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

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.

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

2021 ◽  
Vol 9 (12) ◽  
pp. 2598
Author(s):  
Anton Pembaur ◽  
Erwan Sallard ◽  
Patrick Philipp Weil ◽  
Jennifer Ortelt ◽  
Parviz Ahmad-Nejad ◽  
...  
Keyword(s):  
Point Of Care ◽  
Cost Effective ◽  
Nanopore Sequencing ◽  
Bioinformatics Pipeline ◽  
Sequencing Technologies ◽  
Copy Numbers ◽  
Oxford Nanopore ◽  
Novel Variants ◽  
Primer Sets ◽  
Working Day

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.

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

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

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

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

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

scholarly journals Evaluation of Germline Structural Variant Calling Methods for Nanopore Sequencing Data

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.

scholarly journals Direct RNA Nanopore Sequencing of SARS-CoV-2 Extracted from Critical Material from Swabs

Life ◽  
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.

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

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

scholarly journals De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing

F1000Research ◽  
2018 ◽  
Vol 6 ◽  
pp. 618 ◽  
Author(s):  
Michael Liem ◽  
Hans J. Jansen ◽  
Ron P. Dirks ◽  
Christiaan V. Henkel ◽  
G. Paul H. van Heusden ◽  
...  
Keyword(s):  
De Novo ◽  
Sequence Data ◽  
New Technology ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Short Read ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Before And After ◽  
Long Read

Background: The introduction of the MinION sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. Nanopore sequence data offers great potential for de novo assembly of complex genomes without using other technologies. Furthermore, Nanopore data combined with other sequencing technologies is highly useful for accurate annotation of all genes in the genome. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. We double corrected assemblies from four different assemblers with PILON and assessed sequence correctness before and after PILON correction with a set of 290 Fungi genes using BUSCO. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae (Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. We also compared various technical and software developments for the nanopore sequencing protocol, showing that nanopore-derived assemblies provide the highest contiguity. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.

scholarly journals De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 618 ◽  
Author(s):  
Hans J. Jansen ◽  
Ron P. Dirks ◽  
Michael Liem ◽  
Christiaan V. Henkel ◽  
G. Paul H. van Heusden ◽  
...  
Keyword(s):  
De Novo ◽  
Sequence Data ◽  
New Technology ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Short Read ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Wild Type Yeast

Background: The introduction of the MinIONTM sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. It has been shown that the nanopore sequence data, in combination with other sequencing technologies, is highly useful for accurate annotation of all genes in the genome. However, it also offers great potential for de novo assembly of complex genomes without using other technologies. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae (Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.

scholarly journals New insights on Pseudoalteromonas haloplanktis TAC125 genome organization and benchmarks of genome assembly applications using next and third generation sequencing technologies

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Weihong Qi ◽  
Andrea Colarusso ◽  
Miriam Olombrada ◽  
Ermenegilda Parrilli ◽  
Andrea Patrignani ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Model Organisms ◽  
Nanopore Sequencing ◽  
Third Generation ◽  
Small Plasmid ◽  
Pseudoalteromonas Haloplanktis ◽  
Sequencing Technologies ◽  
Third Generation Sequencing ◽  
Oxford Nanopore ◽  
Generation Sequencing

Abstract Pseudoalteromonas haloplanktis TAC125 is among the most commonly studied bacteria adapted to cold environments. Aside from its ecological relevance, P. haloplanktis has a potential use for biotechnological applications. Due to its importance, we decided to take advantage of next generation sequencing (Illumina) and third generation sequencing (PacBio and Oxford Nanopore) technologies to resequence its genome. The availability of a reference genome, obtained using whole genome shotgun sequencing, allowed us to study and compare the results obtained by the different technologies and draw useful conclusions for future de novo genome assembly projects. We found that assembly polishing using Illumina reads is needed to achieve a consensus accuracy over 99.9% when using Oxford Nanopore sequencing, but not in PacBio sequencing. However, the dependency of consensus accuracy on coverage is lower in Oxford Nanopore than in PacBio, suggesting that a cost-effective solution might be the use of low coverage Oxford Nanopore sequencing together with Illumina reads. Despite the differences in consensus accuracy, all sequencing technologies revealed the presence of a large plasmid, pMEGA, which was undiscovered until now. Among the most interesting features of pMEGA is the presence of a putative error-prone polymerase regulated through the SOS response. Aside from the characterization of the newly discovered plasmid, we confirmed the sequence of the small plasmid pMtBL and uncovered the presence of a potential partitioning system. Crucially, this study shows that the combination of next and third generation sequencing technologies give us an unprecedented opportunity to characterize our bacterial model organisms at a very detailed level.

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