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 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 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 Consistent ultra-long DNA sequencing with automated slow pipetting

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Trent M. Prall ◽  
Emma K. Neumann ◽  
Julie A. Karl ◽  
Cecilia G. Shortreed ◽  
David A. Baker ◽  
...  
Keyword(s):  
Low Cost ◽  
Volumetric Flow Rate ◽  
Read Length ◽  
Great Length ◽  
Nanopore Sequencing ◽  
Library Preparation ◽  
Average Read Length ◽  
Oxford Nanopore ◽  
Long Read ◽  
Significant Length

Abstract Background Oxford Nanopore Technologies’ instruments can sequence reads of great length. Long reads improve sequence assemblies by unambiguously spanning repetitive elements of the genome. Sequencing reads of significant length requires the preservation of long DNA template molecules through library preparation by pipetting reagents as slowly as possible to minimize shearing. This process is time-consuming and inconsistent at preserving read length as even small changes in volumetric flow rate can result in template shearing. Results We have designed SNAILS (Slow Nucleic Acid Instrument for Long Sequences), a 3D-printable instrument that automates slow pipetting of reagents used in long read library preparation for Oxford Nanopore sequencing. Across six sequencing libraries, SNAILS preserved more reads exceeding 100 kilobases in length and increased its libraries’ average read length over manual slow pipetting. Conclusions SNAILS is a low-cost, easily deployable solution for improving sequencing projects that require reads of significant length. By automating the slow pipetting of library preparation reagents, SNAILS increases the consistency and throughput of long read Nanopore sequencing.

scholarly journals A comparative analysis of computational tools for the prediction of epigenetic DNA methylation from long-read sequencing data

2021 ◽  
Author(s):  
Shruta Sandesh Pai ◽  
Aimee Rachel Mathew ◽  
Roy Anindya
Keyword(s):  
Dna Methylation ◽  
Dna Modification ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Computational Tools ◽  
Specific Location ◽  
Oxford Nanopore ◽  
Long Read ◽  
Dna Methylations ◽  
Higher Eukaryotes

AbstractRecent development of Oxford Nanopore long-read sequencing has opened new avenues of identifying epigenetic DNA methylation. Among the different epigenetic DNA methylations, N6-methyladenosine is the most prevalent DNA modification in prokaryotes and 5-methylcytosine is common in higher eukaryotes. Here we investigated if N6-methyladenosine and 5-methylcytosine modifications could be predicted from the nanopore sequencing data. Using publicly available genome sequencing data of Saccharomyces cerevisiae, we compared the open-access computational tools, including Tombo, mCaller, Nanopolish and DeepSignal for predicting 6mA and 5mC. Our results suggest that Tombo and mCaller can predict DNA N6-methyladenosine modifications at a specific location, whereas, Tombo dampened fraction, Nanopolish methylation likelihood and DeepSignal methylation probability have comparable efficiency for 5-methylcytosine prediction from Oxford Nanopore sequencing data.

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 Consistent ultra-long DNA sequencing with automated slow pipetting

2020 ◽  
Author(s):  
Trent M. Prall ◽  
Emma K. Neumann ◽  
Julie A. Karl ◽  
Cecilia G. Shortreed ◽  
David A. Baker ◽  
...  
Keyword(s):  
Low Cost ◽  
Volumetric Flow Rate ◽  
Read Length ◽  
Great Length ◽  
Nanopore Sequencing ◽  
Library Preparation ◽  
Average Read Length ◽  
Oxford Nanopore ◽  
Long Read ◽  
Significant Length

AbstractBackgroundOxford Nanopore Technologies’ instruments can sequence reads of great length. Long reads improve sequence assemblies by unambiguously spanning repetitive elements of the genome. Sequencing reads of significant length requires the preservation of long DNA template molecules through library preparation by pipetting reagents as slowly as possible in order to minimizing shearing. This process is time consuming and inconsistent at preserving read length as even small changes in volumetric flow rate can result in template shearing.ResultsWe have designed SNAILS (Slow Nucleic Acid Instrument for Long Sequences), a 3D-printable instrument that automates slow pipetting of reagents used in long read library preparation for Oxford Nanopore sequencing. Across six sequencing libraries, SNAILS preserved more reads exceeding one hundred kilobases in length and increased the average read length of its libraries over manual slow pipetting.ConclusionsSNAILS is a low-cost, easily deployable solution for improving sequencing projects that require reads of significant length. By automating the slow pipetting of library preparation reagents, SNAILS both increases the consistency and throughput of long read Nanopore sequencing.

scholarly journals SMURF-seq for fast, multiplexed copy number profiling with long-read sequencers

2018 ◽  
Author(s):  
Rishvanth K. Prabakar ◽  
Liya Xu ◽  
James Hicks ◽  
Andrew D. Smith
Keyword(s):  
Copy Number ◽  
Read Length ◽  
Multiple Copy ◽  
Dna Molecules ◽  
Oxford Nanopore ◽  
Long Read ◽  
Oxford Nanopore Technologies ◽  
Reduced Time

We present SMURF-seq, a protocol to efficiently sequence short DNA molecules on a long-read sequencer by randomly ligating them to form long molecules. Applying SMURF-seq using Oxford Nanopore Technologies MinION yields up to 30 countable fragments per read at present, which generates multiple copy number profiles in a single run at a reduced time and cost. More broadly, SMURF-seq expands the utility of long-read sequencers for read-counting applications, which do not benefit from increased read length.

scholarly journals NanoSplicer: Accurate identification of splice junctions using Oxford Nanopore sequencing

2021 ◽  
Author(s):  
Yupei You ◽  
Michael B. Clark ◽  
Heejung Shim
Keyword(s):  
Cancer Cell Line ◽  
Splice Junction ◽  
Electrical Current ◽  
Lung Cancer Cell Line ◽  
Nanopore Sequencing ◽  
Lung Cancer Cell ◽  
Accurate Identification ◽  
Oxford Nanopore ◽  
Long Read ◽  
Splice Junctions

Motivation: Long read sequencing methods have considerable advantages for characterising RNA isoforms. Oxford nanopore sequencing records changes in electrical current when nucleic acid traverses through a pore. However, basecalling of this raw signal (known as a squiggle) is error prone, making it challenging to accurately identify splice junctions. Existing strategies include utilising matched short-read data and/or annotated splice junctions to correct nanopore reads but add expense or limit junctions to known (incomplete) annotations. Therefore, a method that could accurately identify splice junctions solely from nanopore data would have numerous advantages. Results: We developed "NanoSplicer" to identify splice junctions using raw nanopore signal (squiggles). For each splice junction the observed squiggle is compared to candidate squiggles representing potential junctions to identify the correct candidate. Measuring squiggle similarity enables us to compute the probability of each candidate junction and find the most likely one. We tested our method using 1. synthetic mRNAs with known splice junctions 2. biological mRNAs from a lung-cancer cell-line. The results from both datasets demonstrate NanoSplicer improves splice junction identification, especially when the basecalling error rate near the splice junction is elevated. Our method is implemented in the software package NanoSplicer, available at https://github.com/shimlab/NanoSplicer.

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]

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