scholarly journals Snake venom gland cDNA sequencing using the Oxford Nanopore MinION portable DNA sequencer

2015 ◽  
Author(s):  
John F Mulley ◽  
Adam D Hargreaves
Keyword(s):  
Error Correction ◽  
Single Molecule ◽  
De Novo ◽  
Venom Gland ◽  
Sequencing Error ◽  
Cdna Sequencing ◽  
Venom Toxin ◽  
Oxford Nanopore ◽  
Illumina Data ◽  
Corrected Data

Portable DNA sequencers such as the Oxford Nanopore MinION device have the potential to be truly disruptive technologies, facilitating new approaches and analyses and, in some cases, taking sequencing out of the lab and into the field. However, the capabilities of these technologies are still being revealed. Here we show that single-molecule cDNA sequencing using the MinION accurately characterises venom toxin-encoding genes in the painted saw-scaled viper, Echis coloratus. We find the raw sequencing error rate to be around 12%, improved to 0-2% with hybrid error correction and 3% with de novo error correction. Our corrected data provides full coding sequences and 5' and 3' UTRs for 29 of 33 candidate venom toxins detected, far superior to Illumina data (13/40 complete) and Sanger-based ESTs (15/29). We suggest that, should the current pace of improvement continue, the MinION will become the default approach for cDNA sequencing in a variety of species.

scholarly journals Assessing the utility of the Oxford Nanopore MinION for snake venom gland cDNA sequencing

PeerJ ◽  
2015 ◽  
Vol 3 ◽  
pp. e1441 ◽  
Author(s):  
Adam D. Hargreaves ◽  
John F. Mulley
Keyword(s):  
Error Correction ◽  
Single Molecule ◽  
De Novo ◽  
Venom Gland ◽  
Sequencing Error ◽  
Cdna Sequencing ◽  
Venom Toxin ◽  
Oxford Nanopore ◽  
Illumina Data ◽  
Corrected Data

Portable DNA sequencers such as the Oxford Nanopore MinION device have the potential to be truly disruptive technologies, facilitating new approaches and analyses and, in some cases, taking sequencing out of the lab and into the field. However, the capabilities of these technologies are still being revealed. Here we show that single-molecule cDNA sequencing using the MinION accurately characterises venom toxin-encoding genes in the painted saw-scaled viper,Echis coloratus. We find the raw sequencing error rate to be around 12%, improved to 0–2% with hybrid error correction and 3% withde novoerror correction. Our corrected data provides full coding sequences and 5′ and 3′ UTRs for 29 of 33 candidate venom toxins detected, far superior to Illumina data (13/40 complete) and Sanger-based ESTs (15/29). We suggest that, should the current pace of improvement continue, the MinION will become the default approach for cDNA sequencing in a variety of species.

Overlap detection on long, error-prone sequencing reads via smooth q-gram

2020 ◽  
Vol 36 (19) ◽  
pp. 4838-4845
Author(s):  
Yan Song ◽  
Haixu Tang ◽  
Haoyu Zhang ◽  
Qin Zhang
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Error Rates ◽  
Supplementary Information ◽  
Sequencing Error ◽  
Fragment Assembly ◽  
Detection Algorithms ◽  
Third Generation Sequencing ◽  
Oxford Nanopore ◽  
Assembly Algorithms

Abstract Motivation Third generation sequencing techniques, such as the Single Molecule Real Time technique from PacBio and the MinION technique from Oxford Nanopore, can generate long, error-prone sequencing reads which pose new challenges for fragment assembly algorithms. In this paper, we study the overlap detection problem for error-prone reads, which is the first and most critical step in the de novo fragment assembly. We observe that all the state-of-the-art methods cannot achieve an ideal accuracy for overlap detection (in terms of relatively low precision and recall) due to the high sequencing error rates, especially when the overlap lengths between reads are relatively short (e.g. <2000 bases). This limitation appears inherent to these algorithms due to their usage of q-gram-based seeds under the seed-extension framework. Results We propose smooth q-gram, a variant of q-gram that captures q-gram pairs within small edit distances and design a novel algorithm for detecting overlapping reads using smooth q-gram-based seeds. We implemented the algorithm and tested it on both PacBio and Nanopore sequencing datasets. Our benchmarking results demonstrated that our algorithm outperforms the existing q-gram-based overlap detection algorithms, especially for reads with relatively short overlapping lengths. Availability and implementation The source code of our implementation in C++ is available at https://github.com/FIGOGO/smoothq. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Oxford Nanopore Sequencing, Hybrid Error Correction, and de novo Assembly of a Eukaryotic Genome

2015 ◽  
Author(s):  
Sara Goodwin ◽  
James Gurtowski ◽  
Scott Ethe-Sayers ◽  
Panchajanya Deshpande ◽  
Michael Schatz ◽  
...  
Keyword(s):  
Error Correction ◽  
De Novo Assembly ◽  
De Novo ◽  
Correction Algorithm ◽  
Membrane Pore ◽  
Complete Representation ◽  
Oxford Nanopore ◽  
Long Read ◽  
Error Correction Algorithm ◽  
Sequencing Instrument

