scholarly journals Performance of neural network basecalling tools for Oxford Nanopore sequencing

2019 ◽  
Author(s):  
Ryan R. Wick ◽  
Louise M. Judd ◽  
Kathryn E. Holt
Keyword(s):  
Neural Network ◽  
Electrical Signal ◽  
Specific Data ◽  
Consensus Sequences ◽  
Additional Signal ◽  
Oxford Nanopore ◽  
Majority Rules ◽  
Sequencing Platforms ◽  
Oxford Nanopore Technologies ◽  
Level Analysis

AbstractBasecalling, the computational process of translating raw electrical signal to nucleotide sequence, is of critical importance to the sequencing platforms produced by Oxford Nanopore Technologies (ONT). Here we examine the performance of different basecalling tools, looking at accuracy at the level of bases within individual reads and at majority-rules consensus basecalls in an assembly. We also investigate some additional aspects of basecalling: training using a taxon-specific dataset, using a larger neural network model and improving consensus basecalls in an assembly via additional signal-level analysis with Nanopolish. Training basecallers on taxon-specific data resulted in a significant boost in consensus accuracy, mostly due to the reduction of errors in methylation motifs. A larger neural network was able to improve both read and consensus accuracy, but at a cost to speed. Improving consensus sequences (‘polishing’) with Nanopolish somewhat negates the accuracy differences in basecallers, but pre-polish accuracy does have an effect on post-polish accuracy, so basecaller choice is still relevant even when Nanopolish is used.

scholarly journals Updated Genome Sequence for the Probiotic Bacterium Bifidobacterium animalis subsp. lactis BB-12

2021 ◽  
Vol 10 (27) ◽  
Author(s):  
Kristian Jensen ◽  
Kosai Al-Nakeeb ◽  
Anna Koza ◽  
Ahmad A. Zeidan
Keyword(s):  
Genome Sequence ◽  
Probiotic Bacterium ◽  
Content Type ◽  
Short Read Sequencing ◽  
Bifidobacterium Animalis ◽  
Oxford Nanopore ◽  
Hybrid Genome ◽  
Long Read ◽  
Sequencing Platforms ◽  
Oxford Nanopore Technologies

The genome of Bifidobacterium animalis subsp. lactis BB-12 was sequenced using Oxford Nanopore Technologies long-read and Illumina short-read sequencing platforms. A hybrid genome assembly approach was used to construct an updated complete genome sequence for BB-12 containing 1,944,152 bp, with a G+C content of 60.5% and 1,615 genes.

scholarly journals Methplotlib: analysis of modified nucleotides from nanopore sequencing

2019 ◽  
Author(s):  
Wouter De Coster ◽  
Mojca Strazisar
Keyword(s):  
Gene Expression Regulation ◽  
Command Line ◽  
Nanopore Sequencing ◽  
Modified Nucleotides ◽  
Oxford Nanopore ◽  
Command Line Tool ◽  
Within Subjects ◽  
Allele Specific ◽  
Sequencing Platforms ◽  
Oxford Nanopore Technologies

AbstractSummaryModified nucleotides play a crucial role in gene expression regulation. Here we describe methplotlib, a tool developed for the visualization of modified nucleotides detected from Oxford Nanopore Technologies sequencing platforms, together with additional scripts for statistical analysis of allele specific modification within subjects and differential modification frequency across subjects.Availability and implementationThe methplotlib command-line tool is written in Python3, is compatible with Linux, Mac OS and the MS Windows 10 Subsystem for Linux and released under the MIT license. The source code can be found at https://github.com/wdecoster/methplotlib and can be installed from PyPI and bioconda. Our repository includes test data and the tool is continuously tested at [email protected]

scholarly journals Complete Circular Genome Sequences of Brachyspira hyodysenteriae Isolates of the Four Different Sequence Types Causing Swine Dysentery in Switzerland

2021 ◽  
Vol 10 (39) ◽  
Author(s):  
Ana B. García-Martín ◽  
Sarah Schmitt ◽  
Friederike Zeeh ◽  
Vincent Perreten
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Hybrid Assembly ◽  
Swine Dysentery ◽  
Content Type ◽  
Brachyspira Hyodysenteriae ◽  
Oxford Nanopore ◽  
Sequencing Platforms ◽  
Sequence Types ◽  
Oxford Nanopore Technologies

The complete genomes of four Brachyspira hyodysenteriae isolates of the four different sequence types (STs) (ST6, ST66, ST196, and ST197) causing swine dysentery in Switzerland were generated by whole-genome sequencing and de novo hybrid assembly of reads obtained from second (Illumina) and third (Oxford Nanopore Technologies and Pacific Biosciences) high-throughput sequencing platforms.

scholarly journals Recovery of small plasmid sequences via Oxford Nanopore sequencing

