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 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.

scholarly journals Rapid and Inexpensive Whole-Genome Sequencing of SARS-CoV-2 using 1200 bp Tiled Amplicons and Oxford Nanopore Rapid Barcoding

2020 ◽  
Author(s):  
Nikki E. Freed ◽  
Markéta Vlková ◽  
Muhammad B. Faisal ◽  
Olin K. Silander
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Sequence Data ◽  
Variant Calling ◽  
Whole Genome ◽  
Oxford Nanopore ◽  
Cost Efficient ◽  
Primer Sets ◽  
Library Preparation Method

AbstractRapid and cost-efficient whole-genome sequencing of SARS-CoV-2, the virus that causes COVID-19, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10,000 reads (approximately 5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.

scholarly journals Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Nikki E Freed ◽  
Markéta Vlková ◽  
Muhammad B Faisal ◽  
Olin K Silander
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Sequence Data ◽  
Variant Calling ◽  
Whole Genome ◽  
Oxford Nanopore ◽  
Cost Efficient ◽  
Primer Sets ◽  
Library Preparation Method

Abstract Rapid and cost-efficient whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10 000 reads (∼5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.

scholarly journals An explanatory study of the use of e-mail investor communication by South African listed companies

2016 ◽  
Vol 18 (1) ◽  
Author(s):  
Roelof Baard ◽  
George Nel
Keyword(s):  
Listed Companies ◽  
Future Research ◽  
Contact Method ◽  
Interactive Communication ◽  
Handling Performance ◽  
Corporate Websites ◽  
Use Of Social Media ◽  
Almost All ◽  
The Cost ◽  
E Mail

Background: Although research shows that almost all listed companies have corporate websites with dedicated investor relations (IR) sections that enable companies to ‘push’ information to investors, it was argued that such an asymmetrical approach to communication is insufficient for companies wishing to exercise good IR. The purpose of this study was to test the effectiveness of the Internet to act as a mechanism to achieve more interactive communication between companies and investors.Objectives: The objectives of the study were to measure the responsiveness, timeliness and relevance of companies’ responses to e-mail requests, and to test for the determinants (size, market-to-book ratio, profitability, leverage and liquidity) thereof.Method: The mystery investor approach and a content analysis were used to study the e-mail handling performance of companies. The associations between company-specific characteristics were statistically tested.Results: It was found that the e-mail handling performance of companies in this study was poor compared with previous studies. Significant relationships between company size and responsiveness and relevance, and between market-to-book ratio and relevance were reported, as well as between the contact method used to request information and relevance and the use of social media and timeliness.Conclusion: Specific areas where companies could improve their investor communications were identified. The need for further research was discussed to explain some of the relationships found, as well as those not found, in contrast to what was expected. Future research is warranted to examine the relationship between the e-mail handling performance of companies and information asymmetry and the cost of equity of companies.

scholarly journals Comparison of LPWAN Technologies: Cost Structure and Scalability

2021 ◽  
Author(s):  
Mohammad Istiak Hossain ◽  
Jan I. Markendahl
Keyword(s):  
Communication Technology ◽  
Service Providers ◽  
Communication Technologies ◽  
Cost Structure ◽  
Small Scale ◽  
Area Network ◽  
Wide Area Network ◽  
Generic Framework ◽  
Cost Efficient ◽  
The Cost

AbstractSmall-scale commercial rollouts of Cellular-IoT (C-IoT) networks have started globally since last year. However, among the plethora of low power wide area network (LPWAN) technologies, the cost-effectiveness of C-IoT is not certain for IoT service providers, small and greenfield operators. Today, there is no known public framework for the feasibility analysis of IoT communication technologies. Hence, this paper first presents a generic framework to assess the cost structure of cellular and non-cellular LPWAN technologies. Then, we applied the framework in eight deployment scenarios to analyze the prospect of LPWAN technologies like Sigfox, LoRaWAN, NB-IoT, LTE-M, and EC-GSM. We consider the inter-technology interference impact on LoRaWAN and Sigfox scalability. Our results validate that a large rollout with a single technology is not cost-efficient. Also, our analysis suggests the rollout possibility of an IoT communication Technology may not be linear to cost-efficiency.

scholarly journals Development of an RNA Extraction Protocol for a Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR

Foods ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1804
Author(s):  
Daniel Plante ◽  
Julio Alexander Bran Barrera ◽  
Maude Lord ◽  
Irène Iugovaz ◽  
Neda Nasheri
Keyword(s):  
Hepatitis A Virus ◽  
Rna Extraction ◽  
Complex Nature ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Pcr Inhibitors ◽  
Qrt Pcr ◽  
Extraction Protocol ◽  
Viral Particles ◽  
Hands On

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

scholarly journals Cost Analysis of a Novel Method for Ecological Compensation—A Study of the Translocation of Dead Wood

2021 ◽  
Vol 13 (11) ◽  
pp. 6075
Author(s):  
Ola Lindroos ◽  
Malin Söderlind ◽  
Joel Jensen ◽  
Joakim Hjältén
Keyword(s):  
Cost Efficiency ◽  
Dead Wood ◽  
Road Transportation ◽  
Ecological Value ◽  
Ecological Compensation ◽  
Work Time ◽  
Delivery Times ◽  
Novel Method ◽  
Cost Efficient ◽  
The Cost

Translocation of dead wood is a novel method for ecological compensation and restoration that could, potentially, provide a new important tool for biodiversity conservation. With this method, substrates that normally have long delivery times are instantly created in a compensation area, and ideally many of the associated dead wood dwelling organisms are translocated together with the substrates. However, to a large extent, there is a lack of knowledge about the cost efficiency of different methods of ecological compensation. Therefore, the costs for different parts of a translocation process and its dependency on some influencing factors were studied. The observed cost was 465 SEK per translocated log for the actual compensation measure, with an additional 349 SEK/log for work to enable evaluation of the translocation’s ecological results. Based on time studies, models were developed to predict required work time and costs for different transportation distances and load sizes. Those models indicated that short extraction and insertion distances for logs should be prioritized over road transportation distances to minimize costs. They also highlighted a trade-off between costs and time until a given ecological value is reached in the compensation area. The methodology used can contribute to more cost-efficient operations and, by doing so, increase the use of ecological compensation and the benefits from a given input.

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