scholarly journals Halcyon: an accurate basecaller exploiting an encoder–decoder model with monotonic attention

2020 ◽  
Author(s):  
Hiroki Konishi ◽  
Rui Yamaguchi ◽  
Kiyoshi Yamaguchi ◽  
Yoichi Furukawa ◽  
Seiya Imoto
Keyword(s):  
Dna Sequences ◽  
Third Party ◽  
Nanopore Sequencing ◽  
Sequencing Technology ◽  
Structural Variations ◽  
Haplotype Phasing ◽  
Oxford Nanopore ◽  
Input Signals ◽  
Long Read ◽  
Oxford Nanopore Technologies

Abstract Motivation In recent years, nanopore sequencing technology has enabled inexpensive long-read sequencing, which promises reads longer than a few thousand bases. Such long-read sequences contribute to the precise detection of structural variations and accurate haplotype phasing. However, deciphering precise DNA sequences from noisy and complicated nanopore raw signals remains a crucial demand for downstream analyses based on higher-quality nanopore sequencing, although various basecallers have been introduced to date. Results To address this need, we developed a novel basecaller, Halcyon, that incorporates neural-network techniques frequently used in the field of machine translation. Our model employs monotonic-attention mechanisms to learn semantic correspondences between nucleotides and signal levels without any pre-segmentation against input signals. We evaluated performance with a human whole-genome sequencing dataset and demonstrated that Halcyon outperformed existing third-party basecallers and achieved competitive performance against the latest Oxford Nanopore Technologies’ basecallers. Availabilityand implementation The source code (halcyon) can be found at https://github.com/relastle/halcyon. Contact [email protected]

scholarly journals Evaluating nanopore sequencing data processing pipelines for structural variation identification

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Anbo Zhou ◽  
Timothy Lin ◽  
Jinchuan Xing
Keyword(s):  
Detection Accuracy ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Sequencing Technology ◽  
Structural Variations ◽  
Human Genomes ◽  
Data Assessment ◽  
Machine Learning Approach ◽  
Long Read ◽  
The Impact

Abstract Background Structural variations (SVs) account for about 1% of the differences among human genomes and play a significant role in phenotypic variation and disease susceptibility. The emerging nanopore sequencing technology can generate long sequence reads and can potentially provide accurate SV identification. However, the tools for aligning long-read data and detecting SVs have not been thoroughly evaluated. Results Using four nanopore datasets, including both empirical and simulated reads, we evaluate four alignment tools and three SV detection tools. We also evaluate the impact of sequencing depth on SV detection. Finally, we develop a machine learning approach to integrate call sets from multiple pipelines. Overall SV callers’ performance varies depending on the SV types. For an initial data assessment, we recommend using aligner minimap2 in combination with SV caller Sniffles because of their speed and relatively balanced performance. For detailed analysis, we recommend incorporating information from multiple call sets to improve the SV call performance. Conclusions We present a workflow for evaluating aligners and SV callers for nanopore sequencing data and approaches for integrating multiple call sets. Our results indicate that additional optimizations are needed to improve SV detection accuracy and sensitivity, and an integrated call set can provide enhanced performance. The nanopore technology is improving, and the sequencing community is likely to grow accordingly. In turn, better benchmark call sets will be available to more accurately assess the performance of available tools and facilitate further tool development.

scholarly journals Robust long-read native DNA sequencing using the ONT CsgG Nanopore system

2017 ◽  
Vol 2 ◽  
pp. 23 ◽  
Author(s):  
Jean-Michel Carter ◽  
Shobbir Hussain
Keyword(s):  
Dna Sequencing ◽  
Cancer Cell Line ◽  
Read Length ◽  
Nanopore Sequencing ◽  
Computational Tools ◽  
Practical Applications ◽  
Oxford Nanopore ◽  
Sequencing Method ◽  
Long Read ◽  
Oxford Nanopore Technologies

Background: The ability to obtain long read lengths during DNA sequencing has several potentially important practical applications. Especially long read lengths have been reported using the Nanopore sequencing method, currently commercially available from Oxford Nanopore Technologies (ONT). However, early reports have demonstrated only limited levels of combined throughput and sequence accuracy. Recently, ONT released a new CsgG pore sequencing system as well as a 250b/s translocation chemistry with potential for improvements. Methods: We made use of such components on ONTs miniature ‘MinION’ device and sequenced native genomic DNA obtained from the near haploid cancer cell line HAP1. Analysis of our data was performed utilising recently described computational tools tailored for nanopore/long-read sequencing outputs, and here we present our key findings. Results: From a single sequencing run, we obtained ~240,000 high-quality mapped reads, comprising a total of ~2.3 billion bases. A mean read length of 9.6kb and an N50 of ~17kb was achieved, while sequences mapped to reference with a mean identity of 85%. Notably, we obtained ~68X coverage of the mitochondrial genome and were able to achieve a mean consensus identity of 99.8% for sequenced mtDNA reads. Conclusions: With improved sequencing chemistries already released and higher-throughput instruments in the pipeline, this early study suggests that ONT CsgG-based sequencing may be a useful option for potential practical long-read applications.