Monitoring the progress of DNA molecules through a membrane pore has been postulated as a method for sequencing DNA for several decades. Recently, a nanopore-based sequencing instrument, the Oxford Nanopore MinION, has become available that we used for sequencing the S. cerevisiae genome. To make use of these data, we developed a novel open-source hybrid error correction algorithm Nanocorr (https://github.com/jgurtowski/nanocorr) specifically for Oxford Nanopore reads, as existing packages were incapable of assembling the long read lengths (5-50kbp) at such high error rate (between ~5 and 40% error). With this new method we were able to perform a hybrid error correction of the nanopore reads using complementary MiSeq data and produce a de novo assembly that is highly contiguous and accurate: the contig N50 length is more than ten-times greater than an Illumina-only assembly (678kb versus 59.9kbp), and has greater than 99.88% consensus identity when compared to the reference. Furthermore, the assembly with the long nanopore reads presents a much more complete representation of the features of the genome and correctly assembles gene cassettes, rRNAs, transposable elements, and other genomic features that were almost entirely absent in the Illumina-only assembly.

scholarly journals MECAT: an ultra-fast mapping, error correction andde novoassembly tool for single-molecule sequencing reads

2016 ◽  
Author(s):  
Chuan-Le Xiao ◽  
Ying Chen ◽  
Shang-qian Xie ◽  
Kai-Ning Chen ◽  
Yan Wang ◽  
...  
Keyword(s):  
Error Correction ◽  
Single Molecule ◽  
De Novo ◽  
Computational Cost ◽  
Pairwise Alignment ◽  
Global Alignment ◽  
Chinese Han ◽  
Celera Assembler ◽  
Reference Quality ◽  
Molecular Sequencing

ABSTRACTThe high computational cost of current assembly methods for the long, noisy single molecular sequencing (SMS) reads has prevented them from assembling large genomes. We introduce an ultra-fast alignment method based on a novel global alignment score. For large human SMS data, our method is 7X faster than MHAP for pairwise alignment and 15X faster than BLASR for reference mapping. We develop a Mapping, Error Correction and de novo Assembly Tool (MECAT) by integrating our new alignment and error correction methods, with the Celera Assembler. MECAT is capable of producing high qualityde novoassembly of large genome from SMS reads with low computational cost. MECAT produces reference-quality assemblies ofSaccharomyces cerevisiae,Arabidopsis thaliana,Drosophila melanogasterand reconstructs the human CHM1 genome with 15% longer NG50 in only 7600 CPU core hours using 54X SMS reads and a Chinese Han genome in 19200 CPU core hours using 102X SMS reads.

scholarly journals Fast and accurate de novo genome assembly from long uncorrected reads

2016 ◽  
Author(s):  
Robert Vaser ◽  
Ivan Sović ◽  
Niranjan Nagarajan ◽  
Mile Šikić
Keyword(s):  
Error Correction ◽  
De Novo ◽  
High Quality ◽  
De Novo Genome Assembly ◽  
Consensus Sequences ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Order Of Magnitude ◽  
Correction Step ◽  
Consensus Module

The assembly of long reads from Pacific Biosciences and Oxford Nanopore Technologies typically requires resource intensive error correction and consensus generation steps to obtain high quality assemblies. We show that the error correction step can be omitted and high quality consensus sequences can be generated efficiently with a SIMD accelerated, partial order alignment based stand-alone consensus module called Racon. Based on tests with PacBio and Oxford Nanopore datasets we show that Racon coupled with Miniasm enables consensus genomes with similar or better quality than state-of-the-art methods while being an order of magnitude faster.Racon is available open source under the MIT license at https://github.com/isovic/racon.git.

scholarly journals Genomic Surveillance for Antimicrobial Resistance inMannheimia haemolyticaUsing Nanopore Single Molecule Sequencing Technology

2018 ◽  
Author(s):  
Alexander Lim ◽  
Bryan Naidenov ◽  
Haley Bates ◽  
Karyn Willyerd ◽  
Timothy Snider ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Antimicrobial Resistance ◽  
Single Molecule ◽  
Resistant Strain ◽  
De Novo ◽  
Gene Annotation ◽  
Cost Effective ◽  
Multidrug Resistant ◽  
Oxford Nanopore ◽  
Long Read

