Performance of Targeted Library Preparation Solutions for SARS-CoV-2 Whole Genome Analysis

Klempt, Petr; Brož, Petr; Kašný, Martin; Novotny, Ádám; Kvapilová, Kateřina; Kvapil, Petr

doi:10.3390/diagnostics10100769

Cited by 23 publications

(27 citation statements)

References 20 publications

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“…Additional modifications such as DNA-rehybridization steps 25 or slower temperature ramp speeds had no significant effects (Sup Fig 4c, 4d) . We found that an alternative primer design using shorter (~150bp) amplicons, the Paragon Genomics CleanPlex SARS-CoV-2 panel, did recover more complete genomes in very low viral load samples (CT >35), but had lower accuracy and drops in coverage even in high viral load samples which resulted in missed SNV calls ( Sup Fig 3a, 3b ), consistent with previous reports 12,26 . In our hands, a 40 cycle PCR utilizing ARTIC primers without alterations to the ramp temperatures or speeds provided the greatest amplification without deleterious effects of increasing unfounded SNV calls.…”

Section: Resultssupporting

confidence: 90%

DNA spike-ins enable confident interpretation of SARS-CoV-2 genomic data from amplicon-based sequencing

Lagerborg

Normandin

Bauer

et al. 2021

Preprint

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The rapid global spread and continued evolution of SARS-CoV-2 has highlighted an unprecedented need for viral genomic surveillance and clinical viral sequencing. Amplicon-based sequencing methods provide a sensitive, low-cost and rapid approach but suffer a high potential for contamination, which can undermine lab processes and results. This challenge will only increase with expanding global production of sequences by diverse research groups for epidemiological and clinical interpretation. We present a novel approach using synthetic DNA spike-ins (SDSIs) to track samples through a sequencing workflow and detect inter-sample contamination. Applying this approach to the ARTIC Consortium's amplicon design, we define a series of best practices for Illumina-based sequencing and provide a detailed characterization of approaches to increase sensitivity for low-viral load samples incorporating the SDSIs. We demonstrate the utility and efficiency of the SDSI method amidst a real-time investigation of a suspected hospital cluster of SARS-CoV-2 cases.

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Section: Resultssupporting

confidence: 90%

DNA spike-ins enable confident interpretation of SARS-CoV-2 genomic data from amplicon-based sequencing

Lagerborg

Normandin

Bauer

et al. 2021

Preprint

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“…Among the many approaches available for SARS-CoV-2 whole-genome analysis, the amplicon-based sequencing method developed by the ARTIC Network [ 27 ] is currently the most widely used [ 13 , 24 , [28] , [29] , [30] , [31] , [32] ]. Based on the PrimalSeq protocol originally developed for Zika virus [ 23 , 33 ], the ARTIC Network designed a set of 98 tiled amplicons in two PCR pools for the targeted whole-genome amplification of SARS-CoV-2 [ 27 ].…”

Section: Introductionmentioning

confidence: 99%

ACoRE: Accurate SARS-CoV-2 genome reconstruction for the characterization of intra-host and inter-host viral diversity in clinical samples and for the evaluation of re-infections

et al. 2021

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Sequencing the SARS-CoV-2 genome from clinical samples can be challenging, especially in specimens with low viral titer. Here we report Accurate SARS-CoV-2 genome Reconstruction (ACoRE), an amplicon-based viral genome sequencing workflow for the complete and accurate reconstruction of SARS-CoV-2 sequences from clinical samples, including suboptimal ones that would usually be excluded even if unique and irreplaceable. The protocol was optimized to improve flexibility and the combination of technical replicates was established as the central strategy to achieve accurate analysis of low-titer/suboptimal samples. We demonstrated the utility of the approach by achieving complete genome reconstruction and the identification of false-positive variants in >170 clinical samples, thus avoiding the generation of inaccurate and/or incomplete sequences. Most importantly, ACoRE was crucial to identify the correct viral strain responsible of a relapse case, that would be otherwise mis-classified as a re-infection due to missing or incorrect variant identification by a standard workflow.

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“…The sensitivity, specificity and limit of detection are dependent of the chosen method (15). Amplicon-based methods are the most widely used approach for SARS-CoV-2 genotyping due to their relative low cost and simplicity (16-22). Despite their widespread use, amplicon-based technologies are associated with source-specific factors that, if not accounted for, can affect the reliability of assigned genotypes (11-14) and impact, for example, the measurements of viral intra-host variability (23).…”

Section: Introductionmentioning

confidence: 99%

Guidelines for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

Kubik

Marques

Xing

et al. 2020

Preprint

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Background: SARS-CoV-2 genotyping has been instrumental to monitor virus evolution and transmission during the pandemic. The reliability of the information extracted from the genotyping efforts depends on a number of aspects, including the quality of the input material, applied technology and potential laboratory-specific biases. These variables must be monitored to ensure genotype reliability. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in studies of viral spread and evolution. Results: We used clinical samples and synthetic viral genomes to evaluate the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination using an amplicon-based approach. We found that at least 1000 viral genomes are necessary to confidently detect variants in the genome at frequencies of 10% or higher. The broad applicability of our recommendations was validated in >200 clinical samples from six independent laboratories. The genotypes of clinical isolates with viral load above the recommended threshold cluster by sampling location and period. Our analysis also supports the rise in frequency of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was favoured by travelling during the summer 2020. Conclusions: We present much-needed recommendations for reliable determination of SARS-CoV-2 genome sequence and demonstrate their broad applicability in a large cohort of clinical samples.

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Performance of Targeted Library Preparation Solutions for SARS-CoV-2 Whole Genome Analysis

Cited by 23 publications

References 20 publications

DNA spike-ins enable confident interpretation of SARS-CoV-2 genomic data from amplicon-based sequencing

DNA spike-ins enable confident interpretation of SARS-CoV-2 genomic data from amplicon-based sequencing

ACoRE: Accurate SARS-CoV-2 genome reconstruction for the characterization of intra-host and inter-host viral diversity in clinical samples and for the evaluation of re-infections

Guidelines for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

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