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. D) Agilent Bioanalyzer trace for a library prepared from samples with N1 and N2 Ct values between ~2035 using the tailed amplicon v2 (4 pool amplification) workflow. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. We also provide accurate quantification and sizing of NGS library. Nature. qRT-PCR reactions to identify SARS-CoV-2 positive samples were carried out using a modified version of the Centers for Disease Control and Prevention (CDC) SARS-CoV-2 qRT-PCR assay, as previously described [18]. 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 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/). https://doi.org/10.1093/bioinformatics/btp698. 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. Enhanced virome sequencing using targeted sequence capture. Enriched samples, however, had enough reads to align samples to SC1, SC2 and JXGC3 prophage reference sequences. 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. Bioinformatics. To generate cDNA upstream of SARS-CoV-2 genome amplification, the following reaction was set up: 5L template RNA, 11L nuclease-free water, 4L SuperScript IV VILO master mix (Thermo Fisher Scientific, Waltham, MA). Article For the Illumina DNA Flex Enrichment protocol, SARS-CoV-2 genome coverage was more complete for samples with lower N1 and N2 Cts (ranging from ~2030) at comparable read depths and coverage thresholds than with amplicon approaches, similar to the BEI WA isolate data (Fig. Teixeira Ddo, C. et al. 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. Percentage of genome coverage at 100x at different subsampled read depths for WA1 and UMGC SARS-CoV-2 isolates sequenced the tailed amplicon v1 method amplified for 25 PCR cycles in the first PCR reaction. In the future, it will be interesting to determine the absolute sequencing limit of this method. 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. Positive selection (like the SureSelect method described here) can enrich a target hundreds to thousands fold, making it possible to sequence low titer samples. 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. Trees were generated using RaxML v8.2.10 and visualized using FigTree v1.4.3. A total of 1g input DNA per sample was used for SureSelect library preparation (Agilent, Santa Clara, CA). 2.5L extracted RNA was added to 7.5L qPCR master mix comprised of the following components: 1.55L nuclease-free water, 5L GoTaq Probe qPCR Master Mix with dUTP (2X) (Promega, Madison, WI), 0.2L GoScript RT Mix for 1-Step RT-qPCR (Promega, Madison, WI), 0.75L primer/probe sets for either N1, N2, or RP (IDT, Coralville, IA). Phylogenies were generated with all samples and 11 published genomes (TableS2) using two methods, core SNPs and the pan-genome. (Lonza's FlashGel is a similar system.) Scientific Reports (Sci Rep) The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples. The system integrates an instrument, data processing software, reagents, and ScreenTape devices specific for DNA and RNA. I came from a lab in industry that trialed the BioA, TapeStation, Caliper system and Advanced Analytical fragment analyzer. Phylogenic tree (ML midpoint rooted tree) of 849 core SNVs of Candidatus Liberibacter asiaticus strains generated with Rax Maximum Likelihood method. It's called the. However, for samples with N1 and N2 Ct values greater than approximately 30, the number of sequencing reads were substantially reduced and the proportion of reads mapping to the human genome were substantially increased (Supplemental Fig. The positive enrichment approach described in this study shows a relatively simple and universal CLas genome enrichment method. Read-pairs were stitched together using PEAR [20]. Analytical Validation of a COVID-19 qRT-PCR Detection Assay Using a 384-well Format and Three Extraction Methods. 2019;20:85. https://doi.org/10.1186/s13059-019-1691-6. https://doi.org/10.1038/s41598-019-55144-4, DOI: https://doi.org/10.1038/s41598-019-55144-4. Visit our TapeStation portfolio page and discover how! Data Interpretation | Center for Quantitative Life Sciences | Oregon 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. 3e, Supplemental Fig. A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2. This Agilent tape station can scale easily be. Cycling conditions were: 98C for 30s, followed by 25 or 35cycles of 98C for 15s and 65C for 5min. Adapter-ligated libraries were purified using AMPure XP beads (Beckman Coulter, Inc., Brea, CA, USA), amplified, and then purified.
