Sputum specimens and respective isolates RNA extracts were tested with the SARS-CoV-2 real-time RdRP gene duplex reverse transcription (RT)-PCR developed by the French National Reference Center for Respiratory Viruses and the real-time E gene RT-PCR from the Charité protocol (see WHO Coronavirus disease COVID-19 technical guidance: Laboratory testing for 2019-nCoV in humans, available from https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf). Although whole genome sequencing of viral pathogens has become a routine procedure in epidemiological monitoring and surveillance, these molecular investigations essential for public health support are still challenging in remote areas with poor technical resources. In addition, focusing on variant detection, we … Filtered reads were mapped against SARS-CoV-2 reference (NC_045512) using Burrows-Wheeler Aligner MEM algorithm (BWA-MEM) (v0.7.7). For each specimen, the corresponding median read depth is represented by horizontal lines; solid lines for clinical samples, and dashed lines for isolates, respectively. Lancet 395, 565–574. In general, for analyzing only a few (< 20) targets on a few samples, traditional methods such as Sanger sequencing … Viral consensus genomic sequences were rapidly and easily obtained for the two SARS-CoV-2 clinical specimens and their respective isolates, by using the two different sequencing platforms, MinION and iSeq100TM system. J. Biol. VH, AK, CB, and VC analyzed the results and prepared the manuscript. NEW YORK – Two research teams have compared a multitude of single-cell RNA sequencing approaches and set new benchmarks for researchers who use scRNA-seq in their studies. 1. Data were manually inspected using Tablet (v1.19) (Milne et al., 2013). The MinION and iSeq100TM system bring the power of next-generation sequencing to virtually any laboratory, with amplicon-based whole-genome sequencing protocols rapidly transposable to all molecular investigations of epidemic prone pathogens. Microbiol. Both share five nucleotide mutations (C241T, C3037T, C14408T, A23403G, and G25563T), among which three have led to amino acid mutations P4715L in ORF1ab, D614G in S, and Q57H in ORF3a. In the same way, a 4 h MinION run, applied to the two corresponding isolates CIBU-200107C1 and CIBU-200132C1, produced 107,532 and 120,958 high-quality raw reads, respectively, with an average of 1,022 bp read length (Table 1). (2020). Nanopore or Pac Bio) would be interesting in the future. Concerning the amplicons obtained from both isolates CIBU-200107C1 and CIBU-200132C1, they were also multiplexed and run independently in the same conditions. The reference center in Argentina responsible for analyzing all pathogens that pose a threat to human health is the National Administration of Laboratories and Institutes of Health “Dr. doi: 10.1038/s41586-020-2008-3, Ye, Z.-W., Yuan, S., Yuen, K.-S., Fung, S.-Y., Chan, C.-P., and Jin, D.-Y. Lancet Infect Dis. These molecular investigations also depend on efficient and direct sequencing of viral material from clinical samples, i.e., often with low viral genetic loads and a high concentration of contaminant host nucleic acid background. Darwanto A, Hein AM, Strauss S, Kong Y, Sheridan A, Richards D, Lader E, Ngowe M, Pelletier T, Adams D, Ricker A, Patel N, Kühne A, Hughes S, Shiffman D, Zimmermann D, Te Kaat K, Rothmann T. BMC Cancer. doi: 10.1093/bioinformatics/bty191, Lu, R., Zhao, X., Li, J., Niu, P., Yang, B., Wu, H., et al. (2017). 2020 Dec 17;59(1):e00583-20. The same amplicons, obtained from the two clinical samples CIBU-200107 and CIBU-200132, were multiplexed and sequenced using the Illumina iSeq100TM system during a run of 17 h. Illumina run generated 3,990,761 and 4,046,340 raw reads with a Phred quality score of 28, for the two clinical samples, respectively, with an average of 140 bp read length. In principle, the concepts behind Sanger vs. next-generation sequencing (NGS) technologies are similar. The quantity of amplicons was measured with the Qubit 2.0 fluorometer using the dsDNA HS Assay Kit (Thermo Fisher Scientific, United States), and the quality was assessed by electrophoresis in E-Gel® EX 1% agarose (Invitrogen®) and visualized with E-Gel PowerSnap Electrophoresis System (Invitrogen®). Conversely, the genome coverage for the isolates was quite similar, with at 30X 94.9% and 95.3%, respectively (Table 1). The run yielded a median read depth of 2,979X and 3,607X for the two isolates, respectively (Table 1 and Figure 2). Here, we compared the performance of three commercial clinical decision support tools, that is, NAVIFY … 22:331. doi: 10.3201/eid2202.151796, Houldcroft, C. J., Beale, M. A., and Breuer, J. Supporting a broad range of applications, including gene expression profiling, chromosome counting, detection of epigenetic changes, and molecular analysis, NGS is driving discovery and enabling the future of personalized medicine. By using the Primal scheme software on the first released SARS-CoV-2 reference sequence, we immediately developed an amplicon-based approach to sequence around 1,000 bp amplicons on our small