sars cov 2 variant tracking  (Sanofi)

Journal: Scientific Reports

Article Title: Real-time SARS-CoV-2 diagnostic and variants tracking over multiple candidates using nanopore DNA sequencing

doi: 10.1038/s41598-021-95563-w

Figure Lengend Snippet: Scheme illustrating the proposed real-time SARS-CoV-2 diagnostics and variants tracking strategy. RNA samples isolated from multiple candidates are converted into complementary DNA (cDNA) such that samples associated to each candidate are labelled by molecular DNA barcodes appended to their extremities. Barcoded-cDNA samples are pooled together and PCR amplified in presence of a bridge sequence (blue) allowing to generate concatemers. Long DNA concatemers are loaded into the MinION nanopore sequencer for real-time SARS-CoV-2 diagnostics and variants tracking with the help of RETIVAD.

Article Snippet: Figure 4 SARS-CoV-2 variants tracking within the SPIKE gene. ( A ) Scheme illustrating the SPIKE targeted strategy for variants tracking with Oxford Nanopore technology (ONT) sequencing.

Techniques: Isolation, Amplification, Sequencing

Journal: Scientific Reports

Article Title: Real-time SARS-CoV-2 diagnostic and variants tracking over multiple candidates using nanopore DNA sequencing

doi: 10.1038/s41598-021-95563-w

Figure Lengend Snippet: Dual molecular DNA barcoding and PCR-driven concatemerization applied to SARS-CoV-2 diagnostics. ( A ) Reverse transcription is performed with oligonucleotides targeting a Covid-19 region, but in addition containing a unique molecular barcode per sample and a common adapter sequence (Gibson). Reverse transcription assays (cDNA) performed for multiple candidates are pooled together for amplification. PCR amplification is performed with oligonucleotides targeting the common Gibson sequence and a Covid-19-specific primer hosting a second molecular barcode as well as a complementary Gibson sequence. During PCR cycles, amplified products are able to concatenate thanks to the Gibson flanking sequences. We have called this particular PCR strategy “Concat-PCR”. ( B ) DNA electropherogram (Agilent TapeStation system) illustrating concatemer products obtained with PCR primers at 1uM final concentration. ( C ) DNA electropherogram for a concat-PCR assay performed with PCR primers at 0.1uM final concentration, demonstrating a preferential production of long concatemers in contrast to those observed in ( B ). ( D ) Timeline illustrating the various molecular biology steps required for samples preparation prior engaging samples into MinION DNA sequencing.

Article Snippet: Figure 4 SARS-CoV-2 variants tracking within the SPIKE gene. ( A ) Scheme illustrating the SPIKE targeted strategy for variants tracking with Oxford Nanopore technology (ONT) sequencing.

Techniques: Reverse Transcription, Sequencing, Amplification, Concentration Assay, DNA Sequencing

Journal: Scientific Reports

Article Title: Real-time SARS-CoV-2 diagnostic and variants tracking over multiple candidates using nanopore DNA sequencing

doi: 10.1038/s41598-021-95563-w

Figure Lengend Snippet: SARS-CoV-2 diagnostics assay with Nanopore DNA sequencing. ( A ) Scheme of the bioinformatics pipeline within RETIVAD. Long concatemer sequenced reads are computationally fragmented in monomers with the help of the Gibson sequence location. Monomers are stratified on the basis of flanking barcodes (cDNA and PCR incorporated barcodes) and aligned to the Covid-19 viral reference genome to verify its specificity. Considering that Oxford Nanopore instruments provide sequenced data during sequencing, the aforementioned steps are performed on real-time, such that the number of aligned read counts per candidate (defined by the combination of cDNA & PCR incorporated DNA barcodes) are assessed and displayed during the MinION sequencing process. ( B ) The synthetic SARS-CoV-2 RNA control sample (Twist Bioscience; Wuhan-Hu-1 strain) was subsequently diluted and used for reverse transcription and RT-qPCR assay targeting the E-Sarbeco viral region. ( C ) Reverse transcription assay scheme for 8 conditions corresponding to different viral RNA dilutions (dilution ID corresponding to that presented in ( B )) and targeting the E-Sarbeco region. Each of these reverse transcription assays are identified by the indicated molecular barcodes (BC33–BC40). Note that among them a sample devoid of viral RNA is also included (BC37). All 8 samples are pooled at the end of the assay and concat-PCR amplified with a set of other eight barcoded primers targeting the E-Sarbeco gene (BC1–BC8), providing a combinatorial barcoding strategy corresponding to 64 pseudo-candidates. ( D ) Real-time display illustrating the number of aligned read counts associated to the E-Sarbeco region per dual barcode combination (BC3 vs all cDNA barcodes) assessed during Nanopore sequencing. Inset: Zoom view highlighting that the first seven hours of DNA sequencing are sufficient to reveal read count differences between samples. ( E ) Summary of the bioinformatics pipeline assessed at the end of 4 days of DNA sequencing. Concatenation factor is inferred from the ratio between the number of detected monomers and the initial number of sequenced concatemers. Optimal barcodes correspond to the number of sequences presenting an optimal monomer length (> 100nt; < 250nt) and presenting both cDNA & PCR barcode sequences. ( F ) Boxplots illustrating the total aligned read counts associated to each cDNA barcode (BC33–BC40) and the different PCR barcodes. Each of the boxplots are labelled with their corresponding viral RNA dilution (dil.ID indicated in ( B )). ( G ) Heatmap illustrating the total aligned read counts associated to each cDNA barcode (BC33–BC40) and the different PCR barcodes (BC1–BC8). Note that ( F , G ) reveals a sensitivity of 100 viral copies, since the next RNA dilution condition appears in some cases at the same level of the negative samples.

