HiDRA-seq: High-Throughput SARS-CoV-2 Detection by RNA Barcoding and Amplicon Sequencing

  1. Emilio Yángüez
  2. Griffin White
  3. Susanne Kreutzer
  4. Lennart Opitz
  5. Lucy Poveda
  6. Timothy Sykes
  7. Maria Domenica Moccia
  8. Catharine Aquino
  9. Ralph Schlapbach

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Abstract

The recent outbreak of a new coronavirus that causes a Severe Acute Respiratory Syndrome in humans (SARS-CoV-2) has developed into a global pandemic with over 6 million reported cases and more than 375,000 deaths worldwide. Many countries have faced a shortage of diagnostic kits as well as a lack of infrastructure to perform necessary testing. Due to these limiting factors, only patients showing symptoms indicating infection were subjected to testing, whilst asymptomatic individuals, who are widely believed to be responsible for the fast dispersion of the virus, were largely omitted from the testing regimes. The inability to implement high throughput diagnostic and contact tracing strategies has forced many countries to institute lockdowns with severe economic and social consequences. The World Health Organization (WHO) has encouraged affected countries to increase testing capabilities to identify new cases, allow for a well-controlled lifting of lockdown measures, and prepare for future outbreaks. Here, we propose HiDRA-seq, a rapidly implementable, high throughput, and scalable solution that uses NGS lab infrastructure and reagents for population-scale SARS-CoV-2 testing. This method is based on the use of indexed oligo-dT primers to generate barcoded cDNA from a large number of patient samples. From this, highly multiplexed NGS libraries are prepared targeting SARS-CoV-2 specific regions and sequenced. The low amount of sequencing data required for diagnosis allows the combination of thousands of samples in a sequencing run, while reducing the cost to approximately 2 CHF/EUR/USD per RNA sample. Here, we describe in detail the first version of the protocol, which can be further improved in the future to increase its sensitivity and to identify other respiratory viruses or analyze individual genetic features associated with disease progression.

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  1. ScreenIT

    SciScore for 10.1101/2020.06.02.130484: (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.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Sequencing: The three libraries generated from three different viral amplicons, all in combination with a GAPDH-derived library, were paired-end sequenced together in both an Illumina MiniSeq (R1=16 bp, i7=8 bp, R2=50 bp) and NovaSeq6000 sequencers (data not shown, R1=16 bp, i7=8 bp, R2=150 bp).
    MiniSeq
    suggested: None
    Align Python 3 package (data not shown).
    Python
    suggested: (IPython, RRID:SCR_001658)
    After verifying the quality of our reads, Bowtie2 was used to map all of our samples against the SARS-CoV-2 genomic region of interest (from 29600 bp to 29900 bp, inclusive), and GAPDH.
    Bowtie2
    suggested: (Bowtie 2, RRID:SCR_016368)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Whilst RT-qPCR reactions are generally very sensitive (i.e. able to detect true positive cases) and specific, the technology has inherent limitations with regard to large scale population screening, which has become increasingly important during this pandemic. Additionally, RT-qPCR does not provide any genotypic information regarding a patient’s infection beyond the causal organism. Another advantage of NGS over RT-qPCR is that NGS provides a direct and functional measurement SARS-CoV-2. RT-qPCR generates florescent measurements for individual plate wells, indirectly quantifying the presence of genetic material in relative concentrations. Alternatively, NGS generates thousands of reads, directly measuring the specific sequences present in a sample. The sequence information generated could provide insight into the specific infecting isolate and aid in tracing transmission within communities. HiDRA-seq, built on Next Generation Sequencing technology, has the ability to multiplex thousands of barcoded patient samples, significantly increasing current testing capacity. Our method is designed to be partially performed on a small automated liquid handling machine, so that a single person is able process more than 2,000 RNA samples per day with ease. This results in an overall shorter diagnostic turnaround time, with library preparation and sequencing data obtained in as little as 1.5 days. Our system does not match the speed of RT-qPCR for individual samples. However, it outperform...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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  2. Version published to 10.1101/2020.06.02.130484v1 on bioRxiv