2021 ◽  
Vol 7 (8) ◽  
Author(s):  
Ryan R. Wick ◽  
Louise M. Judd ◽  
Kelly L. Wyres ◽  
Kathryn E. Holt
Keyword(s):  
Whole Genome ◽  
Bacterial Isolates ◽  
Small Plasmid ◽  
Dna Library ◽  
Oxford Nanopore ◽  
Sequencing Platforms ◽  
Plasmid Size ◽  
Plasmid Recovery ◽  
Oxford Nanopore Technologies ◽  
Do So

Oxford Nanopore Technologies (ONT) sequencing platforms currently offer two approaches to whole-genome native-DNA library preparation: ligation and rapid. In this study, we compared these two approaches for bacterial whole-genome sequencing, with a specific aim of assessing their ability to recover small plasmid sequences. To do so, we sequenced DNA from seven plasmid-rich bacterial isolates in three different ways: ONT ligation, ONT rapid and Illumina. Using the Illumina read depths to approximate true plasmid abundance, we found that small plasmids (<20 kbp) were underrepresented in ONT ligation read sets (by a mean factor of ~4) but were not underrepresented in ONT rapid read sets. This effect correlated with plasmid size, with the smallest plasmids being the most underrepresented in ONT ligation read sets. We also found lower rates of chimaeric reads in the rapid read sets relative to ligation read sets. These results show that when small plasmid recovery is important, ONT rapid library preparations are preferable to ligation-based protocols.

scholarly journals Benchmarking of long-read assemblers for prokaryote whole genome sequencing

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 2138 ◽  
Author(s):  
Ryan R. Wick ◽  
Kathryn E. Holt
Keyword(s):  
Data Sets ◽  
Computationally Efficient ◽  
Short Read Sequencing ◽  
Oxford Nanopore ◽  
Long Read ◽  
Sequencing Platforms ◽  
Computational Resources ◽  
Assembly Algorithms ◽  
Oxford Nanopore Technologies ◽  
Multiple Assembly

Background: Data sets from long-read sequencing platforms (Oxford Nanopore Technologies and Pacific Biosciences) allow for most prokaryote genomes to be completely assembled – one contig per chromosome or plasmid. However, the high per-read error rate of long-read sequencing necessitates different approaches to assembly than those used for short-read sequencing. Multiple assembly tools (assemblers) exist, which use a variety of algorithms for long-read assembly. Methods: We used 500 simulated read sets and 120 real read sets to assess the performance of six long-read assemblers (Canu, Flye, Miniasm/Minipolish, Raven, Redbean and Shasta) across a wide variety of genomes and read parameters. Assemblies were assessed on their structural accuracy/completeness, sequence identity, contig circularisation and computational resources used. Results: Canu v1.9 produced moderately reliable assemblies but had the longest runtimes of all assemblers tested. Flye v2.6 was more reliable and did particularly well with plasmid assembly. Miniasm/Minipolish v0.3 was the only assembler which consistently produced clean contig circularisation. Raven v0.0.5 was the most reliable for chromosome assembly, though it did not perform well on small plasmids and had circularisation issues. Redbean v2.5 and Shasta v0.3.0 were computationally efficient but more likely to produce incomplete assemblies. Conclusions: Of the assemblers tested, Flye, Miniasm/Minipolish and Raven performed best overall. However, no single tool performed well on all metrics, highlighting the need for continued development on long-read assembly algorithms.

scholarly journals MSRCall: A Multi-scale Deep Neural Network to Basecall Oxford Nanopore Sequences

2021 ◽  
Author(s):  
Yang-Ming Yeh ◽  
Yi-Chang Lu
Keyword(s):  
Neural Network ◽  
Network Architecture ◽  
Ionic Current ◽  
Scale Structure ◽  
Neural Network Architecture ◽  
Translation Process ◽  
Multi Scale ◽  
Oxford Nanopore ◽  
Rna Fragments ◽  
Oxford Nanopore Technologies

MinION, a third-generation sequencer from Oxford Nanopore Technologies, is a portable device that can provide long nucleotide read data in real-time. It primarily aims to deduce the makeup of nucleotide sequences from the ionic current signals generated when passing DNA/RNA fragments through nanopores charged with a voltage difference. To determine the nucleotides from the measured signals, a translation process known as basecalling is required. However, compared to NGS basecallers, the calling accuracy of MinION still needs to be improved. In this work, a simple but powerful neural network architecture called MSRCall is proposed. MSRCall comprises a multi-scale structure, recurrent layers, a fusion block, and a CTC decoder. To better identify both short-range and long-range dependencies, the recurrent layer is redesigned to capture various time-scale features with a multi-scale structure. The results show that MSRCall outperforms other basecallers in terms of both read and consensus accuracies.

scholarly journals Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

2019 ◽  
Author(s):  
Søren M. Karst ◽  
Ryan M. Ziels ◽  
Rasmus H. Kirkegaard ◽  
Emil A. Sørensen ◽  
Daniel McDonald ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Amplicon Sequencing ◽  
High Accuracy ◽  
Pacbio Sequencing ◽  
Consensus Sequences ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genomic Regions ◽  
Oxford Nanopore Technologies

AbstractHigh-throughput amplicon sequencing of large genomic regions remains challenging for short-read technologies. Here, we report a high-throughput amplicon sequencing approach combining unique molecular identifiers (UMIs) with Oxford Nanopore Technologies or Pacific Biosciences CCS sequencing, yielding high accuracy single-molecule consensus sequences of large genomic regions. Our approach generates amplicon and genomic sequences of >10,000 bp in length with a mean error-rate of 0.0049-0.0006% and chimera rate <0.022%.

