In the future, it will be interesting to determine the absolute sequencing limit of this method. https://doi.org/10.1038/nbt.3601. Samples were eluted in 20L of elution buffer and 10L of each sample was pooled and concentrated to 20L using 0.7x AMPureXP beads (Beckman Coulter, Brea, CA). 3c, Supplemental Fig. Any one have suggestions for alternative systems for analyzing fragment sizes (other than gels)? Cycling conditions were: 98C for 30s, followed by 25 or 35cycles of 98C for 15s and 65C for 5min. Liberibacter. We anticipate that this approach will aid in the genomic surveillance of SARS-CoV-2 as well as studies on viral diversity and evolution, and the influence of virus genetics on transmissibility, virulence, and clinical outcomes. statement and The DV 200 score is a quality score for evaluating quality of RNA derived from formalin-fixed paraffin-embedded (FFPE) samples established by Illumina Inc. in 2016. . 2a-b, Supplemental Table1, Supplemental Table2). These amplicons are then subjected to either Illumina or Oxford Nanopore library preparation, using methods that either directly add adapters to the ends of the amplicons or to fragment them to enable sequencing on a wider variety of Illumina instruments. The second strand synthesis reaction was incubated at 16C for 60min. Genomic regions of high recombination were detected and removed with Gubbins v2.3.129, and filtered polymorphic sites extracted to build phylogenies. Bankevich, A. et al. We benchmark this approach against both the standard ARTIC v3 protocol and a sequence capture approach using clinical samples spanning a range of viral loads. Positive selection (like the SureSelect method described here) can enrich a target hundreds to thousands fold, making it possible to sequence low titer samples. Di Paola N, Sanchez-Lockhart M, Zeng X, Kuhn JH, Palacios G. Viral genomics in Ebola virus research. University of Minnesota Genomics Center, Minneapolis, MN, 55455, USA, Daryl M. Gohl,John Garbe,Patrick Grady,Jerry Daniel,Ray H. B. Watson,Benjamin Auch&Kenneth B. Beckman, Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, 55455, USA, Department of Lab Medicine and Pathology, Division of Molecular Pathology and Genomics, University of Minnesota, Minneapolis, MN, 55455, USA, You can also search for this author in The following recipe was used to set up the PCR reactions: 2.5L template cDNA, 14.75L nuclease-free water, 5L 5x Q5 reaction buffer (New England Biolabs, Ipswich, MA), 0.5L 10mM dNTPs (Kapa Biosystems, Woburn, MA), 0.25L Q5 Polymerase (New England Biolabs, Ipswich, MA), 2L primer pool 1 or 2 (10M) for the tailed v1 protocol. Supplemental Fig. The Agilent system takes 30 minutes to analyse 12 samples per run whereas each ScreenTape for the tapestation can analyze 16 samples (1 sample for the library and 15 samples per tape). Andersen KG, Rambaut A, Lipkin WI, Holmes EC, Garry RF. Read-pairs were stitched together using PEAR [20]. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. This pattern was consistent across different concentrations of the same strain. Although the mapping tracks show some different gaps among different strains suggesting uncovered non-conserved regions, the probes still capture sufficient prophage sequences for diversity analysis. S4. The RNA ScreenTape system is designed for analyzing eukaryote and Physical Specifications Signal- to- noise >3 (single peak) Measured against 2200 TapeStation System Agilent Technologies Storage Conditions Start here to learn about Agilent TapeStation system, an automated electrophoresis system that delivers sample quality control (QC) for DNA & RNA applications. Article analyzed data and helped write the manuscript; P.G., J.D., R.W., and B.A. If you need results sooner, please contact us. Raw reads were trimmed of adapter sequences and beginnings and ends trimmed where quality dropped to 0. Reverse indexing primer: CAAGCAGAAGACGGCATACGAGATXXXXXXXXXXGTCTCGTGGGCTCGG. The coefficient of variation (CV) of the ARTIC v3 sample was 0.49 and the CVs of the tailed amplicon v1 samples were 1.70 and 1.26 for the 25 and 35 PCR cycle samples, respectively. We next tested whether splitting the tailed SARS-CoV-2 primers into 4 PCR reactions based on primer performance in the initial sequencing tests could improve balance with the tailed primer approach. The pan-genome phylogenetic tree based on core genes also demonstrates a similar branching pattern. Percentage of genome coverage at 10x at different subsampled read depths for WA1 and UMGC SARS-CoV-2 isolates sequenced with different methods. 