A number of different approaches have been used to sequence SARS-CoV-2. Genome Announc. 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. Supplemental Table2. Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. Without special enrichment, NGS can rarely detect low copy number pathogen sequences from complex samples due to low pathogen/host nucleic acid ratio. In this article, we focus on metrics relevant to evaluating the success of a Pacific Biosciences (PacBio) sequencing run. Next generation sequencing technologies (NGS) have recently enabled large-scale genomic surveillance of infectious diseases. Since primers cannot capture the very ends of the viral genome, amplicon approaches have the drawback of slightly less complete genome coverage, and mutations in primer binding sites have the potential to disrupt the amplification of the associated amplicon [12]. Methods for SARS-CoV-2 genome sequencing compared in this study. Assefa, S., Keane, T. M., Otto, T. D., Newbold, C. & Berriman, M. ABACAS: algorithm-based automatic contiguation of assembled sequences. We thank California Department of Food and Agriculture (CDFA) for providing the infected citrus samples. W.C., S.N., J.R. and M.S., wrote and revised the manuscript. Quality and quantity of libraries were determined by TapeStation using a D1000 ScreenTape (Agilent). 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. 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. . Here we describe an all-amplicon method for producing SARS-CoV-2 sequencing libraries which simplifies the process and lowers the per sample cost for sequencing SARS-CoV-2 genomes (Fig. Reads were discarded with a mean quality score of less than 10 or when shorter than 200 base pairs, to avoid potential probe contamination, using BBDuk v38.12 (http://bbtools.jgi.doe.gov). 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). The SNP tree clearly shows the separation of LHCA and SGCA strains (Figs. Whole genome sequencing can provide precise molecular characterization of the diversity among CLas populations. Genes | Free Full-Text | Evaluation of the Ion AmpliSeq SARS-CoV-2 Thorvaldsdttir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Huanglongbing (HLB), or citrus greening, is a devastating citrus disease caused by phloem-restricted gram-negative bacteria Candidatus Liberibacter spp1,2. Halbert, S. E. The discovery of huanglongbing in Florida. Use the Previous and Next buttons to navigate the slides or the slide controller buttons at the end to navigate through each slide. Next, we assessed the performance of the different SARS-CoV-2 sequencing approaches on a set of de-identified patient samples. 3a). To determine the prophage content of each sample, we aligned all the reads from enriched samples to SC1, SC2 and JXGC3 prophage reference sequences using bowtie2 plugged in Geneious v 10.2.425, and visualized alignments in Integrated Genome Viewer v2.4.1026,27. 2a-b, Supplemental Tables12). Gnirke, A. et al. 14, 178192 (2013). The first strand synthesis reaction was incubated at 25C for 10min, 42C for 50min, 70C for 15min. Bedford T, Riley S, Barr IG, Broor S, Chadha M, Cox NJ, et al. 2020;30:13461351.e2. Several variants of the ARTIC protocol exist in which the pooled SARS-CoV-2 amplicons from a sample are taken through a NGS library preparation protocol (using either ligation or tagmentation-based approaches) in which sample-specific barcodes are added, and are then sequenced using either short-read (Illumina) or long-read (Oxford Nanopore, PacBio) technologies. Itokawa K, Sekizuka T, Hashino M, Tanaka R, Kuroda M. A proposal of alternative primers for the ARTIC Network's multiplex PCR to improve coverage of SARS-CoV-2 genome sequencing. Alignment files were filtered to remove PCR duplicates, retaining only reads in proper pairs with robust mapping quality (MAPQ10) using Samtools v. 1.728. Grubaugh ND, Gangavarapu K, Quick J, Matteson NL, De Jesus JG, Main BJ, et al. 2). The resulting tree was midpoint rooted and visualized using FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). By using this website, you agree to our Percentage of genome coverage at 100x at different subsampled read depths for each sample when sequenced using the following approaches: c Illumina Nextera DNA Enrichment; d ARTIC v3 with TruSeq library preparation. 9, 357359 (2012). 10L of PCR product for each sample was normalized using a SequalPrep 96-well Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA). The TapeStation System proved to be a reliable . 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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. 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. The overlapping number stands for the same SNPs identified between the different comparisons and the non-overlapping numbers specify the unique SNPs to each sample. 