de novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer

AbstractOxford Nanopore Technologies Ltd (Oxford, UK) have recently commercialized MinION, a small and low-cost single-molecule nanopore sequencer, that offers the possibility of sequencing long DNA fragments. The Oxford Nanopore technology is truly disruptive and can sequence small genomes in a matter of seconds. It has the potential to revolutionize genomic applications due to its portability, low-cost, and ease of use compared with existing long reads sequencing technologies. The MinION sequencer enables the rapid sequencing of small eukaryotic genomes, such as the yeast genome. Combined with existing assembler algorithms, near complete genome assemblies can be generated and comprehensive population genomic analyses can be performed. Here, we resequenced the genome of the Saccharomyces cerevisiae S288C strain to evaluate the performance of nanopore-only assemblers. Then we de novo sequenced and assembled the genomes of 21 isolates representative of the S. cerevisiae genetic diversity using the MinION platform. The contiguity of our assemblies was 14 times higher than the Illumina-only assemblies and we obtained one or two long contigs for 65% of the chromosomes. This high continuity allowed us to accurately detect large structural variations across the 21 studied genomes. Moreover, because of the high completeness of the nanopore assemblies, we were able to produce a complete cartography of transposable elements insertions and inspect structural variants that are generally missed using a short-read sequencing strategy.

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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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MUM&Co: accurate detection of all SV types through whole-genome alignment

Bioinformatics ◽

10.1093/bioinformatics/btaa115 ◽

2020 ◽

Vol 36 (10) ◽

pp. 3242-3243 ◽

Cited By ~ 2

Author(s):

Samuel O’Donnell ◽

Gilles Fischer

Keyword(s):

De Novo ◽

Supplementary Information ◽

Genome Alignment ◽

Whole Genome ◽

Structural Variations ◽

Sequencing Technologies ◽

Third Generation Sequencing ◽

Human Genomes ◽

Whole Genome Alignment ◽

Primary Output

Abstract Summary MUM&Co is a single bash script to detect structural variations (SVs) utilizing whole-genome alignment (WGA). Using MUMmer’s nucmer alignment, MUM&Co can detect insertions, deletions, tandem duplications, inversions and translocations greater than 50 bp. Its versatility depends upon the WGA and therefore benefits from contiguous de-novo assemblies generated by third generation sequencing technologies. Benchmarked against five WGA SV-calling tools, MUM&Co outperforms all tools on simulated SVs in yeast, plant and human genomes and performs similarly in two real human datasets. Additionally, MUM&Co is particularly unique in its ability to find inversions in both simulated and real datasets. Lastly, MUM&Co’s primary output is an intuitive tabulated file containing a list of SVs with only necessary genomic details. Availability and implementation https://github.com/SAMtoBAM/MUMandCo. Supplementary information Supplementary data are available at Bioinformatics online.

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

Scientific Reports ◽

10.1038/s41598-019-55908-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Joseph R. Fauver ◽

John Martin ◽

Gary J. Weil ◽

Makedonka Mitreva ◽

Peter U. Fischer

Keyword(s):

Single Molecule ◽

New Technologies ◽

Reference Genome ◽

De Novo ◽

Complete Mitochondrial Genome ◽

Nuclear Genome ◽

Brugia Malayi ◽

Field Isolates ◽

Sequencing Technologies ◽

Long Reads

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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A Transposon Story: From TE Content to TE Dynamic Invasion of Drosophila Genomes Using the Single-Molecule Sequencing Technology from Oxford Nanopore

Cells ◽

10.3390/cells9081776 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1776

Author(s):

Mourdas Mohamed ◽

Nguyet Thi-Minh Dang ◽

Yuki Ogyama ◽

Nelly Burlet ◽

Bruno Mugat ◽

...

