agilent tapestation alternative

agilent tapestation alternative

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. Rapid, sensitive, full-genome sequencing of severe acute respiratory syndrome coronavirus 2. 10L of PCR product for each sample was normalized using a SequalPrep 96-well Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA). Trees were generated using RaxML v8.2.10 and visualized using FigTree v1.4.3. Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification. 3 and TableS4). Human host DNA was filtered by aligning the stitched reads to the human genome (GRCh38). (a) LHCA samples at different Cq values: Cq 20 (blue), Cq 22 (red), Cq 26 (gray), Cq 28 (yellow). S7). Supplemental Table1. Vanaerschot M, Mann SA, Webber JT, Kamm J, Bell SM, Bell J, et al. Andersen KG, Rambaut A, Lipkin WI, Holmes EC, Garry RF. Visit our TapeStation portfolio page and discover how! 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 The analysis method for amplicon libraries is as follows: Sample quality was assessed with FastQC [19]. 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. 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. Nat Biotechnol. 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. Global circulation patterns of seasonal influenza viruses vary with antigenic drift. Liberibacter americanus and Ca. Huanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) vectored by Asian citrus psyllids. Currently, conserved genomic loci, such as the 16S rRNA gene, are used to define the CLas species but lack the genetic variation to differentiate strains6,7. 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. 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). Google Scholar. Enhanced virome sequencing using targeted sequence capture. Cycling conditions were: 98C for 30s, followed by 10cycles of 98C for 20s, 55C for 15s, 72C for 1min, followed by a final extension at 72C for 5min. Usually it costs at least $1500 to $3000dollars to whole genome sequence one high titer sample, but this was substantially reduced after using SureSelect target enrichment. 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. Int J Syst Evol Microbiol. 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. Bedford T, Greninger AL, Roychoudhury P, Starita LM, Famulare M, Huang M-L, et al. Quality and quantity of libraries were determined by TapeStation using a D1000 ScreenTape (Agilent). 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. Supplemental Fig. It is suitable to analyze size, quantity, and integrity of your samples. Each probe consists of 120 mer RNA and the total probe size is 1.32Mbp (TableS1). 2020:2020.08.25.265074. https://doi.org/10.1101/2020.08.25.265074. Article Genome sequences of the strains sequenced in this study are available in GenBank BioProject PRJNA631042. 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. 2c-d). Di Paola N, Sanchez-Lockhart M, Zeng X, Kuhn JH, Palacios G. Viral genomics in Ebola virus research. PubMed Check out the interactive hotspots below and see what these instruments can do for your lab. Nelson AC, Auch B, Schomaker M, Gohl DM, Grady P, Johnson D, et al. Tape station systems use ScreenTape, that's credit-card-sized . 8-well PCR tube strips or 96-well sample plates are available depending on sample throughput, bringing added flexibility A Rn threshold of 0.5 was selected and set uniformly for all runs. 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. 3(6), https://doi.org/10.1128/genomeA.01508-15 (2015). You are currently viewing the SEQanswers forums as a guest, which limits your access. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Supplemental Fig. If material is not included in the articles Creative Commons license 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. C) Tailed amplicon v1 (2 pool amplification); D) Tailed amplicon v2 (4 pool amplification). Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid of Yersinia pestis from victims of the Black Death. Liberibacter asiaticus (CLas) is the most widespread and is the only species associated with the disease in the United States (U.S.)4. I came from a lab in industry that trialed the BioA, TapeStation, Caliper system and Advanced Analytical fragment analyzer. The tree with the highest likelihood across 10 runs was selected. The released CLas genomes were obtained from either highly infected psyllids or citrus samples (equivalent to 18 to 23 Cq using Li 16S qPCR)14,15,16,17 because the whole genome sequence of CLas can only be obtained using metagenomic sequencing, due to the lack of in vitro culture. In this article, we focus on metrics relevant to evaluating the success of a Pacific Biosciences (PacBio) sequencing run. 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. Positive selection (like the SureSelect method described here) can enrich a target hundreds to thousands fold, making it possible to sequence low titer samples. Check out the interactive hotspots below and see what these instruments can do for your lab. 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. Jagoueix, S., Bov, J. M. & Garnier, M. The phloem-limited bacterium of greening disease of citrus is a member of the subdivision of the Proteobacteria. This work was carried out in part using computing resources at the University of Minnesota Supercomputing Institute. Advanced Analytical is my personal favorite. It's called the. Based on validation experiments for the University of Minnesota qRT-PCR clinical COVID-19 diagnostic assay, we estimate that a Ct value of 30 corresponds to roughly 500 SARS-CoV-2 genome copies and a Ct value of 35 corresponds to roughly 15 SARS-CoV-2 genome copies in the 5L input used for cDNA creation [18]. Percentage of bases covered across fixed depths of coverage based on reference guided assemblies and estimated with samtools depth. Whole genome sequencing can provide precise molecular characterization of the diversity among CLas populations. 