conceived and designed the experiments and helped write the manuscript; J.G. The most divergent region of the CLas genome is the prophage region, where strains can contain one to three prophages, with three prophage types known to date. conducted the experiments and helped write the manuscript; A.N. 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. 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/. Performance metrics for Illumina DNA Flex Enrichment Protocol. A broad range of kits are available allowing you to easily qualify and . S8. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Ghosh, D. K. et al. Coverage metrics by sample for sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows. 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. Number of total reads generated per sample using the Illumina Nextera DNA Flex Enrichment workflow relative to: A) Sample N1 Ct value; B) Sample N2 Ct value. Show more Show more Almost yours: 2 weeks, on us. We could tell you about the benefits our TapeStation systems have to offerbut we thought showing you would be better! 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]. Proc Natl Acad Sci USA 108, E746752 (2011). Phytopathology. More posts you may like r/labrats Join 9 days ago Lab archetypes 512 131 r/labrats Join 28 days ago 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. 8-well PCR tube strips or 96-well sample plates are available depending on sample throughput, bringing added flexibility A pneumonia outbreak associated with a new coronavirus of probable bat origin. The marker is used to align the samples. The ARTIC v3 libraryprepared with TruSeq library preparation achieved 99.60% coverage at a minimum of 10x and 97.31% coverage at a minimum of 100x (Fig. Cite this article. Sign up for the Nature Briefing: Translational Research newsletter top stories in biotechnology, drug discovery and pharma. Targeted genome enrichment specifically enriches sequences of interest within a heterogeneous mixture of DNA samples. Google Scholar. 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. In general, the same regions were not always missing, with only ~2kb shared sites missing across samples. Modern alternatives to Agilent Bioanalyzer. Twenty-five l of the DNA libraries, bound to streptavidin beads, was amplified by PCR using SureSelect post capture primer mix and Herculase II Fusing DNA polymerase. S2-S3). Names of CLas samples were listed on the left. S5. Theyve been used for improving genome assemblies. (a) LHCA samples at different Cq values: Cq 20 (blue), Cq 22 (red), Cq 26 (gray), Cq 28 (yellow). The improvement in genome coverage metrics with the tailed amplicon v2 approach was a function of improved amplicon balance (Fig. California Privacy Statement, At a subsampled read depth of 100,000 reads, the Nextera DNA Flex Enrichment method achieved 99.96% coverage at a minimum of 10x and 99.69% coverage at a minimum of 100x (Fig. Five patient samples with N1 and N2 Ct values ranging from ~2035 and the BEI WA isolate sample were selected for TruSeq library prep and sequencing; one sample (N1 Ct=20, N2 Ct=20.4) was prepared in triplicate. 3f, Supplemental Fig. 14, 178192 (2013). Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Samples with N1 and N2 Ct values ranging from approximately 2040 chosen for testing of SARS-CoV-2 sequencing workflows. It is suitable to analyze size, quantity, and integrity of your samples. Pathogen DNA is enriched from 500- to 45,000-fold compared to non-enriched samples. (Lonza's FlashGel is a similar system.) G) 2% agarose gel showing the presence of primer dimers particularly in high N1/N2 Ct samples when indexed using different PCR cycling conditions. Plant Health Progr, https://doi.org/10.1094/PHP-2007-0906-01-RV (2007). 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. 2c-d). The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples. Sequencing data for this project is available through the National Center for Biotechnology Information (NCBI) Sequence Read Archive BioProject PRJNA631042. Article The following indexing primers were used (X indicates the positions of the 10bp unique dual indices): Forward indexing primer: AATGATACGGCGACCACCGAGATCTACACXXXXXXXXXXTCGTCGGCAGCGTC. Wu, F. et al. Quality and quantity of libraries were determined by TapeStation using a D1000 ScreenTape (Agilent). This led to decreased coverage at a given read depth for the tailed amplicon v1 method relative to ARTIC v3 (Fig. Issue: When using the Agilent 4200, 4150 and 2200 TapeStation systems, the DV 200 of FFPE RNA samples can be calculated within the TapeStation analysis software. Robinson, J. T. et al. