Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

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    Evaluation Summary:

    The ability to fully resolve the whole genome of viral pathogens is often hampered by a multitude of different obstacles, one of which is optimally amplifying different regions of the genome. Part of this challenge lies in designing the best primers pairs that can consistently amplify PCR products despite the presence of changes (mutations) in the genome. The work described by Jaworski and colleagues can potentially provide an alternative approach that does not depend on primer pairs to fully sequence one such viral pathogen, SARS-CoV-2, and can also be applied towards other viral families.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called ‘Tiled-ClickSeq’ , which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5’UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

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  1. Author Response:

    Reviewer #1:

    Click-Seq represents a novel method of sequencing RNA viruses such as SARS-CoV-2, with evidence of successfully sequencing the SARS-CoV-2 genome and identification of recombinations and variants. This does appear to be a potential advantage that needs a direct comparison with existing methods to be fully convincing.

    Thank you for your time and comments on our manuscript and approach.

    Specific comments:

    1. The actual sensitivity in terms of number of copies would be useful to know and tocompare with other methods. Here, cultures are used, not clinical samples that make this even more important

    We now present results from three independent batches of Tiled-ClickSeq libraries of 60 NP swabs obtained through routine diagnostics for COVID19. We compare genome coverage and genome completeness with CT values …

  2. Evaluation Summary:

    The ability to fully resolve the whole genome of viral pathogens is often hampered by a multitude of different obstacles, one of which is optimally amplifying different regions of the genome. Part of this challenge lies in designing the best primers pairs that can consistently amplify PCR products despite the presence of changes (mutations) in the genome. The work described by Jaworski and colleagues can potentially provide an alternative approach that does not depend on primer pairs to fully sequence one such viral pathogen, SARS-CoV-2, and can also be applied towards other viral families.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the …

  3. Reviewer #1 (Public Review):

    Click-Seq represents a novel method of sequencing RNA viruses such as SARS-CoV-2, with evidence of successfully sequencing the SARS-CoV-2 genome and identification of recombinations and variants. This does appear to be a potential advantage that needs a direct comparison with existing methods to be fully convincing.

    Specific comments:

    1. The actual sensitivity in terms of number of copies would be useful to know and tocompare with other methods. Here, cultures are used, not clinical samples that make this even more important.

    2. Is the large difference in coverage across the genome shown in Fig 2B, due to methodological issues to random variation. How would this compare to coverage variation by the ARCTIC protocol by different methods

  4. Reviewer #2 (Public Review):

    The authors present a novel method of sequencing SARS-CoV-2, arguing its overcomes many limitations of other currently used methods, particularly the ARTIC protocol. Generally the method is interesting and encouraging to see these limitations can be overcome. Although the authors walk through evidence that their method can successfully sequence the SARS-CoV-2 genome and use the data to identify minor variants and recombination events, the manuscript doesn't contain any direct comparisons of their method with the ARTIC protocol. Consequently, the assertions made throughout the paper of reduced bias and increased sensitivity and utility are not supported empirically.

    Specific comments:

    For instance, in figure 2, I think it is important to present an equivalent plot to Fig 2A for artic samples with equivalent …

  5. Reviewer #3 (Public Review):

    Strengths. While current NGS method(s), namely the ARTIC protocol, has made phenomenal contributions to resolving the genome of SARS-CoV-2, there is room for improvement. Towards this end, Jaworski and company have devised an alternative approach that utilizes a one-step RT PCR that combines ClickSeq with tiled amplification of the viral genome. This negates the use of primer pairs, which may encounter problems with amplification of structural variants. The method appears to be straightforward and amendable for sequencing on Illumina and Oxford platforms. The results generated do support the claims of the authors and have the potential to contribute significantly to understanding the evolutionary dynamics of SARS-CoV-2.

    Weaknesses. The main shortcoming of the manuscript in its current form is that the …

  6. SciScore for 10.1101/2021.03.10.434828: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Viruses and RNA extraction: For WRCEVA isolates, viral RNA was obtained from supernatant materials of viral isolates amplified on Vero cells originally obtained from nasopharyngeal swab samples that tested positive in clinical laboratory assays for SARS-CoV-2 RNA, as described previously (26).
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Wild-type and mutant SARS-CoV-2 were titrated and propagated on Vero E6 cells.
    Vero E6
    sug…