Consistent ultra-long DNA sequencing with automated slow pipetting

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.

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Exploring Quantitative Metagenomics Studies using Oxford Nanopore Sequencing: A Computational and Experimental Protocol

10.21203/rs.3.rs-131495/v1 ◽

2020 ◽

Author(s):

Rohia ALILI ◽

Eugeni BELDA ◽

Karine CLEMENT ◽

Phuong Le ◽

Edi PRIFTI ◽

...

Keyword(s):

Dna Extraction ◽

Gut Microbiome ◽

Large Scale ◽

Low Cost ◽

Read Length ◽

Whole Genome ◽

Nanopore Sequencing ◽

Experimental Protocol ◽

Microbial Composition ◽

Oxford Nanopore

Abstract Background: The gut microbiome plays a major role in chronic diseases, several of which are characterized by an altered diversity and composition of bacterial communities. Large-scale sequencing projects allowed the characterization of these microbial community perturbations. However, a gap remains in how these discoveries can be translated into clinical applications. To facilitate routine implementation of microbiome profiling in clinical settings, portable, real-time, and low-cost sequencing technologies are needed.Results: Here, we propose a computational and experimental protocol for whole genome quantitative metagenomics studies of the human gut microbiome with Oxford Nanopore sequencing technology (ONT). We developed a bioinformatic pipeline to process ONT sequences based on the evaluation of different alignment parameters in the estimation of microbial diversity and composition. We also optimized stool collection and DNA extraction methods to maximize read length, a critical parameter for the sequence alignment and classification. Our analytical pipeline was evaluated using simulations of metagenomic communities to reflect naturally occuring compositional variations. We then validated our experimental and analytical pipeline with stool samples from a bariatric surgery cohort sequenced with ONT and Illumina, revealing comparable diversity and microbial composition profiles. These results were compared to those previously obtained with SOLiD sequencing, where differences were observed, possibly explained by variations in library preparation steps. Finally, we found that sequences obtained with ONT allowed assembly of complete genomes for disease-related species.Conclusion: This protocol can be implemented in the clinical or individual setting, bringing rapid personalized whole genome profiling of target microbiome species. Keywords: quantitative metagenomics, microbiome, obesity, gut microbiota, microbial DNA extraction, sequencing, Simulation, Oxford Nanopore Technologies, MinION.

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

Wellcome Open Research ◽

10.12688/wellcomeopenres.11246.1 ◽

2017 ◽

Vol 2 ◽

pp. 23 ◽

Cited By ~ 12

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.

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Improved assembly of noisy long reads by k-mer validation

10.1101/053256 ◽

2016 ◽

Author(s):

A. Bernardo Carvalho ◽

Eduardo G Dupim ◽

Gabriel Nassar

Keyword(s):

Low Cost ◽

Error Rates ◽

Read Length ◽

Celera Assembler ◽

Short Reads ◽

Sequencing Errors ◽

Long Reads ◽

Oxford Nanopore ◽

Long Read ◽

Validation Procedure

Genome assembly depends critically on read length. Two recent technologies, PacBio and Oxford Nanopore, produce read lengths above 20 kb, which yield genome assemblies that are vastly superior to those based on Sanger or short-reads. However, the very high error rates of both technologies (around 15%-20%) makes assembly computationally expensive and imprecise at repeats longer than the read length. Here we show that the efficiency and quality of the assembly of these noisy reads can be significantly improved at a minimal cost, by leveraging on the low error rate and low cost of Illumina short reads. Namely, k-mers from the PacBio raw reads that are not present in the Illumina reads (which account for ~95% of the distinct k-mers) are deemed as sequencing errors and ignored at the seed alignment step. By focusing on ~5% of the k-mers which are error-free, read overlap sensitivity is dramatically increased. Equally important, the validation procedure can be extended to exclude repetitive k-mers, which avoids read miscorrection at repeats and further improve the resulting assemblies. We tested the k-mer validation procedure in one long-read technology (PacBio) and one assembler (MHAP/ Celera Assembler), but is likely to yield analogous improvements with alternative long-read technologies and overlappers, such as Oxford Nanopore and BLASR/DAligner.

