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 De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing

F1000Research ◽  
2018 ◽  
Vol 6 ◽  
pp. 618 ◽  
Author(s):  
Michael Liem ◽  
Hans J. Jansen ◽  
Ron P. Dirks ◽  
Christiaan V. Henkel ◽  
G. Paul H. van Heusden ◽  
...  
Keyword(s):  
De Novo ◽  
Sequence Data ◽  
New Technology ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Short Read ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Before And After ◽  
Long Read

Background: The introduction of the MinION sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. Nanopore sequence data offers great potential for de novo assembly of complex genomes without using other technologies. Furthermore, Nanopore data combined with other sequencing technologies is highly useful for accurate annotation of all genes in the genome. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. We double corrected assemblies from four different assemblers with PILON and assessed sequence correctness before and after PILON correction with a set of 290 Fungi genes using BUSCO. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae (Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. We also compared various technical and software developments for the nanopore sequencing protocol, showing that nanopore-derived assemblies provide the highest contiguity. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.

scholarly journals De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 618 ◽  
Author(s):  
Hans J. Jansen ◽  
Ron P. Dirks ◽  
Michael Liem ◽  
Christiaan V. Henkel ◽  
G. Paul H. van Heusden ◽  
...  
Keyword(s):  
De Novo ◽  
Sequence Data ◽  
New Technology ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Short Read ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Wild Type Yeast

Background: The introduction of the MinIONTM sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. It has been shown that the nanopore sequence data, in combination with other sequencing technologies, is highly useful for accurate annotation of all genes in the genome. However, it also offers great potential for de novo assembly of complex genomes without using other technologies. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae (Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.

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 Perspectives and benefits of high-throughput long-read sequencing in microbial ecology

2021 ◽  
Author(s):  
Leho Tedersoo ◽  
Mads Albertsen ◽  
Sten Anslan ◽  
Benjamin Callahan
Keyword(s):  
Microbial Ecology ◽  
High Throughput ◽  
Single Molecule ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
Nanopore Sequencing ◽  
High Quality ◽  
Short Read ◽  
Sequencing Technologies ◽  
Long Read

Short-read, high-throughput sequencing (HTS) methods have yielded numerous important insights into microbial ecology and function. Yet, in many instances short-read HTS techniques are suboptimal, for example by providing insufficient phylogenetic resolution or low integrity of assembled genomes. Single-molecule and synthetic long-read (SLR) HTS methods have successfully ameliorated these limitations. In addition, nanopore sequencing has generated a number of unique analysis opportunities such as rapid molecular diagnostics and direct RNA sequencing, and both PacBio and nanopore sequencing support detection of epigenetic modifications. Although initially suffering from relatively low sequence quality, recent advances have greatly improved the accuracy of long read sequencing technologies. In spite of great technological progress in recent years, the long-read HTS methods (PacBio and nanopore sequencing) are still relatively costly, require large amounts of high-quality starting material, and commonly need specific solutions in various analysis steps. Despite these challenges, long-read sequencing technologies offer high-quality, cutting-edge alternatives for testing hypotheses about microbiome structure and functioning as well as assembly of eukaryote genomes from complex environmental DNA samples.

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 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 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 Rapid Mycobacterium tuberculosis spoligotyping from uncorrected long reads using Galru

2020 ◽  
Author(s):  
Andrew J. Page ◽  
Nabil-Fareed Alikhan ◽  
Michael Strinden ◽  
Thanh Le Viet ◽  
Timofey Skvortsov
Keyword(s):  
Mycobacterium Tuberculosis ◽  
State Of The Art ◽  
Sequence Data ◽  
Human Pathogen ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing ◽  
Long Reads ◽  
Long Read

AbstractSpoligotyping of Mycobacterium tuberculosis provides a subspecies classification of this major human pathogen. Spoligotypes can be predicted from short read genome sequencing data; however, no methods exist for long read sequence data such as from Nanopore or PacBio. We present a novel software package Galru, which can rapidly detect the spoligotype of a Mycobacterium tuberculosis sample from as little as a single uncorrected long read. It allows for near real-time spoligotyping from long read data as it is being sequenced, giving rapid sample typing. We compare it to the existing state of the art software and find it performs identically to the results obtained from short read sequencing data. Galru is freely available from https://github.com/quadram-institute-bioscience/galru under the GPLv3 open source licence.

scholarly journals Expanding an expanded genome: long-read sequencing ofTrypanosoma cruzi

2018 ◽  
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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