1b). S4). Metagenomic (RNA) sequencing can be used to sequence and assemble the SARS-CoV-2 genome [10]. Schuenemann, V. J. et al. F) Percentage of sequencing adapter observed for samples prepared with the tailed amplicon v2 (4 pool amplification) workflow. Nat Biotechnol. The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples. Provided by the Springer Nature SharedIt content-sharing initiative. The findings and conclusions in this publication are those of the authors and should not be construed to represent any official USDA or U.S. Government determination or policy. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Check out the interactive hotspots below and see what these instruments can do for your lab. Tailed amplicon v1 amplicon relative abundance. Supplemental Fig. Because the E-gel is dry when the sample gets to in the second well it can be pipetted up in water, TE, or other buffer. Article Genome Res. Sign up for the Nature Briefing: Translational Research newsletter top stories in biotechnology, drug discovery and pharma. Tailed amplicon v1 pool primer sequences. Bioinformatics. Eight samples with >1ng/L concentration of target amplicons were selected for downstream library preparation. Target-enrichment strategies for next-generation sequencing. Prophage Diversity of Candidatus Liberibacter asiaticus Strains in California. Comparison of the Agilent 2100 Bioanalyzer and the 4200 TapeStation Phytopathology, https://doi.org/10.1094/PHYTO-06-18-0185-R (2018). 2017;12:12616. 25(15), 19681969 (2009). In the meantime, to ensure continued support, we are displaying the site without styles We performed initial tests of the tailed amplicon v1 protocol by amplifying the samples listed in Fig. FEMTO Pulse System (Agilent) - We use this instrument for high molecular weight (up to 200 kb fragments), very low concentration DNA sizing, or very low concentration RNA quality assessment. Slider with three articles shown per slide. I have used both widely in my lab and they have given me equivalent results. The authors read and approved the final manuscript. a In Illuminas Nextera DNA Flex Enrichment protocol cDNA is tagmented and made into barcoded sequencing libraries, which are then enriched using sequence capture with a respiratory virus panel containing probes against SARS-CoV-2. Library preparation was performed following the standard Illumina TruSeq Nano DNA protocol for 350 base pair libraries (Illumina, San Diego, CA). 2020;26:4502. It is suitable to analyze size, quantity, and integrity of your samples. This was exemplified by the phylogenetic analysis showing samples from two different locations clustering separately from one another (diversity retained), yet sequencing the same sample at different titer levels clustered together (reproducible results). W.C., S.N., J.R. and M.S., wrote and revised the manuscript. Int J Med Microbiol. Internet Explorer). The IRB panel used WORKSHEET: Human Research (HRP-310) to make the determination that this study was exempt as not human research as defined by DHHS regulations. 2a-b, Supplemental Tables14). The same three variants were detected by all four methods tested (Fig. The authors declare that they have no competing interests. Metsky HC, Siddle KJ, Gladden-Young A, Qu J, Yang DK, Brehio P, et al. 25, 19101920 (2015). Theyve been used for improving genome assemblies. We were able to efficiently get 99% coverage of the reference genome with over 70X sequence coverage using fewer than 5 million total reads even with a low to mid-titer pathogen sample (Cq value of 28.52). How to Determine the DV200 of FFPE RNA Samples on the Agilent TapeStation This Information Applies To: 4200, 4150 and 2200 TapeStation, TapeStation analysis software A02.02 or higher. SNPs were determined using Samtools v1.7. Article The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples. 55(Pt 5), 185762 (2005). Agilent 4200 TapeStation System TechWiz4u 41 subscribers Subscribe 20K views 6 years ago New Agilent 4200 TapeStation For RNA and DNA analysis. Find products using our Selection Tool. 19(5), 455477 (2012). Next, we assessed how well enrichment captures the genome diversity of different strains. 77, 19101917 (2011). 2e). Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. The primers for the primary amplification contained both SARS-CoV-2 targeting sequences (derived from the ARTIC v3 designs), as well as adapter tails for adding indices and Illumina flow cell adapters in a secondary amplification. & Stulberg, M. J. Supplemental Fig. While the RNA tapes are still a bit lacking, my favorite is the genomic tape so you can look at much larger sizes. 105(8), 10439 (2015). We designed a series of experiments in order to test a streamlined tailed amplicon method and to compare amplicon and sequence capture based methods for SARS-CoV-2 sequencing (Fig. Zheng, Z. et al. Briefly, three separate 10L RT-qPCR reactions were set up in a 384-well Barcoded plate (Thermo Fisher Scientific, Waltham, MA) for either the N1, N2, or RP primers and probes. TapeStation Data Interpretation Each lane contains a marker along with your sample. S2-S3, Supplemental Tables12). The SARS-CoV-2 genome was amplified using a two-step PCR protocol. 30(9), 13121313 (2014). 3(6), https://doi.org/10.1128/genomeA.01508-15 (2015). A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2, https://doi.org/10.1186/s12864-020-07283-6, https://www.protocols.io/view/sars-cov-2-tailed-amplicon-illumina-sequencing-bipikdke, https://doi.org/10.1186/s13059-018-1618-7, https://doi.org/10.1038/s41579-020-0354-7, https://doi.org/10.1093/bioinformatics/bty407, https://doi.org/10.1016/j.cub.2020.03.022, https://doi.org/10.1101/2020.08.25.265074, https://doi.org/10.1101/2020.03.10.985150, https://doi.org/10.1186/s13059-019-1691-6, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, https://doi.org/10.1093/bioinformatics/btt593, https://doi.org/10.1093/bioinformatics/btp698, http://creativecommons.org/licenses/by/4.0/, http://creativecommons.org/publicdomain/zero/1.0/. A Rn threshold of 0.5 was selected and set uniformly for all runs. Cai, W., Nunziata, S., Rascoe, J. et al. 2020:2020.08.25.265074. https://doi.org/10.1101/2020.08.25.265074. J Plant Pathol 88, 373714 (2006). Hundreds of millions of sequencing reads are needed to get good CLas genome coverage from an infected citrus sample, making CLas genome sequencing challenging and costly18. High quality libraries were identified with an Agilent TapeStation using High Sensitivity D 1000 ScreenTape and then pooled for sequencing. Li Cq 26 and above). A modified non-directional NEBNext Ultra II First and Second Strand (#E7771 and #E6111, New England Biolabs, Ipswich, MA) protocol was used to generate long fragments of double-stranded cDNA as input material for the Nextera DNA Flex Enrichment with respiratory virus panel. Next, we assessed the performance of the different SARS-CoV-2 sequencing approaches on a set of de-identified patient samples. An alternative to the Agilent Bioanalyzer is Biorads Experion system. The slightly lower coverage metrics at a given subsampled read depth for the tailed amplicon v2 method can likely be explained by primer dimer formation during the two-step amplification process, which is more pronounced for higher N1 and N2 Ct samples (Supplemental Fig. We first evaluated the different SARS-CoV-2 sequencing workflows in their performance with a previously sequenced SARS-CoV-2 isolate strain from Washington state (2019-nCoV/USA-WA1/2020) provided by BEI Resources [15]. In this final installment of our series, we ask our participants about one of the most important aspects of data analysis, accuracy and reproducibility. There are three -proteobacteria associated with HLB: Candidatus Liberibacter asiaticus, Ca. The Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California) is a capillary electrophoresis-based system that can analyse DNA, RNA, and proteins. Ca. You are using a browser version with limited support for CSS. Percentage of genome coverage at 10x at different subsampled read depths for WA1 and UMGC SARS-CoV-2 isolates sequenced the tailed amplicon v1 method amplified for 25 PCR cycles in the first PCR reaction. Gohl, D.M., Garbe, J., Grady, P. et al. To obtain Need Help? Samples will be run as scheduling permits, generally within 1-3 business days. Not for use in diagnostic procedures. Emerg Infect Dis. Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. For Research Use Only. Coverage metrics by sample for sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows. Assefa, S., Keane, T. M., Otto, T. D., Newbold, C. & Berriman, M. ABACAS: algorithm-based automatic contiguation of assembled sequences. 