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach. Overall, 12620 RNA probes were designed. The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples. Not for use in diagnostic procedures. Curr Microbiol. Correspondence to Dai, Z. et al. Genetic Diversity of the Indian Populations of Candidatus Liberibacter asiaticus Based on the Tandem Repeat Variability in a Genomic Locus. Genetic diversity of Candidatus Liberibacter asiaticus based on two hypervariable effector genes in Thailand. The system integrates an instrument, data processing software, reagents, and ScreenTape devices specific for DNA and RNA. Thus a targeted genome enrichment method may be useful and necessary. Vanaerschot M, Mann SA, Webber JT, Kamm J, Bell SM, Bell J, et al. The approach we describe is similar to a tailed-amplicon method that we have used to process more than 150,000 microbiome samples in recent years in the University of Minnesota Genomics Center [14], and thus represents a highly scalable method for sequencing large numbers of SARS-CoV-2 genomes in a rapid and cost-effective manner. PubMedGoogle Scholar. Ct values were exported and analyzed in Microsoft Excel. Comparison of sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows on SARS-CoV-2 isolate. Samples were processed as described above for the two-pool tailed amplicon sequencing workflow, with the exception that in the first round of PCR, four separate reactions were set up using primer pools 1.1, 1.2, 2.1, and 2.2 (see Supplemental Data File2 for primer sequences and pool composition) using 2.5L of template cDNA per reaction. Metagenomic (RNA) sequencing can be used to sequence and assemble the SARS-CoV-2 genome [10]. Integrative Genomics Viewer. I am a little hesitant to recommend this as the machine is nearly 2 decades old now. The Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California) is a capillary electrophoresis-based system that can analyse DNA, RNA, and proteins. Andrews S. FastQC A Quality control tool for high throughput sequence data. We tested a tailed amplicon method (tailed amplicon v1) in which the tailed version of the ARTIC v3 primers were pooled into two pools in a similar manner to the ARTIC v3 protocol. The following reaction was set up for non-fragmented priming of RNA: 5L template RNA and 1L NEBNext Random Primers were combined and incubated at 65C for 5min. 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). To obtain Nature. Liberibacter asiaticus was estimated using HLBaspr real-time quantitative PCR, giving a quantification threshold (Cq) value6. Library preparation was performed following the standard Illumina TruSeq Nano DNA protocol for 350 base pair libraries (Illumina, San Diego, CA). (b) SGCA samples at different Cq values: Cq 20 (blue), Cq 22 (red). S.N. Next, 1g of each library was hybridized with the SureSelect capture library. Consistent with previous descriptions of the ARTIC v3 primers, the balance between the tiled amplicons across these samples was relatively even, with a mean CV of 0.61 among the five patient samples tested, and 0.55 for samples with a N1 and N2 Ct of less than 30 (Fig. If you need results sooner, please contact us. Article 2020;30:13461351.e2. J Plant Pathol 88, 373714 (2006). A pan-genome comparative approach could provide enough genetic variation for high strain resolution, but sequencing CLas genomes has been historically difficult. SureSelect targeted enrichment, a new cost effective method for the whole genome sequencing of Candidatus Liberibacter asiaticus. I don't remember off the top of my head, the big brother version was significantly more expensive than the BioA/Tapestation. Supplemental Fig. Science and Technology, Plant Protection and Quarantine, Animal and Plant Health Inspection Service, United States Department of Agriculture, Beltsville, Maryland, United States of America, Weili Cai,Schyler Nunziata,John Rascoe&Michael J. Stulberg, Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, North Carolina, United States of America, You can also search for this author in 2f), consistent with prior comparisons of the USA-WA1/2020 and the Wuhan-Hu-1 reference strain. Genome Biol. 2020;26:4502. Phytopathology, https://doi.org/10.1094/PHYTO-06-18-0185-R (2018). Core SNPs were identified by mapping trimmed and filtered reads, as well as published genomes, against the Psy62 reference genome to create a whole genome alignment (including invariant sites), keeping sites with at least 10x coverage and greater than 90% consensus for each strain using Snippy v4.0 (https://github.com/tseemann/snippy). Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Wylie, T. N., Wylie, K. M., Herter, B. N. & Storch, G. A. Nat Med. The sample pools were diluted to 2nM based on the Qubit measurements and Agilent sizing information, and 10L of the 2nM pool was denatured with 10L of 0.2N NaOH. Slider with three articles shown per slide. Only small portions of the genome were poorly covered, with more than 90% of the regions showing a depth of coverage of at least 20X across all samples (Fig. https://doi.org/10.1126/science.abc0523. 29, 2426 (2011). First, all DNA samples were sheared using a M220 sonicator (Covaris, Woburn, MA) (duty factor 20%, peak/Displayed Power (W) 50 and 200 cycles/burst for 30second duration time), and adaptors were ligated to end repaired DNA. Next, we assessed how well enrichment captures the genome diversity of different strains. Ithaca, NY 14853Email us. The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples. The quality control methods from gDNA input to final library using the Agilent Bioanalyzer System and Agilent TapeStation System were evaluated. 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. There was complete concordance in the variant calls for all samples with N1 and N2 Ct values below 30, but less agreement among variant calls between methods for the sample with N1 and N2 Ct values of approximately 35 (Fig. Shared and unique variants were compared within and between samples using vcftools diff-site function. Of the seven shared sites missing across samples, four were in prophage regions that could reflect sequence diversity, and the remaining three regions only totaled approximately 200bp. Article In this study, the CLas genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16S qPCR). BMC Genomics 21, 863 (2020). Article Start here to learn about Agilent TapeStation system, an automated electrophoresis system that delivers sample quality control (QC) for DNA & RNA applications. Google Scholar. Li, H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The hybridized . Nat Methods. The system integrates an instrument, data processing software, reagents, and ScreenTape devices specific for DNA and RNA. Differentiation of Candidatus Liberibacter asiaticus isolates by variable-number tandem-repeat analysis. For the ARTIC v3 protocol, the average coverage at a subsampled read depth of 100,000 raw reads was 98.97% (10x) and 95.14% (100x) for all five test samples. Extracted RNA from de-identified clinical biospecimens were obtained subsequent to COVID-19 testing at the University of Minnesota for use under the IRB approved protocol Detection of COVID 19 by Molecular Methods (STUDY00009560). 3b, Supplemental Fig. S6. The most divergent region of the CLas genome is the prophage region, where strains can contain one to three prophages (or, in rare instances, none), with three known prophage types. Over the years we have gradually increased our use of it. Supplemental Fig. Metsky HC, Siddle KJ, Gladden-Young A, Qu J, Yang DK, Brehio P, et al. 1c). M.S. In this study, it costs $500 per sample to obtain the whole genome, which includes $300 RNA probe per reaction and $200 sequencing price. Therefore, it could be possible to obtain the whole genome with even lower titer if more reads are used for the sample. Besides the capability to sequencing medium to low titer samples, the total cost was also reduced by using SureSelect for the whole genome sequencing. All other genomes were obtained from NCBI. This research was supported by the intramural research and citrus health response programs of the U.S. Department of Agriculture, Animal and Plant Health Inspection Service. Phytopathology. S8). Agilent offers two instruments that are based on ScreenTape technology, the 4200 TapeStation system that enables the unattended analysis of up to 96 samples loaded from a well plate and the new 4150 TapeStation instrument, which analyses any sample number between 1 and 16. All four LHCA samples are also clustered together. Comparison of the Agilent 2100 Bioanalyzer and the 4200 TapeStation Huanglongbing (HLB), or citrus greening, is a devastating citrus disease caused by phloem-restricted gram-negative bacteria Candidatus Liberibacter spp1,2. 2020;26.1266-73. Ca. Indeed, this mechanical lysis approach has been widely adopted for lysis of both Gram-positive and Gram-negative bacteria within complex matrices. Int J Syst Evol Microbiol. In comparing the sequence capture and amplicon-based methods, there is a trade-off between the completeness of genome coverage and sensitivity (being able to analyze samples with higher N1 and N2 Ct values). 