Keyword(s):

Single Molecule ◽

Wild Type ◽

Sequencing Data ◽

Short Read ◽

Short Read Sequencing ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

In The Wild ◽

Successive Generations ◽

Type Strains

Transposable elements (TEs) are the main components of genomes. However, due to their repetitive nature, they are very difficult to study using data obtained with short-read sequencing technologies. Here, we describe an efficient pipeline to accurately recover TE insertion (TEI) sites and sequences from long reads obtained by Oxford Nanopore Technology (ONT) sequencing. With this pipeline, we could precisely describe the landscapes of the most recent TEIs in wild-type strains of Drosophila melanogaster and Drosophila simulans. Their comparison suggests that this subset of TE sequences is more similar than previously thought in these two species. The chromosome assemblies obtained using this pipeline also allowed recovering piRNA cluster sequences, which was impossible using short-read sequencing. Finally, we used our pipeline to analyze ONT sequencing data from a D. melanogaster unstable line in which LTR transposition was derepressed for 73 successive generations. We could rely on single reads to identify new insertions with intact target site duplications. Moreover, the detailed analysis of TEIs in the wild-type strains and the unstable line did not support the trap model claiming that piRNA clusters are hotspots of TE insertions.

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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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Nanopore native RNA sequencing of a human poly(A) transcriptome: RNA extraction, cDNA conversion and direct RNA and cDNA library preparation for Oxford Nanopore

10.21203/rs.2.12872/v1 ◽

2019 ◽

Author(s):

Rachael E. Workman ◽

Alison D. Tang ◽

Paul S. Tang ◽

Miten Jain ◽

John R. Tyson ◽

...

Keyword(s):

Rna Sequencing ◽

Rna Extraction ◽

Human Cell Line ◽

Cdna Libraries ◽

Library Preparation ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Sequencing Strategy ◽

Oxford Nanopore Technologies ◽

Cdna Library Preparation

Abstract High throughput cDNA sequencing technologies have dramatically advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and because modifications are not carried forward in cDNA. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study focused on poly(A) RNA from the human cell line GM12878, generating 9.9 million aligned sequence reads. These native RNA reads had an aligned N50 length of 1294 bases, and a maximum aligned length of over 21,000 bases. A total of 78,199 high-confidence isoforms were identified by combining long nanopore reads with short higher accuracy Illumina reads. We describe methods for extracting intact RNA, poly-A selection, cDNA conversion for a portion of sample, and library preparation for both direct RNA and cDNA libraries.

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Clover: a clustering-oriented de novo assembler for Illumina sequences

BMC Bioinformatics ◽

10.1186/s12859-020-03788-9 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Ming-Feng Hsieh ◽

Chin Lung Lu ◽

Chuan Yi Tang

Keyword(s):

De Novo Assembly ◽

De Novo ◽

Low Cost ◽

De Bruijn Graph ◽

Illumina Platform ◽

Sequencing Errors ◽

Sequencing Technologies ◽

String Graph ◽

Clustering Approach ◽

De Bruijn

Abstract Background Next-generation sequencing technologies revolutionized genomics by producing high-throughput reads at low cost, and this progress has prompted the recent development of de novo assemblers. Multiple assembly methods based on de Bruijn graph have been shown to be efficient for Illumina reads. However, the sequencing errors generated by the sequencer complicate analysis of de novo assembly and influence the quality of downstream genomic researches. Results In this paper, we develop a de Bruijn assembler, called Clover (clustering-oriented de novo assembler), that utilizes a novel k-mer clustering approach from the overlap-layout-consensus concept to deal with the sequencing errors generated by the Illumina platform. We further evaluate Clover’s performance against several de Bruijn graph assemblers (ABySS, SOAPdenovo, SPAdes and Velvet), overlap-layout-consensus assemblers (Bambus2, CABOG and MSR-CA) and string graph assembler (SGA) on three datasets (Staphylococcus aureus, Rhodobacter sphaeroides and human chromosome 14). The results show that Clover achieves a superior assembly quality in terms of corrected N50 and E-size while remaining a significantly competitive in run time except SOAPdenovo. In addition, Clover was involved in the sequencing projects of bacterial genomes Acinetobacter baumannii TYTH-1 and Morganella morganii KT. Conclusions The marvel clustering-based approach of Clover that integrates the flexibility of the overlap-layout-consensus approach and the efficiency of the de Bruijn graph method has high potential on de novo assembly. Now, Clover is freely available as open source software from https://oz.nthu.edu.tw/~d9562563/src.html.

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