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 Benchmarking of long-read assemblers for prokaryote whole genome sequencing

F1000Research ◽  
2020 ◽  
Vol 8 ◽  
pp. 2138 ◽  
Author(s):  
Ryan R. Wick ◽  
Kathryn E. Holt
Keyword(s):  
Data Sets ◽  
Computationally Efficient ◽  
Oxford Nanopore ◽  
Long Read ◽  
Sequencing Platforms ◽  
Computational Resources ◽  
Assembly Algorithms ◽  
Oxford Nanopore Technologies ◽  
Sequence Errors ◽  
Multiple Assembly

Background: Data sets from long-read sequencing platforms (Oxford Nanopore Technologies and Pacific Biosciences) allow for most prokaryote genomes to be completely assembled – one contig per chromosome or plasmid. However, the high per-read error rate of long-read sequencing necessitates different approaches to assembly than those used for short-read sequencing. Multiple assembly tools (assemblers) exist, which use a variety of algorithms for long-read assembly. Methods: We used 500 simulated read sets and 120 real read sets to assess the performance of eight long-read assemblers (Canu, Flye, Miniasm/Minipolish, NECAT, NextDenovo/NextPolish, Raven, Redbean and Shasta) across a wide variety of genomes and read parameters. Assemblies were assessed on their structural accuracy/completeness, sequence identity, contig circularisation and computational resources used. Results: Canu v2.0 produced reliable assemblies and was good with plasmids, but it performed poorly with circularisation and had the longest runtimes of all assemblers tested. Flye v2.8 was also reliable and made the smallest sequence errors, though it used the most RAM. Miniasm/Minipolish v0.3/v0.1.3 was the most likely to produce clean contig circularisation. NECAT v20200119 was reliable and good at circularisation but tended to make larger sequence errors. NextDenovo/NextPolish v2.3.0/v1.2.4 was reliable with chromosome assembly but bad with plasmid assembly. Raven v1.1.10 was the most reliable for chromosome assembly, though it did not perform well on small plasmids and had circularisation issues. Redbean v2.5 and Shasta v0.5.1 were computationally efficient but more likely to produce incomplete assemblies. Conclusions: Of the assemblers tested, Flye, Miniasm/Minipolish and Raven performed best overall. However, no single tool performed well on all metrics, highlighting the need for continued development on long-read assembly algorithms.

scholarly journals Benchmarking of long-read assemblers for prokaryote whole genome sequencing

F1000Research ◽  
2020 ◽  
Vol 8 ◽  
pp. 2138 ◽  
Author(s):  
Ryan R. Wick ◽  
Kathryn E. Holt
Keyword(s):  
Data Sets ◽  
Computationally Efficient ◽  
Short Read Sequencing ◽  
Oxford Nanopore ◽  
Long Read ◽  
Sequencing Platforms ◽  
Computational Resources ◽  
Assembly Algorithms ◽  
Oxford Nanopore Technologies ◽  
Multiple Assembly

Background: Data sets from long-read sequencing platforms (Oxford Nanopore Technologies and Pacific Biosciences) allow for most prokaryote genomes to be completely assembled – one contig per chromosome or plasmid. However, the high per-read error rate of long-read sequencing necessitates different approaches to assembly than those used for short-read sequencing. Multiple assembly tools (assemblers) exist, which use a variety of algorithms for long-read assembly. Methods: We used 500 simulated read sets and 120 real read sets to assess the performance of seven long-read assemblers (Canu, Flye, Miniasm/Minipolish, NECAT, Raven, Redbean and Shasta) across a wide variety of genomes and read parameters. Assemblies were assessed on their structural accuracy/completeness, sequence identity, contig circularisation and computational resources used. Results: Canu v1.9 produced moderately reliable assemblies but had the longest runtimes of all assemblers tested. Flye v2.7 was more reliable and did particularly well with plasmid assembly. Miniasm/Minipolish v0.3 and NECAT v20200119 were the most likely to produce clean contig circularisation. Raven v0.0.8 was the most reliable for chromosome assembly, though it did not perform well on small plasmids and had circularisation issues. Redbean v2.5 and Shasta v0.4.0 were computationally efficient but more likely to produce incomplete assemblies. Conclusions: Of the assemblers tested, Flye, Miniasm/Minipolish and Raven performed best overall. However, no single tool performed well on all metrics, highlighting the need for continued development on long-read assembly algorithms.

Sign in / Sign up

Export Citation Format

Share Document