2019;20:8. https://doi.org/10.1186/s13059-018-1618-7. Tape station systems use ScreenTape, that's credit-card-sized . The four PCR reactions were combined in a 1:1:1:1 ratio after an initial PCR amplification of 35cycles and a 1:100 dilution of the combined PCRs for each sample was indexed according to the process described above. Daryl M. Gohl. Adapter-ligated libraries were purified using AMPure XP beads (Beckman Coulter, Inc., Brea, CA, USA), amplified, and then purified. The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples. Used Tapestation for sale. Agilent - Keysight equipment & more - Machinio To confirm the expected library size of approximately 550bp, pooled libraries were run on either an Agilent Bioanalyzer or TapeStation (Agilent, Santa Clara, CA). Gohl DM, Vangay P, Garbe J, MacLean A, Hauge A, Becker A, et al. 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. S2-S3, Supplemental Tables12). All four LHCA samples are also clustered together. All authors reviewed and approved the manuscript. We have the Tape Station for Agilent. California Privacy Statement, Start here to learn about Agilent TapeStation system, an automated electrophoresis system that delivers sample quality control (QC) for DNA & RNA applications. 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). Coverage metrics by method for sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows. The proximal origin of SARS-CoV-2. Mol Plant Microbe Interact. S1. 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. 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. This page was generated at 12:51 AM. 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]. 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. The poorer performance with respect to coverage metrics with the tailed amplicon v1 protocol was due to substantially worse balance between the different tiled amplicons compared with the ARTIC v3 (untailed) primers (Fig. SNPs were determined based on the alignment profile to Psy62. Zheng, Z., Deng, X., & Chen, J. and W.C., Conceived and designed the experiments. 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. Free software from Agilent is available to view your data on a PC. Sufficient amplification to carry out TruSeq library prep was seen for samples with Cts of around 35 or less. Hence, non-target enrichment of samples still makes CLas genome sequencing quite difficult and costly, and is not suitable for sequencing low titer samples (e.g. 3b, Supplemental Fig. Data Interpretation | Center for Quantitative Life Sciences | Oregon Genome Res. While the RNA tapes are still a bit lacking, my favorite is the genomic tape so you can look at much larger sizes. (b) SGCA samples at different Cq values: Cq 20 (blue), Cq 22 (red). In summary, our data suggest that SureSelect-based target enrichment system is an excellent and cost effective method for CLas whole genome sequencing from infected citrus samples, including those with pathogen titer far lower than those used in previous studies. 2a-b, Supplemental Tables14). S6. In this study, it costs $500 per sample to obtain the whole genome, which includes $300 RNA probe per reaction and $200 sequencing price. 1b), in which cDNA is made from SARS-CoV-2 positive samples and amplified using primers that generate tiled PCR products are being used to sequence SARS-CoV-2 [3]. Liberibacter asiaticus was estimated using HLBaspr real-time quantitative PCR, giving a quantification threshold (Cq) value6. Percentage of bases covered across fixed depths of coverage based on reference guided assemblies and estimated with samtools depth. Privacy As of Novemeber 2020, over 225,000 SARS-CoV-2 genome sequences have been deposited in public repositories such as NCBI and GISAID [5, 6]. TapeStation Parts & Accessories | Agilent The first CLas genome sequence was released in 2009, isolated from a single infected psyllid13, and in nearly 10 years since there have been only 14 additional CLas genomes deposited to NCBI (only five are complete). Overall, 12620 RNA probes were designed. 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. Supplemental Fig. 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/. The marker is used to align the samples. Eight samples with >1ng/L concentration of target amplicons were selected for downstream library preparation. For ARTIC v3 tests, based on the N1 and N2 target Ct values from clinical testing, we used either 25, 30, or 35 PCR cycles for the amplification reactions. 1). 3(6), https://doi.org/10.1128/genomeA.01508-15 (2015). Find products using our Selection Tool. 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]. Bead beating is the most common alternative to enzymatic lysis for DNA extraction from stool. With its unique design and intuitive features, common QC bottlenecks are resolved by the automation of key steps such as gel loading and sample injection increasing lab efficiency. This approach has the disadvantage that samples must typically be sequenced very deeply in order to obtain sufficient coverage of the viral genome, and thus the cost of this approach is high relative to more targeted methods. In initial tests, samples with N1 and N2 Ct values greater than 35 yielded poor coverage (~50% genome coverage at 10x) using the tailed amplicon method, did not yield useful data for the Nextera DNA Flex Enrichment protocol, and did not generate enough amplicon template to proceed with library preparation for the ARTIC v3 method (data not shown). 