scholarly journals ntJoin: Fast and lightweight assembly-guided scaffolding using minimizer graphs

2020 ◽  
Author(s):  
Lauren Coombe ◽  
Vladimir Nikolić ◽  
Justin Chu ◽  
Inanc Birol ◽  
René L. Warren
Keyword(s):  
Reference Sequence ◽  
Biological Research ◽  
Closely Related Species ◽  
Draft Assembly ◽  
Short Read ◽  
Sequencing Technologies ◽  
Long Read ◽  
Genome Assemblies ◽  
High Quality Genome ◽  
Reference Human Genome

AbstractSummaryThe ability to generate high-quality genome sequences is cornerstone to modern biological research. Even with recent advancements in sequencing technologies, many genome assemblies are still not achieving reference-grade. Here, we introduce ntJoin, a tool that leverages structural synteny between a draft assembly and reference sequence(s) to contiguate and correct the former with respect to the latter. Instead of alignments, ntJoin uses a lightweight mapping approach based on a graph data structure generated from ordered minimizer sketches. The tool can be used in a variety of different applications, including improving a draft assembly with a reference-grade genome, a short read assembly with a draft long read assembly, and a draft assembly with an assembly from a closely-related species. When scaffolding a human short read assembly using the reference human genome or a long read assembly, ntJoin improves the NGA50 length 23- and 13-fold, respectively, in under 13 m, using less than 11 GB of RAM. Compared to existing reference-guided assemblers, ntJoin generates highly contiguous assemblies faster and using less memory.Availability and implementationntJoin is written in C++ and Python, and is freely available at https://github.com/bcgsc/[email protected]

scholarly journals ntJoin: Fast and lightweight assembly-guided scaffolding using minimizer graphs

2020 ◽  
Vol 36 (12) ◽  
pp. 3885-3887 ◽  
Author(s):  
Lauren Coombe ◽  
Vladimir Nikolić ◽  
Justin Chu ◽  
Inanc Birol ◽  
René L Warren
Keyword(s):  
Reference Sequence ◽  
Supplementary Information ◽  
Biological Research ◽  
Closely Related Species ◽  
Draft Assembly ◽  
Short Read ◽  
Sequencing Technologies ◽  
Long Read ◽  
Genome Assemblies ◽  
High Quality Genome

Abstract Summary The ability to generate high-quality genome sequences is cornerstone to modern biological research. Even with recent advancements in sequencing technologies, many genome assemblies are still not achieving reference-grade. Here, we introduce ntJoin, a tool that leverages structural synteny between a draft assembly and reference sequence(s) to contiguate and correct the former with respect to the latter. Instead of alignments, ntJoin uses a lightweight mapping approach based on a graph data structure generated from ordered minimizer sketches. The tool can be used in a variety of different applications, including improving a draft assembly with a reference-grade genome, a short-read assembly with a draft long-read assembly and a draft assembly with an assembly from a closely related species. When scaffolding a human short-read assembly using the reference human genome or a long-read assembly, ntJoin improves the NGA50 length 23- and 13-fold, respectively, in under 13 m, using <11 GB of RAM. Compared to existing reference-guided scaffolders, ntJoin generates highly contiguous assemblies faster and using less memory. Availability and implementation ntJoin is written in C++ and Python and is freely available at https://github.com/bcgsc/ntjoin. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Read coverage as an indicator of misassembly in a short-read based genome assembly

2019 ◽  
Author(s):  
Peipei Wang ◽  
Fanrui Meng ◽  
Bethany M. Moore ◽  
Shin-Han Shiu
Keyword(s):  
Machine Learning ◽  
Great Majority ◽  
Significant Advance ◽  
High Coverage ◽  
Short Read ◽  
Sequencing Technologies ◽  
Long Read ◽  
Genomic Regions ◽  
Genome Assemblies ◽  
Simple Sequence

ABSTRACTAvailability of genome sequences has led to significant advance in biology. With few exceptions, the great majority of existing genome assemblies are derived from short read sequencing technologies with highly uneven read coverages indicative of sequencing and assembly issues. In tomato, 0.6% (5.1 Mb) and 9.7% (79.6 Mb) of short-read based assembly had significantly higher and lower coverage compared to background, respectively. We established machine learning models capable of predicting genomic regions with variable coverages and found that high coverage regions tend to have lower simple sequence repeat but higher tandem gene densities compared to background regions. To determine if the high coverage regions were misassembled, we examined a recently available long-read based assembly and found that 27.8% (1.41 Mb) of high coverage regions were potentially mis-assembled of duplicate sequences, compared to 1.4% in background regions. In addition, using a machine learning model that can distinguish correctly and incorrectly assembled high coverage regions, we found that misassembled, high coverage regions tend to be flanked by simple sequence repeats, pseudogenes, and transposon elements. Our study provides insights on the causes of variable coverage regions and a quantitative assessment of factors contributing to misassembly when using short reads.

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

2016 ◽  
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.

scholarly journals Comprehensive identification of transposable element insertions using multiple sequencing technologies

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.