scholarly journals Generating long-read sequences using Oxford Nanopore Technology from Diospyros celebica genomic DNA

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Iskandar Zulkarnaen Siregar ◽  
Fifi Gus Dwiyanti ◽  
Rahadian Pratama ◽  
Deden Derajat Matra ◽  
Muhammad Majiidu
Keyword(s):  
Dna Sequences ◽  
Plant Genetic Resources ◽  
Sustainable Use ◽  
Genomic Analysis ◽  
Genomic Research ◽  
Sequencing Technology ◽  
Data Description ◽  
Oxford Nanopore ◽  
Long Read ◽  
The Tropics

Abstract Objectives Development of sequencing technology has opened up vast opportunities for tree genomic research in the tropics. One of the aforesaid technologies named ONT (Oxford Nanopore Technology) has attracted researchers in undertaking testings and experiments due to its affordability and accessibility. To the best of our knowledge, there has been no published reports on the use of ONT for genomic analysis of Indonesian tree species. This progress is promising for further improvement in order to acquire more genomic data for research purposes. Therefore, the present study was carried out to determine the effectiveness of ONT in generating long-read DNA sequences using DNA isolated from leaves and wood cores of Macassar ebony (Diospyros celebica Bakh.). Data description Long-read sequences data of leaves and wood cores of Macassar ebony were generated by using the MinION device and MinKnow v3.6.5 (ONT). The obtained data, as the first long-read sequence dataset for Macassar ebony, is of great importance to conserve the genetic diversity, understanding the molecular mechanism, and sustainable use of plant genetic resources for downstream applications.

scholarly journals Accurate profiling of forensic autosomal STRs using the Oxford Nanopore Technologies MinION device

2021 ◽  
Author(s):  
Courtney L. Hall ◽  
Rupesh K. Kesharwani ◽  
Nicole R. Phillips ◽  
John V. Planz ◽  
Fritz J. Sedlazeck ◽  
...  
Keyword(s):  
Specific Method ◽  
Nanopore Sequencing ◽  
Autosomal Strs ◽  
Str Typing ◽  
Str Loci ◽  
Oxford Nanopore ◽  
Long Read ◽  
Flanking Region ◽  
Sequencing Platforms ◽  
Oxford Nanopore Technologies

The high variability characteristic of short tandem repeat (STR) markers is harnessed for human identification in forensic genetic analyses. Despite the power and reliability of current typing techniques, sequence-level information both within and around STRs are masked in the length-based profiles generated. Forensic STR typing using next generation sequencing (NGS) has therefore gained attention as an alternative to traditional capillary electrophoresis (CE) approaches. In this proof-of-principle study, we evaluate the forensic applicability of the newest and smallest NGS platform available — the Oxford Nanopore Technologies (ONT) MinION device. Although nanopore sequencing on the handheld MinION offers numerous advantages, including on-site sample processing, the relatively high error rate and lack of forensic-specific analysis software has prevented accurate profiling across STR panels in previous studies. Here we present STRspy, a streamlined method capable of producing length- and sequence-based STR allele designations from noisy, long-read data. To demonstrate the capabilities of STRspy, seven reference samples (female: n = 2; male: n = 5) were amplified at 15 and 30 PCR cycles using the Promega PowerSeq 46GY System and sequenced on the ONT MinION device in triplicate. Basecalled reads were processed with STRspy using a custom database containing alleles reported in the STRSeq BioProject NIST 1036 dataset. Resultant STR allele designations and flanking region single nucleotide polymorphism (SNP) calls were compared to the manufacturer-validated genotypes for each sample. STRspy generated robust and reliable genotypes across all autosomal STR loci amplified with 30 PCR cycles, achieving 100% concordance based on both length and sequence. Furthermore, we were able to identify flanking region SNPs with >90% accuracy. These results demonstrate that nanopore sequencing platforms are capable of revealing additional variation in and around STR loci depending on read coverage. As the first long-read platform-specific method to successfully profile the entire panel of autosomal STRs amplified by a commercially available multiplex, STRspy significantly increases the feasibility of nanopore sequencing in forensic applications.