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. Each LHCA sample contained prophages SC1 and SC2, while SGCA samples contained only SC1 (Fig. The primary amplification was carried out in a manner similar to the ARTIC v3 method described above, using two primer pools which tile the SARS-CoV-2 genome. CLas positive leaf samples from grafted trees were collected for genomic DNA extraction. Quality and quantity of libraries were determined by TapeStation using a D1000 ScreenTape (Agilent). Find products using our Selection Tool. 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). Science (80- ). Cai, W., Yan, Z., Rascoe, J. After all wash steps, the beads were suspended in 50l of nuclease free water. Need Help? 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. Successful grafted citrus trees were determined by HLBaspr real-time quantitative PCR from symptomatic leaves. 3f, Supplemental Fig. Sci Rep 9, 18962 (2019). Bedford T, Greninger AL, Roychoudhury P, Starita LM, Famulare M, Huang M-L, et al. Chromatography & Spectroscopy Lab Supplies, GC Calculators & Method Translation Software, BioCalculators / Nucleic Acid Calculators. 2020;579:2659. We generated libraries for all six samples in parallel without enrichment using a TruSeq PCR free DNA library preparation kit (Illumina, San Diego, CA). Optical and PCR duplicates were flagged in alignment files using Picard v.2.10.5 (http://broadinstitute.github.io/picard). All other genomes were obtained from NCBI. 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). 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. A minimum of two no template controls (NTCs) were included on all runs. General. We thank Brandon Vanderbush for conducting QC on the SARS-CoV-2 samples and sequencing libraries. These results indicate that this SureSelect target enrichment method can be used to sequence CLas more efficiently than the canonic NGS method. Samples will be run as scheduling permits, generally within 1-3 business days. For the ARTIC v3, tailed amplicon v1, and tailed amplicon v2 methods, samples were amplified for 35 PCR cycles in the first PCR reaction. As a continuation of our last article, we will be covering important metrics related to long-read sequencing technologies. Characterization of Candidatus Liberibacter asiaticus populations by double-locus analyses. Plant Health Progr, https://doi.org/10.1094/PHP-2007-0906-01-RV (2007). The mean CV of all six patient samples was 0.76 (compared to a CV of 0.61 with ARTIC v3) and 0.52 for samples with a N1 and N2 Ct of less than 30 (compared to 0.55 with the ARTIC v3 protocol; Fig. ADS Nearly all draft genomes come from highly infected citrus or psyllids (usually with a Cq value lower than 23 using Li 16S qPCR), which limits strain diversity and epidemiology studies since not all samples can be sequenced reliably. 1). Integrative Genomics Viewer. We thank Sean Wang and Matt Plumb from the Minnesota Department of Heath for helpful discussions and for sharing ARTIC v3 primers. Select Tape Type D5000 ScreenTape assays is comparable to Bioanalyzer High Sensitivity DNA Chip Full tape and per sample options are available for the High Sensitivity D5000 and Genomic DNA tapes. 77, 19101917 (2011). Researchers have used enrichment strategies to increase the number of target reads in sequencing. and W.C., collected and analyzed data. Trees were generated using RaxML v8.2.10 and visualized using FigTree v1.4.3. S2-S3). Croucher, N. J. et al. The number in each circle represents the number of SNPs between the different comparisons. Phylogenic tree (ML midpoint rooted tree) of 935 core genes of Candidatus Liberibacter asiaticus strains, generated with Rax Maximum Likelihood method. 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. For the ARTIC v3, tailed amplicon v1, and tailed amplicon v2 methods, samples were amplified for 35 PCR cycles in the first PCR reaction. conceived and designed the experiments and helped write the manuscript; J.G. Percentage of genome coverage at 10x at different subsampled read depths for WA1 and UMGC SARS-CoV-2 isolates sequenced with different methods. 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. 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]. Reactions were run on a QuantStudio QS5 (Thermo Fisher Scientific, Waltham, MA) using the following cycling conditions: one cycle of 45C for 15min, followed by one cycle of 95C for 2min, followed by 45cycles of 95C for 15s and 60C for 1min. e Observed read depth for each of the expected amplicons for the BEI WA1 isolate amplified with the tailed amplicon v2 protocol (4 pool amplification) at a subsampled read depth of 100,000 raw reads. 