One-Seq: A Highly Scalable Sequencing-Based Diagnostic for SARS-CoV-2 and Other Single-Stranded Viruses
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Abstract
The management of pandemics such as COVID-19 requires highly scalable and sensitive viral diagnostics, together with variant identification. Next-generation sequencing (NGS) has many attractive features for highly multiplexed testing, however current sequencing-based methods are limited in throughput by early processing steps on individual samples (e.g. RNA extraction and PCR amplification). Here we report a new method, “One-Seq”, that eliminates the current bottlenecks in scalability by enabling early pooling of samples, before any extraction or amplification steps. To enable early pooling, we developed a one-pot reaction for efficient reverse transcription (RT) and upfront barcoding in extraction-free clinical samples, and a “protector” strategy in which carefully designed competing oligonucleotides prevent barcode crosstalk and preserve detection of the high dynamic range of viral load in clinical samples. This method is highly sensitive, achieving a limit of detection (LoD) down to 2.5 genome copy equivalent (gce) in contrived RT samples, 10 gce in multiplexed sequencing, and 2-5 gce with multi-primer detection, suggesting an LoD of 200-500 gce/ml for clinical testing. In clinical specimens, One-Seq showed quantitative viral detection against clinical Ct values with 6 logs of linear dynamic range and detection of SARS-CoV-2 positive samples down to ∼360 gce/ml. In addition, One-Seq reports a number of hotspot viral mutations at equal scalability at no extra cost. Scaling up One-Seq would allow a throughput of 100,000-1,000,000 tests per day per single clinical lab, at an estimated amortized reagent cost of $1.5 per test and turn-around time of 7.5-15 hr.
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SciScore for 10.1101/2021.04.12.21253357: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources The purified library sample was then normalized using either Qubit or Agilent TapeStation before proceeding to sequencing run. Agilent TapeStationsuggested: (Agilent TapeStation Laptop, RRID:SCR_019547)Sequencing analysis: The bioinformatic analysis of sequencing results was performed in a few steps: FASTQ generation and adapter trimming (Illumina BaseSpace), sequence alignment (bowtie2), demultiplexing and read counting (custom scripts in MATLAB and Excel). MATLABsuggested: …SciScore for 10.1101/2021.04.12.21253357: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources The purified library sample was then normalized using either Qubit or Agilent TapeStation before proceeding to sequencing run. Agilent TapeStationsuggested: (Agilent TapeStation Laptop, RRID:SCR_019547)Sequencing analysis: The bioinformatic analysis of sequencing results was performed in a few steps: FASTQ generation and adapter trimming (Illumina BaseSpace), sequence alignment (bowtie2), demultiplexing and read counting (custom scripts in MATLAB and Excel). MATLABsuggested: (MATLAB, RRID:SCR_001622)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.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.
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