2c-d). PubMedGoogle Scholar. cDNA was used to generate libraries using the Nextera DNA Flex Enrichment protocol (Illumina, San Diego, CA, catalog number 20025524) with the respiratory virus oligo panel including SARS-CoV-2 probes (Illumina, San Diego, CA, catalog number 20042472) according to manufacturers instructions. Percentage of reads aligned to a human reference genome using the Illumina Nextera DNA Flex Enrichment workflow relative to: C) Sample N1 Ct value; D) Sample N2 Ct value. 2020:eabc0523. It is suitable to analyze size, quantity, and integrity of your samples. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. The two SGCA strain samples are clustered together and most closely related to the previously reported SGCA strain, SGCA5. Indeed, this mechanical lysis approach has been widely adopted for lysis of both Gram-positive and Gram-negative bacteria within complex matrices. Four different Cq value (20.1, 22.84, 26.84, and 28.52) LHCA strain samples and two different Cq value (20.61 and 22.16) SGCA samples were selected to assess the sensitivity and selectivity of whole-genome enrichment and sequencing. Li, W., Hartung, J. S. & Levy, L. Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. For samples with N1 and N2 Ct vales of less than 30, average coverage was 99.92% (10x) and 99.62% (100x) at a subsampled read depth of 100,000 raw reads (Supplemental Tables12). Additionally, to study the impact of strain diversity in CLas epidemiology, it is important to include more geographic locations, and newly infected samples often carry a much lower pathogen titer than the successfully sequenced samples. SGCA (20 and 22) and LHCA (26,22,28, and 20) were all sequenced in this study. Genome Biol. Reads that did not align to the host genome were aligned to the reference Wuhan-Hu-1 [5] SARS-CoV-2 genome (MN908947.3) using BWA [21]. Bedford T, Riley S, Barr IG, Broor S, Chadha M, Cox NJ, et al. The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples. These gels can be automatically imaged while running by using a companion light box and camera setups. I don't remember off the top of my head, the big brother version was significantly more expensive than the BioA/Tapestation. All these results suggest that Agilent SureSelect XT HS target enrichment can effectively capture target DNA from complex CLas samples and significantly increase the pathogen DNA ratio. The concentration and sizing is determined from the standard ladder loaded into lane one. Bov, J. M. Huanglongbing: a destructive, newly emerging, century-old disease of citrus. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. We have the Tape Station for Agilent. W.C., conducted the experiments. S6. Nine samples spanning a range of viral loads as assessed by the Ct values of the viral N1 and N2 targets by qRT-PCR were selected for these studies. Variants detected for the indicated sample and sequencing protocol at a read depth of up to 1,000,000 raw reads (or the maximum read depth for the sample if below 1,000,000 reads). a Samples with N1 and N2 Ct values ranging from approximately 2035 chosen for testing of SARS-CoV-2 sequencing workflows. The tree with the highest likelihood across 10 runs was selected. S6, Supplemental Tables14). and JavaScript. Springer Nature. Google Scholar. The Agilent Tapestation provides an automated alternative to traditional gel electrophoresis, allowing researchers to analyze the quantity and size of DNA or RNA samples from only a few microliters. Google Scholar. Proceedings of the 2nd International Citrus Canker and Huanglongbing Research Workshop 2005, Orlando Florida, USA, p50 (2005). The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples. No we just use an Agilent Bioanalyzer purchased back in 2003. qRT-PCR reactions to identify SARS-CoV-2 positive samples were carried out using a modified version of the Centers for Disease Control and Prevention (CDC) SARS-CoV-2 qRT-PCR assay, as previously described [18]. Loop-mediated isothermal amplification (LAMP) assay for specific and rapid detection of Dickeya fangzhongdai targeting a unique genomic region, Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions, Rapid Detection of Genetic Engineering, Structural Variation, and Antimicrobial Resistance Markers in Bacterial Biothreat Pathogens by Nanopore Sequencing, Multiple internal controls enhance reliability for PCR and real time PCR detection of Rathayibacter toxicus, Targeted enrichment outperforms other enrichment techniques and enables more multi-species RNA-Seq analyses, Metagenomic sequencing for detection and identification