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

Wellcome Open Research ◽

10.12688/wellcomeopenres.11246.3 ◽

2018 ◽

Vol 2 ◽

pp. 23 ◽

Cited By ~ 5

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.

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

Wellcome Open Research ◽

10.12688/wellcomeopenres.11246.2 ◽

2017 ◽

Vol 2 ◽

pp. 23 ◽

Cited By ~ 5

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.

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Exploring Semi-Quantitative Metagenomic Studies Using Oxford Nanopore Sequencing: A Computational and Experimental Protocol

Genes ◽

10.3390/genes12101496 ◽

2021 ◽

Vol 12 (10) ◽

pp. 1496

Author(s):

Rohia Alili ◽

Eugeni Belda ◽

Phuong Le ◽

Thierry Wirth ◽

Jean-Daniel Zucker ◽

...

Keyword(s):

Gut Microbiome ◽

Large Scale ◽

Low Cost ◽

Extraction Methods ◽

Read Length ◽

Whole Genome ◽

Nanopore Sequencing ◽

Experimental Protocol ◽

Microbial Composition ◽

Oxford Nanopore

The gut microbiome plays a major role in chronic diseases, of which several are characterized by an altered composition and diversity of bacterial communities. Large-scale sequencing projects allowed for characterizing the perturbations of these communities. However, translating these discoveries into clinical applications remains a challenge. To facilitate routine implementation of microbiome profiling in clinical settings, portable, real-time, and low-cost sequencing technologies are needed. Here, we propose a computational and experimental protocol for whole-genome semi-quantitative metagenomic studies of human gut microbiome with Oxford Nanopore sequencing technology (ONT) that could be applied to other microbial ecosystems. We developed a bioinformatics protocol to analyze ONT sequences taxonomically and functionally and optimized preanalytic protocols, including stool collection and DNA extraction methods to maximize read length. This is a critical parameter for the sequence alignment and classification. Our protocol was evaluated using simulations of metagenomic communities, which reflect naturally occurring compositional variations. Next, we validated both protocols using stool samples from a bariatric surgery cohort, sequenced with ONT, Illumina, and SOLiD technologies. Results revealed similar diversity and microbial composition profiles. This protocol can be implemented in a clinical or research setting, bringing rapid personalized whole-genome profiling of target microbiome species.

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Exploring Quantitative Metagenomics Studies Using Oxford Nanopore Sequencing: A Computational and Experimental Protocol

10.20944/preprints202108.0104.v1 ◽

2021 ◽

Author(s):

Rohia Alili ◽

Eugeni Belda ◽

Phuong Le ◽

Thierry Wirth ◽

Jean-Daniel Zucker ◽

...

Keyword(s):

Gut Microbiome ◽

Large Scale ◽

Low Cost ◽

Extraction Methods ◽

Read Length ◽

Whole Genome ◽

Nanopore Sequencing ◽

Experimental Protocol ◽

Microbial Composition ◽

Oxford Nanopore

Background: The gut microbiome plays a major role in chronic diseases, of which several are characterized by an altered composition and diversity of bacterial communities. Large-scale sequencing projects allowed characterizing the perturbations of these communities. However, translating these discoveries into clinical applications remains a challenges. To facilitate routine implementation of microbiome profiling in clinical settings, portable, real-time, and low-cost sequencing technologies are needed. Results: Here, we propose a computational and experimental protocol for whole genome quantitative metagenomics studies of human gut microbiome with Oxford Nanopore sequencing technology (ONT) that could be applied to other microbial ecosystems. We developed a bioinformatic protocol to analyse ONT sequences taxonomically and functionally and optimized pre-analytic protocols including stool collection and DNA extraction methods to maximize read length. This is a critical parameter for the sequence alignment and classification. Our protocol was evaluated using simulations of metagenomic communities which reflect naturally occuring compositional variations. Next, we validated both protocols using stool samples from a bariatric surgery cohort, sequenced with ONT, Illumina and SOLiD technologies. Results revealed similar diversity and microbial composition profiles. Conclusion: This protocol can be implemented in the clinical or research setting, bringing rapid personalized whole genome profiling of target microbiome species.