portable sequencers. | SAMtools (v1.9) were used to sort the aligned BAM files, to obtain coverage data and a consensus sequence. As soon as the first SARS-CoV-2 reference genome was released in Genbank (NC_045512), we designed two sets of primers to generate a tiling path along the genome using the Primal Scheme tool. Total RNA from clinical samples was extracted using the NucleoMag kit on the KingFisher automate (Macherey Nagel, Germany) and from isolates using the NucleoSpin DX Virus (Macherey Nagel, Germany), following the manufacturer’s instructions. Nat. Int. Thus, the mapping against Wuhan SARS-CoV-2 reference (NC_045512), by using Minimap2, retrieved 43,952 (96.3%) and 122,108 (99.9%) reads, respectively, for the two clinical samples (Table 1). Microbiol. However, it is also essential that this approach is accessible at the pen-side of infected humans to investigate outbreaks in remote areas. It enables scientists to analyze the entire human genome in a single sequencing … Recently, in late December 2019, a novel Betacoronavirus, SARS-CoV-2, originating from the Chinese city of Wuhan, emerged and was then identified as the causative agent of a new severe form of pneumonia, COVID-19. Data were demultiplexed using the guppy_barcoder with the option require_barcodes_both_ends to ensure barcodes are present at each end of the fragment. In contrast to the Sanger method of chain termination followed by capillary electrophoresis, NGS technology typically uses sequencing … For each specimen, the corresponding median read depth is represented by horizontal lines; solid lines for clinical samples and dashed lines for isolates, respectively. doi: 10.1038/nprot.2017.066, Song, Z., Xu, Y., Bao, L., Zhang, L., Yu, P., Qu, Y., et al. For the clinical sample with the lower viral load CIBU-200107, the genome coverage was significantly improved by the isolation step, less so for the second clinical sample with a very low Ct value (Figure 2). Three decades later, the introduction of next-generation sequencing resulted in dramatic decreases in sequencing costs while greatly increasing the volume and complexity of sequence data produced. (2016). The implementation of short turnaround time and user-friendly sequencing tools in affected regions to track the spread, analyze variants, and identify clusters of transmission of emerging viruses, like SARS-CoV-2, is becoming essential. Comparison of genome coverage profiles obtained for the clinical samples (CIBU-200107 in green, CIBU-200132 in red) and their respective isolates (CIBU-200107C1 in black, CIBU-200132C1 in blue) using Illumina iSeq100TM system. The genome coverage variation between both clinical samples reflected the initial viral load difference, which was even more marked at 100X (Table 1), as is the difference between the median read depths. Next-generation sequencing (NGS) is a high-throughput methodology that enables rapid sequencing of the base pairs in DNA or RNA samples. In both NGS and Sanger sequencing (also known as dideoxy or capillary electrophoresis sequencing), DNA polymerase adds fluorescent nucleotides one by one onto a growing DNA template strand. Mobile Next-Generation Sequencing Platforms Comparison for SARS-CoV-2 Genome Procurement. The libraries were qualified on an Agilent Technologies 2100 Bioanalyzer using a high-sensitivity DNA chip following the manufacturer’s instructions and quantified with the Qubit 2.0 fluorometer using the dsDNA HS Assay Kit (Thermo Fisher Scientific, United States). We also evaluated the performance of these two sequencers, in terms of ease of use, hands-on time, simplification of the sequencing process, and capacity for real-time genome sequencing in settings lacking laboratory resources for outbreak response. DUBLIN--(BUSINESS WIRE)--Mar 2, 2020--The “Next Generation Sequencing (NGS) Market, 2020-2030: Service Providers (Whole Genome, Whole Exome and Targeted Sequencing) and Technology Platforms” report has been added to ResearchAndMarkets.com’s offering. In outbreak situations, these two sequencers therefore make rapid sequencing accessible to a very large number of laboratories, requiring a minimum of molecular biology skills. Comparison of Four Next Generation Sequencing Platforms for Fusion Detection: Oncomine by ThermoFisher, AmpliSeq by Illumina, FusionPlex by ArcherDX, and QIAseq by QIAGEN Cancer Genet . Briefly, PCR amplicons pools were end-repaired and dA-tailed using an UltraII End Prep Reaction Module (NEB, United States) followed by ligation of native barcodes using the NEBNext UltraII Ligation module (NEB, United States). ... Illumina iSchool is a free online educational resource to learn about Next-Generation Sequencing (NGS) and its applications. The sequencing-ready libraries were prepared using the Nextera DNA Flex Prep kit (Illumina, United States). Regardless of using a read depth cutoff of 10X or 30X, we obtained around 89 and 94% of SARS-CoV-2 genome coverage, for the clinical samples, respectively. doi: 10.1136/jclinpath-2019-206422. The library was loaded onto an R9.4 flow cell (FLO-MIN106) and sequenced on a MinION Mk1B device within 4 h (Figure 1). (2003). Beginning in January 2008, the data represent the costs of generating DNA sequence using 'second-generation' (or 'next-generation') sequencing platforms. These multiplex primer sets (divided into two separate pools) were designed using the Primal scheme software1 on the SARS-CoV-2 reference sequence (Genbank accession number NC_045512) to sequentially amplify 1,000 bp using the following PCR conditions: 98°C for 30 s, 40 cycles of 98°C for 15 s, 65°C for 5 min, and ended by 65°C for 5 min. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material. Nevertheless, for all the specimens, the extremities of the genome could not be obtained. AK and CB conducted the experiments. Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. Implementation of next generation sequencing technology for somatic mutation detection in routine laboratory practice. Mobile Next-Generation Sequencing Platforms Comparison for SARS-CoV-2 Genome Procurement. Illumina sequencing platforms Our innovative next-generation sequencing (NGS) platforms deliver exceptional data quality and accuracy, at a massive scale. See rights and permissions. ... 2020 … 348, 1967–1976. N. Engl. The full-length genome was amplified directly from the RNA extracts from the clinical samples and the corresponding isolates, to obtain PCR products overlapping by 120 bp and covering the entire 29,903 bp viral genomic sequence. Pools of specific primer sets were used to generate 36 amplicons from the cDNA using the Q5 Hot Start High-Fidelity DNA Polymerase (NEB, United States) (Supplementary Table). SARS-CoV-2 next-generation sequencing workflow, from the sample to sequence analysis. Global human health is increasingly challenged by emerging viral threats, especially those observed over the last 20 years with the Severe Acute Respiratory Syndrome (SARS) and the Middle East Respiratory Syndrome (MERS) caused by coronaviruses. Use of the QIAGEN GeneReader NGS system for detection of KRAS mutations, validated by the QIAGEN Therascreen PCR kit and alternative NGS platform. RT was performed at 48°C for 15 min and 80°C for 5 min. The reads, averaging ~4.6 kb are significantly longer than other sequencing platforms making it ideal for sequencing small genomes such as bacteria or viruses. At the source of viral outbreaks, readily adaptable methods with rapid turn-around times are necessary for pathogen identification, surveillance programs, and public health strategies. VH, AK, CB, JV, and VC conceived the study. The advancements in the next-generation sequencing technologies have brought to the fore several amendments in the sequencing process by offering … Next generation sequencing has become the premier tool in genetic and genomic analysis. Next-generation Sequencing (NGS) Market 2020 report presents you analysis of market size, share, growth, trends, cost structure, statistical and comprehensive data of the global market. 16:e1002794. In this regard, we therefore evaluated two space-saving and easily portable next-generation sequencers, MinION and iSeq100TM system, through the current pandemic of COVID-19. Received: 18 June 2020; Accepted: 02 September 2020;Published: 25 September 2020. Patients and Methods: A total of 472 NSCLC patients were identified as ALK-positive by NGS and/or IHC between March 2014 and February 2020. The average base error rate was around 0.2%. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Comparison of next-generation sequencing and mutation-specific platforms in clinical practice. doi: 10.1038/s41586-020-2012-7, Keywords: COVID-19, real-time genome sequencing, amplicon-based sequencing, iSeq100TM system, MinION, SARS-CoV-2, Citation: Hourdel V, Kwasiborski A, Balière C, Matheus S, Batéjat CF, Manuguerra J-C, Vanhomwegen J and Caro V (2020) Rapid Genomic Characterization of SARS-CoV-2 by Direct Amplicon-Based Sequencing Through Comparison of MinION and Illumina iSeq100TM System. Hinrichs JW, van Blokland WT, Moons MJ, Radersma RD, Radersma-van Loon JH, de Voijs CM, Rappel SB, Koudijs MJ, Besselink NJ, Willems SM, de Weger RA. Next-generation sequencing, in contrast, makes large-scale whole-genome sequencing (WGS) accessible and practical for the average researcher. A Comparison of NGS Platforms. This site needs JavaScript to work properly. Slideshare uses cookies to improve functionality … HHS Its sequencing principle is based on the DNB and the cPAS technologies, which were also used in the previous version of the BGISEQ-500 device. This work was made possible thanks to financial support obtained through the «URGENCE Nouveau Coronavirus» fundraising campaign of Institut Pasteur. These both mobile benchtop instruments: 10.1016/S0140-6736 ( 20 ) 30251-8, Milne, I.,,. Were mapped against SARS-CoV-2 reference ( NC_045512 ) using Minimap2 ( v2.9 ) or... 26,852 and 28,391 bp was generated for the two sputum samples CIBU-200107 and CIBU-200132, were tested with option. Methods for Mycobacterium tuberculosis J Clin Microbiol between 900 and 1,200 base pairs in DNA or RNA.! 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