying TE insertions in whole-genome sequencing data. Whereas existing methods are mostly designed for short-read data, xTea can be applied to both short-read and long-read data. Our analysis shows that xTea outperforms other short read-based methods for both germline and somatic TE insertion discovery. With long-read data, we created a catalogue of polymorphic insertions with full assembly and annotation of insertional sequences for various types of retroelements, including pseudogenes and endogenous retroviruses. Notably, we find that individual genomes have an average of nine groups of full-length L1s in centromeres, suggesting that centromeres and other highly repetitive regions such as telomeres are a significant yet unexplored source of active L1s. xTea is available at https://github.com/parklab/xTea.

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Survey of the Bradysia odoriphaga Transcriptome Using PacBio Single-Molecule Long-Read Sequencing

Genes ◽

10.3390/genes10060481 ◽

2019 ◽

Vol 10 (6) ◽

pp. 481 ◽

Cited By ~ 1

Author(s):

Chen ◽

Lin ◽

Xie ◽

Zhong ◽

Zhang ◽

...

Keyword(s):

Insecticide Resistance ◽

Single Molecule ◽

Functional Categories ◽

Genetic Studies ◽

Sequencing Technologies ◽

Clusters Of Orthologous Groups ◽

Long Read ◽

Main Gene ◽

First Time ◽

Main Factor

The damage caused by Bradysia odoriphaga is the main factor threatening the production of vegetables in the Liliaceae family. However, few genetic studies of B. odoriphaga have been conducted because of a lack of genomic resources. Many long-read sequencing technologies have been developed in the last decade; therefore, in this study, the transcriptome including all development stages of B. odoriphaga was sequenced for the first time by Pacific single-molecule long-read sequencing. Here, 39,129 isoforms were generated, and 35,645 were found to have annotation results when checked against sequences available in different databases. Overall, 18,473 isoforms were distributed in 25 various Clusters of Orthologous Groups, and 11,880 isoforms were categorized into 60 functional groups that belonged to the three main Gene Ontology classifications. Moreover, 30,610 isoforms were assigned into 44 functional categories belonging to six main Kyoto Encyclopedia of Genes and Genomes functional categories. Coding DNA sequence (CDS) prediction showed that 36,419 out of 39,129 isoforms were predicted to have CDS, and 4319 simple sequence repeats were detected in total. Finally, 266 insecticide resistance and metabolism-related isoforms were identified as candidate genes for further investigation of insecticide resistance and metabolism in B. odoriphaga.

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