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). Appl Environ Microbiol. Right primers: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG . The SARS-CoV-2 genome was amplified using a two-step PCR protocol. The quality control methods from gDNA input to final library using the Agilent Bioanalyzer System and Agilent TapeStation System were evaluated. statement and Halbert, S. E. The discovery of huanglongbing in Florida. https://doi.org/10.1093/bioinformatics/btt593. 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. Upon splitting the tailed SARS-CoV-2 primers into 4 PCR reactions based on primer performance in the initial sequencing tests, the tailed amplicon v2 method had much improved amplicon balance. 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. Introduction of a bead clean-up step between the first and second PCRs can also help reduce the proportion of adapter dimers when using the tailed amplicon v2 protocol (Amy Kistler, personal communication). 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. Chromatography & Spectroscopy Lab Supplies, GC Calculators & Method Translation Software, BioCalculators / Nucleic Acid Calculators. 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. Indeed, this mechanical lysis approach has been widely adopted for lysis of both Gram-positive and Gram-negative bacteria within complex matrices. To download or contribute to the package, please see its page on GitHub. e Tailed amplicon v1 (2 pool amplification); f Tailed amplicon v2 (4 pool amplification). 19(5), 455477 (2012). Paden C, Tao Y, Queen K, Zhang J, Li Y, Uehara A, et al. I have used both widely in my lab and they have given me equivalent results. Supplemental Table3. S2-S3, Supplemental Tables12). 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. The two SGCA strain samples are clustered together and most closely related to the previously reported SGCA strain, SGCA5. https://doi.org/10.1038/s41579-020-0354-7. 7(2), 1118 (2010). 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). and S.Y. Supplemental Fig. Proc Natl Acad Sci USA 108, E746752 (2011). Library preparation was performed following the standard Illumina TruSeq Nano DNA protocol for 350 base pair libraries (Illumina, San Diego, CA). Read-pairs were stitched together using PEAR [20]. Devices from other companies that anyone can recommend? The Agilent TapeStation is used for DNA analysis. Nucleic acids research. Li H, Durbin R. Fast and accurate long-read alignment with burrowswheeler transform. The system integrates an instrument, data processing software, reagents, and ScreenTape devices specific for DNA and RNA. 22, 10111020 (2009). Tailed amplicon v2 amplicon relative abundance. My Agilent Bioanalyzer is giving me fits lately! 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. The concentration of Ca. 1c). Theyve been used for improving genome assemblies. 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. New! $12,500 USD. 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. Genome Announc. However, NGS technology has significant limitations when performing pathogen diagnostics in complex metagenomic samples. 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. 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 overlapping number stands for the same SNPs identified between the different comparisons and the non-overlapping numbers specify the unique SNPs to each sample. PubMed Central A detailed protocol is available on protocols.io: https://www.protocols.io/view/sars-cov-2-tailed-amplicon-illumina-sequencing-bipikdke. All samples should meet the following criteria: Provide at least 2uL of each sample. 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. 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). Terms and Conditions, 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). S8. The RNA probe price can drop further to around $100 dollar per sample if it is bulk order (96 reactions each order instead of 16). https://doi.org/10.1038/nbt.3601. The secondary amplification was done using the following recipe: 5L template DNA (1:100 dilution of the first PCR reaction), 0.7L nuclease-free water, 2L 5x Q5 reaction buffer (New England Biolabs, Ipswich, MA), 0.2L 10mM dNTPs (Kapa Biosystems, Woburn, MA, 0.1L Q5 Polymerase (New England Biolabs, Ipswich, MA), 0.5L forward primer (10M), 0.5L reverse primer (10M). Percentage of genome coverage at 10x at different subsampled read depths for each sample when sequenced using the following approaches: A) Illumina Nextera DNA Enrichment; B) ARTIC v3 with TruSeq library preparation. A total of 1g input DNA per sample was used for SureSelect library preparation (Agilent, Santa Clara, CA). Wylie, T. N., Wylie, K. M., Herter, B. N. & Storch, G. A. Provided by the Springer Nature SharedIt content-sharing initiative. A minimum of two no template controls (NTCs) were included on all runs. Analytical Validation of a COVID-19 qRT-PCR Detection Assay Using a 384-well Format and Three Extraction Methods. 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/. 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]. Click here to register now, and join the discussion. S1. 1). 1a) can be used to enrich for viral sequences in order to lower sequencing costs and are being employed to sequence SARS-CoV-2 [11]. 6(25), https://doi.org/10.1128/genomeA.00554-18 (2018). Cai, W., Yan, Z., Rascoe, J. 