of the boxwood blight pathogen Calonectria pseudonaviculata, De novo assembly and annotation of three Leptosphaeria genomes using Oxford Nanopore MinION sequencing, Evaluation of Oxford Nanopores MinION Sequencing Device for Microbial Whole Genome Sequencing Applications, Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens, https://www.aphis.usda.gov/plant_health/plant_pest_info/citrus_greening/downloads/pdf_files/nationalquarantinemap.pdf, http://tree.bio.ed.ac.uk/software/figtree/, https://doi.org/10.1094/PHP-2007-0906-01-RV, https://doi.org/10.1371/journal.pone.0112968, https://doi.org/10.1094/PHYTO-08-17-0282-R, https://doi.org/10.1094/PHYTO-06-18-0185-R, http://creativecommons.org/licenses/by/4.0/, Sign up for Nature Briefing: Translational Research. 2019;37:1608. The genetic identity of strains found in new locations or with varying aggressiveness can help inform the effectiveness of quarantine programs and provide researchers with data to search for virulence-associated genetic elements. As a continuation of our last article, we will be covering important metrics related to long-read sequencing technologies. Importantly, the RNA probe design of this positive capture method ensures retention of strain diversity, which other positive selection methods using primers run a risk of losing. Without special enrichment, NGS can rarely detect low copy number pathogen sequences from complex samples due to low pathogen/host nucleic acid ratio. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. However, NGS technology has significant limitations when performing pathogen diagnostics in complex metagenomic samples. The hybridized . Samples are colored as in panels c-f. b Evenness of representation of amplicons for different workflows as a function of sample N1 Ct value. Targeted genome enrichment specifically enriches sequences of interest within a heterogeneous mixture of DNA samples. The Wuhan-Hu-1 SARS-CoV-2 reference genome (Accession number: MN908947) and the human GRCh38 reference genome primary assembly (Accession number: GCA_000001405.28) used in this study were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/). 2f), consistent with prior comparisons of the USA-WA1/2020 and the Wuhan-Hu-1 reference strain. Prior to this work, obtaining a CLas whole genome sequence was a challenge. Over the years we have gradually increased our use of it. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. In addition, two SARS-CoV-2 negative samples were selected to assess cross-contamination or other sequencing artifacts. Sequencing of SureSelect enriched and non-enriched libraries was performed on an Illumina MiSeq platform (Illumina) on two separate v3 600-cycle cartridges (2300bp). PacBio has become synonymous with their High Fidelity (HiFi) sequencing. The Agilent TapeStation 2200 is an intuitive system for automating RNA, DNA, and protein sample quality control and reliable electrophoresis. Correspondence to Mass spectrometry, chromatography, spectroscopy, software, dissolution, sample handling and vacuum technologies courses, Live or on-demand webinars on product introductions, applications and software enhancements, Worldwide trade shows, conferences, local seminars and user group meetings, Service Plans, On Demand Repair, Preventive Maintenance, and Service Center Repair, Software to manage instrument access, sample processing, inventories, and more, Instrument/software qualifications, consulting, and data integrity validations, Learn essential lab skills and enhance your workflows, Instrument & equipment deinstallation, transportation, and reinstallation, CrossLab Connect services use laboratory data to improve control and decision-making, Advance lab operations with lab-wide services, asset management, relocation, Shorten the time it takes to start seeing the full value of your instrument investment. 20, 1239 (2012). The 4-pool amplification scheme (tailed amplicon v2) achieved coverage metrics close to the untailed ARTIC v3 approach at comparable read depths with 99.60% coverage at a minimum of 10x and 95.64% coverage at a minimum of 100x (Fig. E) Mean read 1 quality score for samples prepared with the tailed amplicon v2 (4 pool amplification) workflow. M.S. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. A minimum of two no template controls (NTCs) were included on all runs. Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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