Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology

The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Among available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, as it is mobile, scalable, and cost-effective. Therefore, streamlined nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification. We adapted and simplified existing workflows using the ‘midnight’ 1200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost the entire SARS-CoV-2 genome. Subsequently, we applied Oxford Nanopore Rapid Barcoding and the portable MinION Mk1C sequencer combined with the interARTIC bioinformatics pipeline. We tested a simplified and less time-consuming workflow using SARS-CoV-2-positive specimens from clinical routine and identified the CT value as a useful pre-analytical parameter, which may help to decrease sequencing failures rates. Complete pipeline duration was approx. 7 h for one specimen and approx. 11 h for 12 multiplexed barcoded specimens. The adapted protocol contains fewer processing steps and can be completely conducted within one working day. Diagnostic CT values deduced from qPCR standardization experiments can act as principal criteria for specimen selection. As a guideline, SARS-CoV-2 genome copy numbers lower than 4 × 106 were associated with a coverage threshold below 20-fold and incompletely assembled SARS-CoV-2 genomes. Thus, based on the described thermocycler/chemistry combination, we recommend CT values of ~26 or lower to achieve full and high-quality SARS-CoV-2 (+)RNA genome coverage.

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Simplified point-of-care full SARS-CoV-2 genome sequencing using nanopore technology

10.1101/2021.07.08.21260171 ◽

2021 ◽

Author(s):

Anton Pembaur ◽

Erwan Sallard ◽

Patrick Philipp Weil ◽

Jennifer Ortelt ◽

Parviz Ahmad-Nejad ◽

...

Keyword(s):

Point Of Care ◽

Warning System ◽

Nanopore Sequencing ◽

Bioinformatics Pipeline ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Pcr Multiplex ◽

Primer Sets ◽

Working Day ◽

Almost All

Background: The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment a global decentralized surveillance and warning system to recognize local outbreaks and the emergence of novel variants-of-concern. Among the available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, since it is mobile, scalable and acquisition investments are comparably low. Therefore, streamlined and efficient nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification, in particular for smaller hospital laboratories with lower throughput. Results: We adapted and simplified existing workflows using the midnight 1,200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost all of the SARS-CoV-2 genome. Subsequently, we applied the Oxford Nanopore Rapid barcoding protocol and the portable MinION Mk1C sequencer in combination with the ARTIC bioinformatics pipeline. We tested the simplified and less time-consuming workflow on confirmed SARS-CoV-2-positive specimens from clinical routine and identified pre-analytical parameters, which may help to decrease the rate of sequencing failures. Duration of the complete pipeline was approx. 7 hrs for one specimen and approx. 11 hrs for 12 multiplexed barcoded specimens. Conclusions: The adapted protocol contains less processing steps. Diagnostic CT values are the principal criteria for specimen selection. Subsequent to diagnostic qRT-PCR, multiplex library preparation, quality controls, nanopore sequencing and the bioinformatic pipeline can be completely conducted within one working-day.

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nanopore nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon, combined RT-PCR) v2

10.17504/protocols.io.b3bcqiiw ◽

2021 ◽

Author(s):

Anton Pembaur ◽

Erwan Sallard ◽

Patrick Weil ◽

Jennifer Ortelt ◽

Parviz Ahmad-Nejad ◽

...

Keyword(s):

Rna Extraction ◽

Bioinformatics Pipeline ◽

Bioinformatic Pipeline ◽

Oxford Nanopore ◽

Hands On ◽

Cost Efficient ◽

Primer Sets ◽

Working Day ◽

Almost All ◽

The Cost

We established a protocol for fast, cost efficient Sars-CoV-2 sequencing with little as possible hands-on time (around 3h in total, excluding RNA extraction). The whole Sequencing can be done in one working day, including the bioinformatic pipeline. The cost per sample accumulates at around 40$, with already isolated RNA. We adapted and simplified existing workflows using the ‘midnight’ 1,200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost all of the SARS-CoV-2 genome. Subsequently, we applied the Oxford Nanopore Rapid barcoding protocol and the portable MinION Mk1C sequencer in combination with the ARTIC bioinformatics pipeline. We tested the simplified and less time-consuming workflow on confirmed SARS-CoV-2-positive specimens from clinical routine and identified pre-analytical parameters, which may help to decrease the rate of sequencing failures. Duration of the complete pipeline was approx. 7 hrs for one specimen and approx. 11 hrs for 12 multiplexed barcoded specimens. This protocol is a modified version of Nikki Freed and Olin Silanders protocol. To get information such as Primers, visit their protocol. Nikki Freed, Olin Silander 2020. nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon).doi: 10.1093/biomethods/bpaa014 Our peer-reviewed paper is available here: https://www.mdpi.com/2076-2607/9/12/2598

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nanopore nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon, combined RT-PCR) v2

10.17504/protocols.io.bx72prqe ◽

2021 ◽

Author(s):

Anton not provided Pembaur ◽

Erwan not provided Sallard ◽

Patrick Weil ◽

Jennifer Ortelt ◽

Parviz Ahmad-Nejad ◽

...