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. CAS A) Agilent TapeStation trace for a library prepared from samples with N1 and N2 Ct values between ~2040 using the tailed amplicon v1 (2 pool amplification) workflow. 2015;523:21720. The system integrates an instrument, data processing software, reagents, and ScreenTape devices specific for DNA and RNA. Seemann, T. Prokka: rapid prokaryotic genome annotation. The final pooled sample was quantified using a Qubit Fluorometer and High Sensitivity DNA assay (Thermo Fisher Scientific, Waltham, MA). W.C., S.N., J.R. and M.S., wrote and revised the manuscript. Thorvaldsdttir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. These results indicate that this SureSelect target enrichment method can be used to sequence CLas more efficiently than the canonic NGS method. Nat Rev Microbiol. 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). While the RNA tapes are still a bit lacking, my favorite is the genomic tape so you can look at much larger sizes. Teixeira Ddo, C. et al. Variants located outside of the region targeted by the amplicon panel were filtered out (reference genome positions 154 and 29,83629,903), and consensus sequences bases corresponding to those regions were trimmed. Cryptic transmission of SARS-CoV-2 in Washington state. africanus1,3. 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. Next, 1 g of each library was hybridized with the SureSelect capture library. A pan-genome comparative approach could provide enough genetic variation for high strain resolution, but sequencing CLas genomes has been historically difficult. It does use gel capillaries and the array lasts for only 6 months at a time so if you are not a high volume user, it might not be as cost effective as it is for me. 2020;2019:2020.04.02.022186. All raw read files were deposited to the SRA public database under BioProject ID PRJNA540608. Profiles of CLas MiSeq reads mapping in reference to prophage SCI, SC2 and JXGC-3. Int J Med Microbiol. Grubaugh ND, Gangavarapu K, Quick J, Matteson NL, De Jesus JG, Main BJ, et al. 2020:2020.03.10.985150. https://doi.org/10.1101/2020.03.10.985150. Cornell Visualization and Imaging Partnership, Ask Us Anything About Your Needs or Projects. Genome Biol. We performed initial tests of the tailed amplicon v1 protocol by amplifying the samples listed in Fig. Nature Biotechnology. 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. Probable Pangolin Origin of SARS-CoV-2 Associated with the COVID-19 Outbreak. There are also smaller, 12-lane E-Gels that can be used for sample recovery. The tailed amplicon v2 protocol had an average coverage at a subsampled read depth of 100,000 raw reads of 97.54% (10x) and 87.17% (100x) for all six test samples (Supplemental Tables12). Use the Previous and Next buttons to navigate the slides or the slide controller buttons at the end to navigate through each slide. 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. Bedford T, Riley S, Barr IG, Broor S, Chadha M, Cox NJ, et al. Nature. For pan-genome generation, reads mapping to the Psy62 reference genome were extracted and assembled using SPAdes v3.12.0 with k-mer lengths of 21, 33, 55, 77, 99, and 12731. Quick J, Grubaugh ND, Pullan ST, Claro IM, Smith AD, Gangavarapu K, et al. f Variants detected for the BEI WA1 isolate 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). The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples. 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. Are there any alternatives to this that anyone can recommend that is more modern tech? Nat Protoc. These amplification primers had the following structure (see Supplemental Data File1 for primer sequences): Left primers: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG . In this final installment of our series, we ask our participants about one of the most important aspects of data analysis, accuracy and reproducibility. Scientific Reports (Sci Rep) PubMedGoogle Scholar. For samples with Ct vales of less than 30, average coverage was 98.81% (10x) and 94.72% (100x) at a subsampled read depth of 100,000 raw reads (Fig. Article CAS https://doi.org/10.1093/bioinformatics/bty407. 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. 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. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Amplicon libraries (ARTIC v3, Tailed v1, Tailed v2) were diluted to 8 pM in Illuminas HT1 buffer, spiked with 5% PhiX, and sequenced using a MiSeq 600cycle v3 kit (Illumina, San Diego, CA). Features 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. 2). All authors reviewed and approved the manuscript. 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. Wu, F. et al. This is exemplified by the CLas genome of the lowest titer sample (equivalent to 28.52 Cq using Li 16S qPCR) being easily obtained with just 3.2 million total reads. bioRxiv. 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]. The system integrates an instrument, data processing software, reagents, and ScreenTape devices specific for DNA and RNA. Samples with N1 and N2 Ct values ranging from approximately 2040 chosen for testing of SARS-CoV-2 sequencing workflows. 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. Manage cookies/Do not sell my data we use in the preference centre. S2, Supplemental Tables14). 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]. Li, W., Hartung, J. S. & Levy, L. Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. The second strand synthesis reaction was incubated at 16C for 60min. The proximal origin of SARS-CoV-2. 4 and 5). 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.

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