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High resolution species detection: accurate long read eDNA metabarcoding of North Sea fish using Oxford Nanopore sequencing

10.22541/au.163828284.40261296/v1 ◽

2021 ◽

Author(s):

Karlijn Doorenspleet ◽

Lara Jansen ◽

Saskia Oosterbroek ◽

Oscar Bos ◽

Pauline Kamermans ◽

...

Keyword(s):

High Resolution ◽

North Sea ◽

Fish Species ◽

16S Rrna Genes ◽

Rrna Genes ◽

Nanopore Sequencing ◽

Nature Restoration ◽

Oxford Nanopore ◽

Field Samples ◽

Long Read

To monitor the effect of nature restoration projects in North Sea ecosystems, accurate and intensive biodiversity assessments are vital. DNA based techniques and especially environmental DNA (eDNA) metabarcoding from seawater is becoming a powerful monitoring tool. However, current approaches are based on genetic target regions of <500 nucleotides, which offer limited taxonomic resolution. This study aims to develop and validate a long read nanopore sequencing method for eDNA that enables improved identification of fish species. We designed a universal primer pair targeting a 2kb region covering the 12S and 16S rRNA genes of fish mitochondria. eDNA was amplified and sequenced using the Oxford Nanopore MiniON. Sequence data was processed using the new pipeline Decona, and accurate consensus identities of above 99.9% were retrieved. The primer set efficiency was tested with eDNA from a 3.000.000 L zoo aquarium with 31 species of bony fish and elasmobranchs. Over 55% of the species present were identified on species level and over 75% on genus level. Next, our long read eDNA metabarcoding approach was applied to North Sea eDNA field samples collected at ship wreck sites, the Gemini Offshore Wind Farm, the Borkum Reef Grounds and a bare sand bottom. Here, location specific fish and vertebrate communities were obtained. Incomplete reference databases still form a major bottleneck in further developing high resolution long read metabarcoding. Yet, the method has great potential for rapid and accurate fish species monitoring in marine field studies.

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Detection of single nucleotide and copy number variants in the Fabry disease-associated GLA gene using nanopore sequencing

Scientific Reports ◽

10.1038/s41598-021-01749-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Albina Nowak ◽

Omer Murik ◽

Tzvia Mann ◽

David A. Zeevi ◽

Gheona Altarescu

Keyword(s):

Fabry Disease ◽

Copy Number ◽

New Technology ◽

Copy Number Variants ◽

Cost Effective ◽

Nanopore Sequencing ◽

Oxford Nanopore ◽

Long Read ◽

Sanger Sequence ◽

Gla Gene

AbstractMore than 900 variants have been described in the GLA gene. Some intronic variants and copy number variants in GLA can cause Fabry disease but will not be detected by classical Sanger sequence. We aimed to design and validate a method for sequencing the GLA gene using long-read Oxford Nanopore sequencing technology. Twelve Fabry patients were blindly analyzed, both by conventional Sanger sequence and by long-read sequencing of a 13 kb PCR amplicon. We used minimap2 to align the long-read data and Nanopolish and Sniffles to call variants. All the variants detected by Sanger (including a deep intronic variant) were also detected by long-read sequencing. One patient had a deletion that was not detected by Sanger sequencing but was detected by the new technology. Our long-read sequencing-based method was able to detect missense variants and an exonic deletion, with the added advantage of intronic analysis. It can be used as an efficient and cost-effective tool for screening and diagnosing Fabry disease.

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