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 Contiguity: Contig adjacency graph construction and visualisation

2015 ◽  
Author(s):  
Mitchell J Sullivan ◽  
Nouri L Ben Zakour ◽  
Brian M Forde ◽  
Mitchell Stanton-Cook ◽  
Scott A Beatson
Keyword(s):  
De Novo ◽  
Reference Sequence ◽  
De Bruijn Graph ◽  
Interactive Software ◽  
Graph Exploration ◽  
Adjacency Graph ◽  
Highly Sensitive ◽  
Long Read ◽  
Genome Assemblies ◽  
Adjacency Graphs

Contiguity is an interactive software for the visualization and manipulation of de novo genome assemblies. Contiguity creates and displays information on contig adjacency which is contextualized by the simultaneous display of a comparison between assembled contigs and reference sequence. Where scaffolders allow unambiguous connections between contigs to be resolved into a single scaffold, Contiguity allows the user to create all potential scaffolds in ambiguous regions of the genome. This enables the resolution of novel sequence or structural variants from the assembly. In addition, Contiguity provides a sequencing and assembly agnostic approach for the creation of contig adjacency graphs. To maximize the number of contig adjacencies determined, Contiguity combines information from read pair mappings, sequence overlap and De Bruijn graph exploration. We demonstrate how highly sensitive graphs can be achieved using this method. Contig adjacency graphs allow the user to visualize potential arrangements of contigs in unresolvable areas of the genome. By combining adjacency information with comparative genomics, Contiguity provides an intuitive approach for exploring and improving sequence assemblies. It is also useful in guiding manual closure of long read sequence assemblies. Contiguity is an open source application, implemented using Python and the Tkinter GUI package that can run on any Unix, OSX and Windows operating system. It has been designed and optimized for bacterial assemblies. Contiguity is available at http://mjsull.github.io/Contiguity .

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 Complete Genome Resequencing of Thermus thermophilus Strain TMY by Hybrid Assembly of Long- and Short-Read Sequencing Technologies

2021 ◽  
Vol 10 (46) ◽  
Author(s):  
Kentaro Miyazaki ◽  
Natsuko Tokito
Keyword(s):  
Complete Genome ◽  
Thermus Thermophilus ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Hybrid Assembly ◽  
Genome Resequencing ◽  
Short Read ◽  
Content Type ◽  
Sequencing Technologies ◽  
Long Read

Complete genome resequencing was conducted for Thermus thermophilus strain TMY by hybrid assembly of Oxford Nanopore Technologies long-read and MGI short-read data. Errors in the previously reported genome sequence determined by PacBio technology alone were corrected, allowing for high-quality comparative genomic analysis of closely related T. thermophilus genomes.

scholarly journals Education in the genomics era: Generating high-quality genome assemblies in university courses

GigaScience ◽  
2020 ◽  
Vol 9 (6) ◽  
Author(s):  
Stefan Prost ◽  
Sven Winter ◽  
Jordi De Raad ◽  
Raphael T F Coimbra ◽  
Magnus Wolf ◽  
...  
Keyword(s):  
Low Cost ◽  
Genomic Data ◽  
Master's Level ◽  
Genome Data ◽  
Sequencing Technologies ◽  
Third Generation Sequencing ◽  
University Courses ◽  
Hands On ◽  
Genome Assemblies ◽  
High Quality Genome

Abstract Recent advances in genome sequencing technologies have simplified the generation of genome data and reduced the costs for genome assemblies, even for complex genomes like those of vertebrates. More practically oriented genomic courses can prepare university students for the increasing importance of genomic data used in biological and medical research. Low-cost third-generation sequencing technology, along with publicly available data, can be used to teach students how to process genomic data, assemble full chromosome-level genomes, and publish the results in peer-reviewed journals, or preprint servers. Here we outline experiences gained from 2 master's-level courses and discuss practical considerations for teaching hands-on genome assembly courses.

scholarly journals A high-quality genome assembly from a single, field-collected spotted lanternfly (Lycorma delicatula) using the PacBio Sequel II system

GigaScience ◽  
2019 ◽  
Vol 8 (10) ◽  
Author(s):  
Sarah B Kingan ◽  
Julie Urban ◽  
Christine C Lambert ◽  
Primo Baybayan ◽  
Anna K Childers ◽  
...  
Keyword(s):  
Invasive Species ◽  
Genome Assembly ◽  
De Novo ◽  
Fragment Size ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Lycorma Delicatula ◽  
Long Read ◽  
Genome Assemblies ◽  
High Quality Genome

ABSTRACT Background A high-quality reference genome is an essential tool for applied and basic research on arthropods. Long-read sequencing technologies may be used to generate more complete and contiguous genome assemblies than alternate technologies; however, long-read methods have historically had greater input DNA requirements and higher costs than next-generation sequencing, which are barriers to their use on many samples. Here, we present a 2.3 Gb de novo genome assembly of a field-collected adult female spotted lanternfly (Lycorma delicatula) using a single Pacific Biosciences SMRT Cell. The spotted lanternfly is an invasive species recently discovered in the northeastern United States that threatens to damage economically important crop plants in the region. Results The DNA from 1 individual was used to make 1 standard, size-selected library with an average DNA fragment size of ∼20 kb. The library was run on 1 Sequel II SMRT Cell 8M, generating a total of 132 Gb of long-read sequences, of which 82 Gb were from unique library molecules, representing ∼36× coverage of the genome. The assembly had high contiguity (contig N50 length = 1.5 Mb), completeness, and sequence level accuracy as estimated by conserved gene set analysis (96.8% of conserved genes both complete and without frame shift errors). Furthermore, it was possible to segregate more than half of the diploid genome into the 2 separate haplotypes. The assembly also recovered 2 microbial symbiont genomes known to be associated with L. delicatula, each microbial genome being assembled into a single contig. Conclusions We demonstrate that field-collected arthropods can be used for the rapid generation of high-quality genome assemblies, an attractive approach for projects on emerging invasive species, disease vectors, or conservation efforts of endangered species.

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