AbstractDisruptive innovations in long-range, cost-effective direct template nucleic acid sequencing are transforming clinical and diagnostic medicine. A multidrug resistant strain and a pan-susceptible strain ofMannheimia haemolytica, isolated from pneumonic bovine lung samples, were respectively sequenced at 146x and 111x coverage with Oxford Nanopore Technologies MinION.De novoassembly produced a complete genome for the non-resistant strain and a nearly complete assembly for the drug resistant strain. Functional annotation using RAST (Rapid Annotations using Subsystems Technology), CARD (Comprehensive Antibiotic Resistance Database) and ResFinder databases identified genes conferring resistance to different classes of antibiotics including beta lactams, tetracyclines, lincosamides, phenicols, aminoglycosides, sulfonamides and macrolides. Antibiotic resistance phenotypes of theM. haemolyticastrains were confirmed with minimum inhibitory concentration (MIC) assays. The sequencing capacity of highly portable MinION devices was verified by sub-sampling sequencing reads; potential for antimicrobial resistance determined by identification of resistance genes in the draft assemblies with as little as 5,437 MinION reads corresponded to all classes of MIC assays. The resulting quality assemblies and AMR gene annotation highlight efficiency of ultra long-read, whole-genome sequencing (WGS) as a valuable tool in diagnostic veterinary medicine.

scholarly journals Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome

2015 ◽  
Vol 25 (11) ◽  
pp. 1750-1756 ◽  
Author(s):  
Sara Goodwin ◽  
James Gurtowski ◽  
Scott Ethe-Sayers ◽  
Panchajanya Deshpande ◽  
Michael C. Schatz ◽  
...  
Keyword(s):  
Error Correction ◽  
De Novo Assembly ◽  
De Novo ◽  
Eukaryotic Genome ◽  
Nanopore Sequencing ◽  
Oxford Nanopore

scholarly journals lra: A long read aligner for sequences and contigs

2021 ◽  
Vol 17 (6) ◽  
pp. e1009078
Author(s):  
Jingwen Ren ◽  
Mark J. P. Chaisson
Keyword(s):  
Dynamic Programming ◽  
Single Molecule ◽  
De Novo Assembly ◽  
De Novo ◽  
Concave Function ◽  
Single Molecule Sequencing ◽  
Link Type ◽  
Oxford Nanopore ◽  
Concave Cost ◽  
Long Read

It is computationally challenging to detect variation by aligning single-molecule sequencing (SMS) reads, or contigs from SMS assemblies. One approach to efficiently align SMS reads is sparse dynamic programming (SDP), where optimal chains of exact matches are found between the sequence and the genome. While straightforward implementations of SDP penalize gaps with a cost that is a linear function of gap length, biological variation is more accurately represented when gap cost is a concave function of gap length. We have developed a method, lra, that uses SDP with a concave-cost gap penalty, and used lra to align long-read sequences from PacBio and Oxford Nanopore (ONT) instruments as well as de novo assembly contigs. This alignment approach increases sensitivity and specificity for SV discovery, particularly for variants above 1kb and when discovering variation from ONT reads, while having runtime that are comparable (1.05-3.76×) to current methods. When applied to calling variation from de novo assembly contigs, there is a 3.2% increase in Truvari F1 score compared to minimap2+htsbox. lra is available in bioconda (https://anaconda.org/bioconda/lra) and github (https://github.com/ChaissonLab/LRA).

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

2016 ◽  
Author(s):  
Benjamin Istace ◽  
Anne Friedrich ◽  
Léo d’Agata ◽  
Sébastien Faye ◽  
Emilie Payen ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Low Cost ◽  
Ease Of Use ◽  
Yeast Genome ◽  
Structural Variations ◽  
Sequencing Technologies ◽  
Population Genomic ◽  
Oxford Nanopore ◽  
Sequencing Strategy

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.

scholarly journals Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation

2016 ◽  
Author(s):  
Sergey Koren ◽  
Brian P. Walenz ◽  
Konstantin Berlin ◽  
Jason R. Miller ◽  
Nicholas H. Bergman ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Error Rates ◽  
Celera Assembler ◽  
Oxford Nanopore ◽  
Long Read ◽  
Reference Quality ◽  
Order Of Magnitude ◽  
Assembly Algorithms ◽  
Oxford Nanopore Technologies

AbstractLong-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. However, given the relatively high error rates of such technologies, efficient and accurate assembly of large repeats and closely related haplotypes remains challenging. We address these issues with Canu, a successor of Celera Assembler that is specifically designed for noisy single-molecule sequences. Canu introduces support for nanopore sequencing, halves depth-of-coverage requirements, and improves assembly continuity while simultaneously reducing runtime by an order of magnitude on large genomes versus Celera Assembler 8.2. These advances result from new overlapping and assembly algorithms, including an adaptive overlapping strategy based on tf-idf weighted MinHash and a sparse assembly graph construction that avoids collapsing diverged repeats and haplotypes. We demonstrate that Canu can reliably assemble complete microbial genomes and near-complete eukaryotic chromosomes using either PacBio or Oxford Nanopore technologies, and achieves a contig NG50 of greater than 21 Mbp on both human and Drosophila melanogaster PacBio datasets. For assembly structures that cannot be linearly represented, Canu provides graph-based assembly outputs in graphical fragment assembly (GFA) format for analysis or integration with complementary phasing and scaffolding techniques. The combination of such highly resolved assembly graphs with long-range scaffolding information promises the complete and automated assembly of complex genomes.

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