308(2), 256262 (2018). Roary: rapid large-scale prokaryote pan genome analysis. However, the relatively low-cost of amplicon methods make them a good choice for population-scale viral surveillance and such approaches have recently been used successfully to monitor the spread of viruses such as Zika and Ebola [2,3,4]. The LHCA strain clusters most closely to the other reported California strains, AHCA1 and SGCA5, however it does form its own distinct clade from those strains too. Tailed amplicon v1 pool primer sequences. The concentration of Ca. Supported on their Sequel II and IIe instruments, and now expanded to their latest Revio sequencer, HiFi sequencing is built, Long-read technologies have repeatedly demonstrated their value in genomics research. Target enrichment efficiency was estimated by aligning trimmed and quality filtered reads to the CLas strain Psy62 reference genome and comparing alignment rate between enriched and non-enriched samples (Table1). Thorvaldsdttir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Systematic improvement of amplicon marker gene methods for increased accuracy in microbiome studies. It is suitable to analyze size, quantity, and integrity of your samples. Katoh, H. et al. Article Here we compare sequence capture and amplicon-based methods for sequencing SARS-CoV-2 and describe a streamlined tailed amplicon method for cost-effective and highly scalable SARS-CoV-2 sequencing. The authors declare no competing interests. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. VCF files were filtered to retain only variants sequenced to a minimum depth of coverage of 10 in enriched samples, and 3 in non-enriched samples. Sci Rep 9, 18962 (2019). A-F) Observed read depth for each of the expected amplicons for the indicated sample amplified with the tailed amplicon v1 protocol at a subsampled read depth of 100,000 raw reads. Not surprisingly, we got the same prophage pattern for the SGCA strain sequenced in this study as SGCA5 (SC1 only), another strain from the same location14. I see there a quite a few machines made for high throughput for NGS workflows, but I only need low throughput. As expected, since the amplicon approaches are unable to cover sequences at the ends of the SARS-CoV-2 genome, the DNA Flex Enrichment sequence capture method produced the highest genome coverage. 2020:eabc0523. The system includes instrument, software, reagents, and ScreenTape devices to analyze size, quantity, and integrity of your DNA and RNA sample. 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). Eight samples with >1ng/L concentration of target amplicons were selected for downstream library preparation. 99(2), 13944 (2009). Teixeira Ddo, C. et al. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Cai, W., Yan, Z., Rascoe, J. Researchers have used enrichment strategies to increase the number of target reads in sequencing. 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. This method has been widely used to capture and enrich targeted DNA from complex biological samples, but is not commonly used to recover plant pathogens from a plant host background21,22,23. PacBio has become synonymous with their High Fidelity (HiFi) sequencing. Coverage metrics by method for sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows. Such high pathogen titer samples are needed because a low percentage of sequencing reads belonging to CLas are present in a metagenomic sample, primarily because of large genome size difference between pathogen and host and relative low copy number of pathogen DNA. It has a sample throughput of 12 samples per run, and results are generated in approximately 30 minutes [ 11 ]. Samples will be run as scheduling permits, generally within 1-3 business days. Enriched samples, however, had enough reads to align samples to SC1, SC2 and JXGC3 prophage reference sequences. The probe set here use the SC1, SC2 and JXGC-3 as three prophage reference genomes, but we anticipate that it would capture all type 1, type 2 and type 3 prophage sequences if present in the samples. Candidatus Liberibacter americanus, associated with citrus huanglongbing (greening disease) in So Paulo State, Brazil. We thank Sean Wang and Matt Plumb from the Minnesota Department of Heath for helpful discussions and for sharing ARTIC v3 primers. The same three variants were detected by all four methods tested (Fig. These results indicate that this SureSelect target enrichment method can be used to sequence CLas more efficiently than the canonic NGS method. Check out the interactive hotspots below and see what these instruments can do for your lab. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. Genome Announc, https://doi.org/10.1128/genomeA.00999-14 (2014). While adjusting the primer concentration for over-represented amplicons did lower the CV of the tailed amplicon pool, amplicon balance was still substantially worse than with the untailed ARTIC v3 primers (data not shown). 130 Biotechnology Building Draft Genome Sequence of Candidatus Liberibacter asiaticus from California. De-identified clinical biospecimens were obtained subsequent to COVID-19 testing at the University of Minnesota under a protocol approved by the University of Minnesota Institutional Review Board (FWA number 00000312): Detection of COVID 19 by Molecular Methods (STUDY00009560). For samples with N1 and N2 Ct vales of less than 30, average coverage was 98.99% (10x) and 96.45% (100x) at a subsampled read depth of 100,000 raw reads (Fig. S7. Finally, amplicon approaches (Fig. With positive target selection, the probe-bound DNA is eluted and collected for further NGS application, and often has much higher target DNA concentration than the original input samples19,20. S5). Trees were generated using RaxML v8.2.10 and visualized using FigTree v1.4.3. The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples. Comparison of sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows on clinical specimens spanning a range of viral loads. While this issue can be overcome by increased sequencing depth, future optimizations aimed at reducing primer dimer contamination such as more stringent size selection or sequencing on an instrument with less size bias, such as the NovaSeq [16] could reduce this effect. General. Article Article Thus, for testing the tailed amplicon v2 approach, and comparing among all four methods, we used a subset of these patient samples with N1 and N2 Ct values ranging from ~2035 (Fig. After SureSelect enrichment, both of these samples had 99% genome coverage with at least 250X depth of coverage. CAS cDNA synthesis reactions were incubated at: 25C for 10min, followed by 50C for 10min and 85C for 5min. Probable Pangolin Origin of SARS-CoV-2 Associated with the COVID-19 Outbreak. 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. Sequencing of SureSelect enriched and non-enriched libraries was performed on an Illumina MiSeq platform (Illumina) on two separate v3 600-cycle cartridges (2300bp). Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. Zheng, Z. et al. To further analyze the repeatability and specificity of this method, we identified and compared the SNPs of these two strains at different Cq values. Zheng, Z., Deng, X., & Chen, J. Several large-scale consortia in the UK (COG-UK: COVID-19 Genomics UK), Canada (CanCOGeN: Canadian COVID Genomics Network), and the United States (CDC SPHERES: SARS-CoV-2 Sequencing for Public Health Emergency Response, Epidemiology, and Surveillance) have begun coordinated efforts to sequence large numbers of SARS-CoV-2 genomes. Bioinformatics. Sequencing-based genomic surveillance has been applied to both endemic disease, such as seasonal influenza [1], and to emerging disease outbreaks such as Zika and Ebola [2,3,4]. Double-stranded cDNA size was determined using Bioanalyzer high sensitivity DNA assay (Agilent, Santa Clara, CA) and quantified with Qubit Fluorometer and High Sensitivity DNA assay (Thermo Fisher Scientific, Waltham, MA). By using this website, you agree to our In addition, we included two patient negative samples in these experiments. The number in each circle represents the number of SNPs between the different comparisons. c Observed read depth for each of the expected amplicons for the BEI WA1 isolate amplified with the ARTIC v3 protocol at a subsampled read depth of 100,000 raw reads. The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples. 2a-b, Supplemental Tables14). e Tailed amplicon v1 (2 pool amplification); f Tailed amplicon v2 (4 pool amplification). Google Scholar. I use the Qiaxcel system. C) Tailed amplicon v1 (2 pool amplification); D) Tailed amplicon v2 (4 pool amplification). W.C., S.N., J.R. and M.S., wrote and revised the manuscript. The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: Genomic RNA from SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-52285. Genome Biol. Google Scholar. 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.
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