Article Snippet: Figure 4 SARS-CoV-2 variants tracking within the SPIKE gene. ( A ) Scheme illustrating the SPIKE targeted strategy for variants tracking with Oxford Nanopore technology (ONT) sequencing.

Techniques: DNA Sequencing, Sequencing, Reverse Transcription, Quantitative RT-PCR, Amplification, Nanopore Sequencing

Journal: Scientific Reports

Article Title: Real-time SARS-CoV-2 diagnostic and variants tracking over multiple candidates using nanopore DNA sequencing

doi: 10.1038/s41598-021-95563-w

Figure Lengend Snippet: SARS-CoV-2 variants tracking within the SPIKE gene. ( A ) Scheme illustrating the SPIKE targeted strategy for variants tracking with Oxford Nanopore technology (ONT) sequencing. Reverse transcription is performed with random hexamer primers presenting unique molecular barcodes and the Gibson adapter sequence. This approach, allows to generate cDNA products of variable length covering the whole viral but also the human genome. Barcoded cDNA samples are PCR amplified with four SPIKE-specific primers (each of them including a common molecular DNA barcode and spaced by ~ 1 kb distance) and one Gibson primer targeting the adapter sequence introduced during reverse transcription. For facilitating the assay, all four SPIKE and Gibson primers are used within an equimolar pool. PCR amplified samples are analyzed by Oxford Nanopore MinION DNA sequencing. ( B ) Genome browser view displaying the aligned read counts obtained after ONT sequencing. Location of each of the SPIKE-targeting primers (P1-P4) is displayed and corresponds to regions presenting the highest read counts within the SPIKE gene. ( C ) Summary of the used synthetic SARS-CoV-2 viral RNA samples, corresponding to the Wuhan-Hu-1 control sample (reference strain) and two described variant strains (HF2393 and B.1.1.7). ( D ) Reverse transcription assay scheme for 7 conditions corresponding to different dilutions of viral RNA issued from the strains illustrated in ( C ) (BC37-BC43) and a control sample devoid of viral RNA (BC44). In all cases, 15 ng of human RNA material—issued from cell lines in culture—has been also introduced. All 8 samples are pooled at the end of the reverse transcription assay and PCR amplified with either four pools of barcoded primers targeting the SPIKE gene (BC1–BC4) or four barcoded primers targeting the E-Sarbeco region (BC5–BC8), providing a combinatorial barcoding strategy corresponding to 32 pseudo-candidates. ( E ) Quantitative PCR targeting either the human RNAse-P gene or the viral E-Sarbeco region, performed over the reverse transcription assays described in ( D ). ( F ) Real-time Covid-19 diagnostic performed by RETIVAD displaying the number of aligned read counts per barcoded cDNA sample (described in ( D )) for the gene SPIKE (BC1) or the E-Sarbeco region (BC5). ( G ) Boxplots illustrating the total aligned read counts associated to the gene SPIKE or the E-Sarbeco region for each of the conditions described in ( D ). ( H ) Variant analysis performed by RETIVAD over the barcoded sample 37 (BC37) associated to the corresponding PCR barcodes (BC1–BC4). The displayed genome browser view reveals the presence of multiple nucleotide variants (colored bars), among them seven being retrieved on all four samples (BC1-BC4) and with a significant read coverage. Five of them (A23,063T; C23,271A; C23,604A; C23,709T; T24,506G) correspond to missense mutations that were described within the B.1.1.7 UK variant strain (blue). In addition, two other missense mutations were also detected; the D614G (previously described within SARS-CoV-2 European strains) and the H1058G. ( I ) Similar to (H) but for the sample BC40, corresponding to a tenfold lower RNA titer than that used for sample BC37. Note that the same missense mutations retrieved in (H) are observed (colored bars), and this in despite of the lower aligned read counts. ( J ) Variants detection assay over the sampled BC38, corresponding to the SARS-CoV-2 variant strain HF2393, described in France in 2020. In this case, only the missense mutation D614G is retrieved among all four samples (BC1–BC4).

Article Snippet: Figure 4 SARS-CoV-2 variants tracking within the SPIKE gene. ( A ) Scheme illustrating the SPIKE targeted strategy for variants tracking with Oxford Nanopore technology (ONT) sequencing.

Techniques: Sequencing, Reverse Transcription, Random Hexamer Labeling, Amplification, DNA Sequencing, Variant Assay, Real-time Polymerase Chain Reaction, Diagnostic Assay, Detection Assay, Mutagenesis