REMBRANDT: A high-throughput barcoded sequencing approach for COVID-19 screening

  1. Dario Palmieri
  2. Jalal Siddiqui
  3. Anne Gardner
  4. Richard Fishel
  5. Wayne O. Miles

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Abstract

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV), is a highly infectious RNA virus. A still-debated percentage of patients develop coronavirus disease 2019 (COVID-19) after infection, whose symptoms include fever, cough, shortness of breath and fatigue. Acute and life-threatening respiratory symptoms are experienced by 10-20% of symptomatic patients, particularly those with underlying medical conditions that includes diabetes, COPD and pregnancy. One of the main challenges in the containment of COVID-19 is the identification and isolation of asymptomatic/pre-symptomatic individuals. As communities re-open, large numbers of people will need to be tested and contact-tracing of positive patients will be required to prevent additional waves of infections and enable the continuous monitoring of the viral loads COVID-19 positive patients. A number of molecular assays are currently in clinical use to detect SARS-CoV-2. Many of them can accurately test hundreds or even thousands of patients every day. However, there are presently no testing platforms that enable more than 10,000 tests per day. Here, we describe the foundation for the REcombinase Mediated BaRcoding and AmplificatioN Diagnostic Tool (REMBRANDT), a high-throughput Next Generation Sequencing-based approach for the simultaneous screening of over 100,000 samples per day. The REMBRANDT protocol includes direct two-barcoded amplification of SARS-CoV-2 and control amplicons using an isothermal reaction, and the downstream library preparation for Illumina sequencing and bioinformatics analysis. This protocol represents a potentially powerful approach for community screening, a major bottleneck for testing samples from a large patient population for COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2020.05.16.099747: (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
    Repeat the wash step.
    Repeat
    suggested: (ProRepeat, RRID:SCR_006113)
    Bioinformatics (B): B1: De-multiplexing reads B2: Build index for mapping reads using RSubread B3:
    RSubread
    suggested: (Rsubread, RRID:SCR_016945)
    Construct SAF annotation file for RSubread alignment and featureCounts formatted as below: B4: Map de-multiplexed reads to reference index using align() function align.stat <-align(index = “./reference_index”, readfile1 = reads1, readfile2 = reads2, output_file = “./Rsubread_alignment.
    featureCounts
    suggested: (featureCounts, RRID:SCR_012919)

    Results from OddPub: Thank you for sharing your code.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    One of the major current limitations of population testing is the availability of reagents (swabs, viral transport medium, RNA isolation kits, probes and other molecular biology reagents). The current clinical standard for COVID-19 diagnosis (qRT-PCR) requires suitable equipment for the amplification of the viral RNA and the detection of the infection. Each of these steps significantly slows sample processing and limits the number of tests that can be performed in a day. For this reason, new diagnostic tools are needed that are fast and efficient as well as scalable to population sized numbers and based on readily available reagents. To address this need, the scientific community has delivered a remarkable and unprecedented number of assays to diagnose COVID-19. Several diagnostic approaches that can provide fast and cheap tests have been developed. One of the leading approaches uses blood tests to identify IgG and IgM antibodies against SARS-CoV-2 proteins (15). These tests show great promise. However, this assay can only examine the immunological status of the patient and cannot determine the viral load in exhaled respiratory particles, the major source of disease spread. These diagnostic kits will therefore be important to identify patients which may have developed immunity against the virus, but are not appropriate to test the ability of a patient to infect others. A number of groups have been working to develop novel tools to detect the genetic material of SARS-CoV-2 wit...

    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.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1101/2020.05.16.099747v1 on bioRxiv