scholarly journals Salmonella Paratyphi Infection: Use of Nanopore Sequencing as a Vivid Alternative for the Identification of Invading Bacteria

2021 ◽  
Vol 122 (2) ◽  
pp. 96-105
Author(s):  
Martin Chmel ◽  
Oldřich Bartoš ◽  
Ondřej Beran ◽  
Petr Pajer ◽  
Jiří Dresler ◽  
...  
Keyword(s):  
Stool Culture ◽  
Reference Laboratory ◽  
Nanopore Sequencing ◽  
Data Generation ◽  
Sequencing Technology ◽  
Time Data ◽  
Serological Tests ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies ◽  
Identification And Characterization

In our study we present an overview of the use of Oxford Nanopore Technologies (ONT) sequencing technology on the background of Enteric fever. Unlike traditional methods (e.g., qPCR, serological tests), the nanopore sequencing technology enables virtually real-time data generation and highly accurate pathogen identification and characterization. Blood cultures were obtained from a 48-year-old female patient suffering from a high fever, headache and diarrhea. Nevertheless, both the initial serological tests and stool culture appeared to be negative. Therefore, the bacterial isolate from blood culture was used for nanopore sequencing (ONT). This technique in combination with subsequent bioinformatic analyses allowed for prompt identification of the disease-causative agent as Salmonella enterica subsp. enterica serovar Paratyphi A. The National Reference Laboratory for Salmonella (NIPH) independently reported this isolate also as serovar Paratyphi A on the basis of results of biochemical and agglutination tests. Therefore, our results are in concordance with certified standards. Furthermore, the data enabled us to assess some basic questions concerning the comparative genomics, i.e., to describe whether the isolated strain differs from the formerly published ones or not. Quite surprisingly, these results indicate that we have detected a novel and so far, unknown variety of this bacteria.

scholarly journals Robust long-read native DNA sequencing using the ONT CsgG Nanopore system

2018 ◽  
Vol 2 ◽  
pp. 23 ◽  
Author(s):  
Jean-Michel Carter ◽  
Shobbir Hussain
Keyword(s):  
Dna Sequencing ◽  
Cancer Cell Line ◽  
Read Length ◽  
Nanopore Sequencing ◽  
Computational Tools ◽  
Practical Applications ◽  
Oxford Nanopore ◽  
Sequencing Method ◽  
Long Read ◽  
Oxford Nanopore Technologies

Background: The ability to obtain long read lengths during DNA sequencing has several potentially important practical applications. Especially long read lengths have been reported using the Nanopore sequencing method, currently commercially available from Oxford Nanopore Technologies (ONT). However, early reports have demonstrated only limited levels of combined throughput and sequence accuracy. Recently, ONT released a new CsgG pore sequencing system as well as a 250b/s translocation chemistry with potential for improvements. Methods: We made use of such components on ONTs miniature ‘MinION’ device and sequenced native genomic DNA obtained from the near haploid cancer cell line HAP1. Analysis of our data was performed utilising recently described computational tools tailored for nanopore/long-read sequencing outputs, and here we present our key findings. Results: From a single sequencing run, we obtained ~240,000 high-quality mapped reads, comprising a total of ~2.3 billion bases. A mean read length of 9.6kb and an N50 of ~17kb was achieved, while sequences mapped to reference with a mean identity of 85%. Notably, we obtained ~68X coverage of the mitochondrial genome and were able to achieve a mean consensus identity of 99.8% for sequenced mtDNA reads. Conclusions: With improved sequencing chemistries already released and higher-throughput instruments in the pipeline, this early study suggests that ONT CsgG-based sequencing may be a useful option for potential practical long-read applications with relevance to complex genomes.

scholarly journals Oxford Nanopore and Bionano Genomics technologies evaluation for plant structural variation detection

2021 ◽  
Author(s):  
Aurélie Canaguier ◽  
Romane Guilbaud ◽  
Erwan Denis ◽  
Ghislaine Magdelenat ◽  
Caroline Belser ◽  
...  
Keyword(s):  
Large Scale ◽  
Optical Mapping ◽  
Sequencing Error ◽  
Structural Variations ◽  
The Public ◽  
Oxford Nanopore ◽  
A Genome ◽  
Long Read ◽  
Optical Maps ◽  
Oxford Nanopore Technologies