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. We also provide accurate quantification and sizing of NGS library. ARTIC v3 amplicon relative abundance. Sign up for the Nature Briefing: Translational Research newsletter top stories in biotechnology, drug discovery and pharma. Internet Explorer). The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in a credit line to the material. All other genomes were obtained from NCBI. The authors read and approved the final manuscript. and JavaScript. Bioinformatics. Genomic regions of high recombination were detected and removed with Gubbins v2.3.129, and filtered polymorphic sites extracted to build phylogenies. Quick J, Grubaugh ND, Pullan ST, Claro IM, Smith AD, Gangavarapu K, et al. We carried out initial tests of the Nextera DNA Flex Enrichment protocol, the tailed amplicon v1 approach, and the ARTIC v3 approach using this sample set. Need Help? Nucleic acids research. Supplier: Agilent Technologies Accessories and spare parts for the 4150 and 4200 TapeStation systems like plates and foil seals, loading tips, TapeStation Test Tape, Needle Cartridge. Differentiation of Candidatus Liberibacter asiaticus isolates by variable-number tandem-repeat analysis. Cai, W., Nunziata, S., Rascoe, J. et al. https://doi.org/10.1038/s41579-020-0354-7. Nature. Agilent has a new system that fills the same space as the BioAnalyzer but is reportedly simpler and faster. Agilent Bioanalyzer alternatives? - SEQanswers 3e, Supplemental Fig. Chromatography & Spectroscopy Lab Supplies, GC Calculators & Method Translation Software, BioCalculators / Nucleic Acid Calculators. Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid of Yersinia pestis from victims of the Black Death. Agilent 4200 TapeStation System TechWiz4u 41 subscribers Subscribe 20K views 6 years ago New Agilent 4200 TapeStation For RNA and DNA analysis. Bov, J. M. Huanglongbing: a destructive, newly emerging, century-old disease of citrus. Wylie, T. N., Wylie, K. M., Herter, B. N. & Storch, G. A. Comparison of the Agilent 2100 Bioanalyzer and the 4200 TapeStation We reasoned that reducing the concentration of the primers that were over-represented in the initial round of sequencing may improve balance. 2200 TapeStation Parts & Accessories - Agilent Technologies The advantage to negative selection is it allows for the identification of new, large DNA insertions or mutations. 2016;34:9429. 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. Gohl DM, Magli A, Garbe J, Becker A, Johnson DM, Anderson S, et al. 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. If you have a disability and are having trouble accessing information on this website or need materials in an alternate format, contact web-accessibility@cornell.edu for assistance. 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. To download or contribute to the package, please see its page on GitHub. The system integrates an instrument, data processing software, reagents, and ScreenTape devices specific for DNA and RNA. 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/). 2010;26:58995. Venn diagrams show the overlapping of SNPs (single nucleotide polymorphisms) from different samples. Automation of PacBio SMRTbell NGS library preparation for - PubMed Vanaerschot M, Mann SA, Webber JT, Kamm J, Bell SM, Bell J, et al. New! The system integrates an instrument, data processing software, reagents, and ScreenTape devices specific for DNA and RNA. The alignment is generated using bowtie2 plugged in Geneious v 10.2.4, and visualized in Integrated Genome Viewer v2.4.10. It works for me as well as the Bioanalyzer but the sample cost is about 4 times lower. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. Without enrichment, LHCA-20 and SGCA-20, the highest pathogen concentration samples, had genome coverage of 65 and 60%, respectively, both with 1x depth of coverage (Table1). Each probe consists of 120 mer RNA and the total probe size is 1.32Mbp (TableS1). Gohl, D.M., Garbe, J., Grady, P. et al. The pan-genome phylogenetic tree based on core genes also demonstrates a similar branching pattern. 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 reaction was set up to create cDNA using the ARTIC v3 protocol: 5L template RNA, 11L nuclease-free water, 4L SuperScript IV VILO master mix (Thermo Fisher Scientific, Waltham, MA). bioRxiv. We describe a modified workflow for SARS-CoV-2 sequencing which builds on the tiled amplicon approach developed by the ARTIC consortium and currently employed by many labs around the world. After size selection and an initial size distribution quantification with an Agilent TapeStation (see . The system integrates an instrument, data processing software, reagents, and ScreenTape devices specific for DNA and RNA. We could tell you about the benefits our TapeStation systems have to offerbut we thought showing you would be better! Complete genome sequence of citrus huanglongbing bacterium, Candidatus Liberibacter asiaticus obtained through metagenomics. CLas associated HLB was first found in Florida in early September, 20055 and was vectored by the Asian citrus psyllid (Diaphorina citri), which had been introduced into Florida in the late 1990s. Pathogen DNA is enriched from 500- to 45,000-fold compared to non-enriched samples. Nat Biotechnol. Core alignments of 935 genes were extracted and used to estimate a maximum likelihood tree using RaxML, as outlined above. The positions of all variants detected in this study are shown and bases where the sample matches the Wuhan-Hu-1 reference areshown in grey. 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. 2020:eabc0523. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.