Keyword(s):

Rna Extraction ◽

Bioinformatics Pipeline ◽

Bioinformatic Pipeline ◽

Oxford Nanopore ◽

Hands On ◽

Cost Efficient ◽

Primer Sets ◽

Working Day ◽

Almost All ◽

The Cost

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NanoHIV: A Bioinformatics Pipeline for Producing Accurate, Near Full-Length HIV Proviral Genomes Sequenced Using the Oxford Nanopore Technology

Cells ◽

10.3390/cells10102577 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2577

Author(s):

Imogen A. Wright ◽

Kayla E. Delaney ◽

Mary Grace K. Katusiime ◽

Johannes C. Botha ◽

Susan Engelbrecht ◽

...

Keyword(s):

Pcr Amplification ◽

Cost Effective ◽

Full Length ◽

Sequencing Error ◽

Consensus Building ◽

Genome Sequences ◽

Bioinformatics Pipeline ◽

Oxford Nanopore ◽

Single Genome ◽

Hiv 1

HIV-1 proviral single-genome sequencing by limiting-dilution polymerase chain reaction (PCR) amplification is important for differentiating the sequence-intact from defective proviruses that persist during antiretroviral therapy (ART). Intact proviruses may rebound if ART is interrupted and are the barrier to an HIV cure. Oxford Nanopore Technologies (ONT) sequencing offers a promising, cost-effective approach to the sequencing of long amplicons such as near full-length HIV-1 proviruses, but the high diversity of HIV-1 and the ONT sequencing error render analysis of the generated data difficult. NanoHIV is a new tool that uses an iterative consensus generation approach to construct accurate, near full-length HIV-1 proviral single-genome sequences from ONT data. To validate the approach, single-genome sequences generated using NanoHIV consensus building were compared to Illumina® consensus building of the same nine single-genome near full-length amplicons and an average agreement of 99.4% was found between the two sequencing approaches.

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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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Evaluation of Germline Structural Variant Calling Methods for Nanopore Sequencing Data

Frontiers in Genetics ◽

10.3389/fgene.2021.761791 ◽

2021 ◽

Vol 12 ◽

Author(s):

Davide Bolognini ◽

Alberto Magi

Keyword(s):

Variant Calling ◽

Research Report ◽

Nanopore Sequencing ◽

Sequencing Data ◽

Factors Affecting ◽

Sequencing Technologies ◽

Long Reads ◽

Oxford Nanopore ◽

Sequencing Studies ◽

Long Read

Structural variants (SVs) are genomic rearrangements that involve at least 50 nucleotides and are known to have a serious impact on human health. While prior short-read sequencing technologies have often proved inadequate for a comprehensive assessment of structural variation, more recent long reads from Oxford Nanopore Technologies have already been proven invaluable for the discovery of large SVs and hold the potential to facilitate the resolution of the full SV spectrum. With many long-read sequencing studies to follow, it is crucial to assess factors affecting current SV calling pipelines for nanopore sequencing data. In this brief research report, we evaluate and compare the performances of five long-read SV callers across four long-read aligners using both real and synthetic nanopore datasets. In particular, we focus on the effects of read alignment, sequencing coverage, and variant allele depth on the detection and genotyping of SVs of different types and size ranges and provide insights into precision and recall of SV callsets generated by integrating the various long-read aligners and SV callers. The computational pipeline we propose is publicly available at https://github.com/davidebolo1993/EViNCe and can be adjusted to further evaluate future nanopore sequencing datasets.

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Detection of Single Nucleotide and Copy Number Variants in the Fabry Disease-associated GLA Gene Using Nanopore Sequencing

10.21203/rs.3.rs-579267/v1 ◽

2021 ◽

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

Abstract Introduction: More than one thousand 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.Aims: We aimed to design and validate a method for sequencing the GLA gene using long read Oxford Nanopore sequencing technology.Methods: Twelve Fabry patients were blindly analyzed, both by conventional Sanger sequence and by long read sequencing of a 13kb PCR amplicon. We used minimap2 to align the long read data and Nanopolish and Sniffles to call variants.Results: 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.Conclusions: 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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Direct RNA Nanopore Sequencing of SARS-CoV-2 Extracted from Critical Material from Swabs

Life ◽

10.3390/life12010069 ◽

2022 ◽

Vol 12 (1) ◽

pp. 69

Author(s):

Davide Vacca ◽

Antonino Fiannaca ◽

Fabio Tramuto ◽

Valeria Cancila ◽

Laura La Paglia ◽

...