AbstractBackgroundStructural Variations (SVs) are very diverse genomic rearrangements. In the past, their detection was restricted to cytological approaches, then to NGS read size and partitionned assemblies. Due to the current capabilities of technologies such as long read sequencing and optical mapping, larger SVs detection are becoming more and more accessible.This study proposes a comparison in SVs detection and characterization from long-read sequencing obtained with the MinION device developed by Oxford Nanopore Technologies and from optical mapping produced by the Saphyr device commercialized by Bionano Genomics. The genomes of the two Arabidopsis thaliana ecotypes Columbia-0 (Col-0) and Landsberg erecta 1 (Ler-1) were chosen to guide the use of one or the other technology.ResultsWe described the SVs detected from the alignment of the best ONT assembly and DLE-1 optical maps of A. thaliana Ler-1 on the public reference Col-0 TAIR10.1. After filtering, 1 184 and 591 Ler-1 SVs were retained from ONT and BioNano technologies respectively. A total of 948 Ler-1 ONT SVs (80.1%) corresponded to 563 Bionano SVs (95.3%) leading to 563 common locations in both technologies. The specific locations were scrutinized to assess improvement in SV detection by either technology. The ONT SVs were mostly detected near TE and gene features, and resistance genes seemed particularly impacted.ConclusionsStructural variations linked to ONT sequencing error were removed and false positives limited, with high quality Bionano SVs being conserved. When compared with the Col-0 TAIR10.1 reference, most of detected SVs were found in same locations. ONT assembly sequence leads to more specific SVs than Bionano one, the later being more efficient to characterize large SVs. Even if both technologies are obvious complementary approaches, ONT data appears to be more adapted to large scale populations study, while Bionano performs better in improving assembly and describing specificity of a genome compared to a reference.

scholarly journals Robust long-read native DNA sequencing using the ONT CsgG Nanopore system

2017 ◽  
Vol 2 ◽  
pp. 23 ◽  
Author(s):  
Jean-Michel Carter ◽  
Shobbir Hussain
Keyword(s):  
Dna Sequencing ◽  
Cancer Cell Line ◽  
Read Length ◽  
Nanopore Sequencing ◽  
Computational Tools ◽  
Practical Applications ◽  
Oxford Nanopore ◽  
Sequencing Method ◽  
Long Read ◽  
Oxford Nanopore Technologies

Background: The ability to obtain long read lengths during DNA sequencing has several potentially important practical applications. Especially long read lengths have been reported using the Nanopore sequencing method, currently commercially available from Oxford Nanopore Technologies (ONT). However, early reports have demonstrated only limited levels of combined throughput and sequence accuracy. Recently, ONT released a new CsgG pore sequencing system as well as a 250b/s translocation chemistry with potential for improvements. Methods: We made use of such components on ONTs miniature ‘MinION’ device and sequenced native genomic DNA obtained from the near haploid cancer cell line HAP1. Analysis of our data was performed utilising recently described computational tools tailored for nanopore/long-read sequencing outputs, and here we present our key findings. Results: From a single sequencing run, we obtained ~240,000 high-quality mapped reads, comprising a total of ~2.3 billion bases. A mean read length of 9.6kb and an N50 of ~17kb was achieved, while sequences mapped to reference with a mean identity of 85%. Notably, we obtained ~68X coverage of the mitochondrial genome and were able to achieve a mean consensus identity of 99.8% for sequenced mtDNA reads. Conclusions: With improved sequencing chemistries already released and higher-throughput instruments in the pipeline, this early study suggests that ONT CsgG-based sequencing may be a useful option for potential practical long-read applications with relevance to complex genomes.

scholarly journals Genome and transcriptome of a pathogenic yeast, Candida nivariensis

2021 ◽  
Author(s):  
Yunfan Fan ◽  
Andrew N Gale ◽  
Anna Bailey ◽  
Kali Barnes ◽  
Kiersten Colotti ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Glabrata ◽  
Nanopore Sequencing ◽  
Cdna Sequencing ◽  
Pathogenic Yeast ◽  
Mitochondrial Sequence ◽  
Dna And Rna ◽  
Oxford Nanopore ◽  
Candida Nivariensis ◽  
Oxford Nanopore Technologies

Abstract We present a highly contiguous genome and transcriptome of the pathogenic yeast, Candida nivariensis. We sequenced both the DNA and RNA of this species using both the Oxford Nanopore Technologies (ONT) and Illumina platforms. We assembled the genome into an 11.8 Mb draft composed of 16 contigs with an N50 of 886 Kb, including a circular mitochondrial sequence of 28 Kb. Using direct RNA nanopore sequencing and Illumina cDNA sequencing, we constructed an annotation of our new assembly, supplemented by lifting over genes from Saccharomyces cerevisiae and Candida glabrata.

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