Tailed amplicon v1 amplicon relative abundance. Next generation sequencing technologies (NGS) have recently enabled large-scale genomic surveillance of infectious diseases. Find products using our Selection Tool. Supplemental Fig. Mesirov. 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. Page, A. J. et al. It has a sample throughput of 12 samples per run, and results are generated in approximately 30 minutes [ 11 ]. PacBio has become synonymous with their High Fidelity (HiFi) sequencing. c The tailed amplicon approach, developed here, enriches first strand cDNA using ARTIC v3 primers containing adapter tails. This led to decreased coverage at a given read depth for the tailed amplicon v1 method relative to ARTIC v3 (Fig. Anuhea DeLude, Riley Wells, Mohammad Arif, Antonio Rodrguez, Brecht Guillemyn, Mario Vaneechoutte, Amy S. Gargis, Blake Cherney, David Sue, Mohammad Arif, Grethel Y. Busot, James P. Stack, Matthew Chung, Laura Teigen, Julie C. Dunning Hotopp, Shu Yang, Marcela A. Johnson, Boris A. Vinatzer, Fabien Dutreux, Corinne Da Silva, Jean-Marc Aury, Andrea D. Tyler, Laura Mataseje, Cindi R. Corbett, Natacha Couto, Leonard Schuele, John W. Rossen, Scientific Reports Over the past ten years, NGS (next generation sequencing) has been widely applied to identity pathogens, characterize genetic variants, and provide a molecular basis for building additional diagnostic tools. 2020;26.1266-73. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. 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). Input material was not sheared, as the amplicons were already the desired fragment length. 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 SARS-CoV-2 genome was amplified using a two-step PCR protocol. TapeStation Software for NGS Sample Quality Control | Agilent https://doi.org/10.1016/j.cub.2020.03.022. B) Percentage of genome coverage at 100x at different subsampled read depths for the indicated sample when sequenced using the indicated workflow. 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. Michael J. Stulberg. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. 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. These gels can be automatically imaged while running by using a companion light box and camera setups. Genome Announc, https://doi.org/10.1128/genomeA.00999-14 (2014). All extraction methods used 100L of viral transport medium as input and eluted in 100L of appropriate elution buffer as indicated by manufacturer protocols. The number in each circle represents the number of SNPs between the different comparisons. 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. Schuenemann, V. J. et al. 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 system integrates an instrument, data processing software, reagents, and ScreenTape devices specific for DNA and RNA. The analysis method for amplicon libraries is as follows: Sample quality was assessed with FastQC [19]. 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. 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. 2e). Eight samples with >1ng/L concentration of target amplicons were selected for downstream library preparation. d Observed read depth for each of the expected amplicons for the BEI WA1 isolate amplified with the tailed amplicon v1 (2 pool amplification) protocol at a subsampled read depth of 100,000 raw reads. An estimated 10,000 viral genome copies were used as input for cDNA generation. 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. Interested in learning more about the TapeStation systems and how easy-to-use ScreenTape technology can give you a faster time to results and constant per-sample costs in sample quality control? 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. Supplemental Table4. Finally, we examined the variants detected in the patient samples for each of the SARS-CoV-2 sequencing methods. 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. Huanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) vectored by Asian citrus psyllids. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Halbert, S. E. The discovery of huanglongbing in Florida. Article Privacy The first strand synthesis reaction was incubated at 25C for 10min, 42C for 50min, 70C for 15min. PubMed Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Harcourt J, Tamin A, Lu X, Kamili S, Sakthivel SK, Murray J, et al. BMC Microbiol. Cycling conditions were: 98C for 30s, followed by 25 or 35cycles of 98C for 15s and 65C for 5min. 2020;579:2703. 108(4), 454461, https://doi.org/10.1094/PHYTO-08-17-0282-R (2018). 2023 BioMed Central Ltd unless otherwise stated. The cycling conditions were as follows: 98C for 2min; followed by 1624 cycles of 98C for 30s, 60C for 30s, and 72C for 1min; and a final extension at 72C for 5min., using 16 cycles for Cq 20 samples, 18 cycles for Cq 22 samples, and 24 cycles for Cq 26 and Cq 28 samples. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Positive selection (like the SureSelect method described here) can enrich a target hundreds to thousands fold, making it possible to sequence low titer samples. 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. It is suitable to analyze size, quantity, and integrity of your samples. 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. 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. It's called the. 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. 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. Library preparation was performed following the standard Illumina TruSeq Nano DNA protocol for 350 base pair libraries (Illumina, San Diego, CA). The system includes instrument, software, reagents, and ScreenTape devices to analyze size, quantity, and integrity of your DNA and RNA sample. I have used both widely in my lab and they have given me equivalent results. 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). 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. 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. After PCR, streptavidin beads were removed using a magnet stand, and the PCR products were further purified with AMPure XP beads. Find products using our Selection Tool. Show more Show more Almost yours: 2 weeks, on us. 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]. Correspondence to Finally, amplicon approaches (Fig. We generated libraries for all six samples in parallel without enrichment using a TruSeq PCR free DNA library preparation kit (Illumina, San Diego, CA). Enriched samples with the lowest pathogen concentration had 99% genome coverage and at least 70X sequence coverage. Effective disease managing efforts require a greater understanding of the causal agents, which can be achieved through whole genome sequencing. Agilent 4200 TapeStation System - YouTube 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. 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 BEI WA1 isolate strain was amplified for both 25 or 35 PCR cycles, using the same enzymes and PCR conditions used for the ARTIC v3 data set. Article A-F) Observed read depth for each of the expected amplicons for the indicated sample amplified with the ARTIC v3 protocol with TruSeq library preparation at a subsampled read depth of 100,000 raw reads. 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. The following indexing primers were used (X indicates the positions of the 10bp unique dual indices): Forward indexing primer: AATGATACGGCGACCACCGAGATCTACACXXXXXXXXXXTCGTCGGCAGCGTC. 25, 19101920 (2015). J Microbiol Methods 66, 104115 (2006). and S.Y. 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). Capturing sequence diversity in metagenomes with comprehensive and scalable probe design. 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. Need Help? 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. C) Tailed amplicon v1 (2 pool amplification); D) Tailed amplicon v2 (4 pool amplification). 2a-b, Supplemental Tables14). For target selection, pre-designed probes are added to the mixed genomic DNA extracts and capture their complimentary DNA sequences through complimentary hybridization, allowing the uncaptured DNA to be removed during wash steps. After trimming and filtering, 4050% of the enriched reads were discarded due to insufficient read length and suspected probe contamination, while less than 5% of non-enriched reads were discarded (TableS3). Genome Announc. 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. 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. 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. Pathogen DNA is enriched from 500- to 45,000-fold compared to non-enriched samples. Data Interpretation | Center for Quantitative Life Sciences | Oregon The Agilent TapeStation is used for DNA analysis. A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2. Variants detected using different sequencing workflows. . 2010;26:58995. 130 Biotechnology Building Check out the interactive hotspots below and see what these instruments can do for your lab. Performance metrics for Illumina DNA Flex Enrichment Protocol. Proc Natl Acad Sci USA 108, E746752 (2011). 2a-b, Supplemental Table1, Supplemental Table2). In general, the same regions were not always missing, with only ~2kb shared sites missing across samples. The ARTIC primer pools have gone through multiple iterations to improve evenness of coverage [13]. 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. 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]. Nat Methods.