Keyword(s):

Rna Sequencing ◽

Systematic Errors ◽

N Gene ◽

Nanopore Sequencing ◽

Rna Seq ◽

Bioinformatics Pipeline ◽

Critical Material ◽

Oxford Nanopore ◽

Oropharyngeal Swab ◽

Generation Sequencing

In consideration of the increasing prevalence of COVID-19 cases in several countries and the resulting demand for unbiased sequencing approaches, we performed a direct RNA sequencing (direct RNA seq.) experiment using critical oropharyngeal swab samples collected from Italian patients infected with SARS-CoV-2 from the Palermo region in Sicily. Here, we identified the sequences SARS-CoV-2 directly in RNA extracted from critical samples using the Oxford Nanopore MinION technology without prior cDNA retrotranscription. Using an appropriate bioinformatics pipeline, we could identify mutations in the nucleocapsid (N) gene, which have been reported previously in studies conducted in other countries. In conclusion, to the best of our knowledge, the technique used in this study has not been used for SARS-CoV-2 detection previously owing to the difficulties in the extraction of RNA of sufficient quantity and quality from routine oropharyngeal swabs. Despite these limitations, this approach provides the advantages of true native RNA sequencing and does not include amplification steps that could introduce systematic errors. This study can provide novel information relevant to the current strategies adopted in SARS-CoV-2 next-generation sequencing.

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A Multiplex Pharmacogenetics Assay using the MinION Nanopore Sequencing Device

10.1101/563262 ◽

2019 ◽

Author(s):

Yusmiati Liau ◽

Simone L. Cree ◽

Simran Maggo ◽

Allison L. Miller ◽

John F. Pearson ◽

...

Keyword(s):

Dna Sequencing ◽

Genetic Polymorphisms ◽

Point Of Care ◽

Variant Calling ◽

Cost Effective ◽

Response Variability ◽

Multiplex Assay ◽

Nanopore Sequencing ◽

Three Samples ◽

Screening Assays

AbstractAimThe MinION nanopore sequencing device opens the opportunity to cost-effective and point-of-care DNA sequencing. We developed a multiplex assay targeting pharmacogenetic variants related to clopidogrel and warfarin, two commonly used drugs that show response variability due to genetic polymorphisms.Materials & MethodsSix reference and 78 clinical DNA samples were amplified by PCR to generate 15 amplicons targeting key variants. These products were then barcoded to enable sample multiplexing. Three variant calling tools were used to compare genotyping accuracy.Results and ConclusionsAll but three samples were successfully sequenced and genotyped. Nanopolish software achieved accuracy > 90 % for all except one variant. While minor mis-genotyping issues exist, this work demonstrates that drug-specific or broad pharmacogenetic screening assays are possible on the MinION sequencing device.

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Direct RNA nanopore sequencing of SARS-CoV-2 extracted from critical material from swabs

10.1101/2020.12.21.20191346 ◽

2020 ◽

Author(s):

Davide Vacca ◽

Antonino Fiannaca ◽

Fabio Tramuto ◽

Valeria Cancila ◽

Laura La Paglia ◽

...

Keyword(s):

Rna Sequencing ◽

Gene Sequence ◽

N Gene ◽

Nanopore Sequencing ◽

Bioinformatics Pipeline ◽

Critical Material ◽

Oxford Nanopore ◽

Sequencing Experiment ◽

Oropharyngeal Swab ◽

Generation Sequencing

ABSTRACTBackgroundIn consideration of the increasing prevalence of COVID-19 cases in several countries and the resulting demand for unbiased sequencing approaches, we performed a direct RNA sequencing experiment using critical oropharyngeal swab samples collected from Italian patients infected with SARS-CoV-2 from the Palermo region in Sicily.MethodsHere, we identified the sequences SARS-CoV-2 directly in RNA extracted from critical samples using the Oxford Nanopore MinION technology without prior cDNA retro-transcription.ResultsUsing an appropriate bioinformatics pipeline, we could identify mutations in the nucleocapisid (N) gene, which have been reported previously in studies conducted in other countries.ConclusionTo the best of our knowledge, the technique used in this study has not been used for SARS-CoV-2 detection previously owing to the difficulties in the extraction of RNA of sufficient quantity and quality from routine oropharyngeal swabs.Despite these limitations, this approach provides the advantages of true native RNA sequencing, and does not include amplification steps that could introduce systematic errors.This study can provide novel information relevant to the current strategies adopted in SARS-CoV-2 next-generation sequencing.We deposited the gene sequence in the NCBI database under the following URL:https://www.ncbi.nlm.nih.gov/nuccore/MT457389

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