ApharSeq: An Extraction-free Early-Pooling Protocol for Massively Multiplexed SARS-CoV-2 Detection
Bibliographic record
Abstract
Abstract The global SARS-CoV-2 pandemic created a dire need for viral detection tests worldwide. Most current tests for SARS-CoV-2 are based on RNA extraction followed by quantitative reverse-transcription PCR assays. While automation and improved logistics increased the capacity of these tests, they cannot exceed the lower bound dictated by one extraction and one RT-PCR reaction per sample. Multiplexed next generation sequencing (NGS) assays provide a dramatic increase in throughput, and hold the promise of richer information including viral strains, host immune response, and multiple pathogens. Here, we establish a significant improvement of existing RNA-seq detection protocols. Our workflow, ApharSeq, includes a fast and cheap RNA capture step, that is coupled to barcoding of individual samples, followed by sample-pooling prior to the reverse transcription, PCR and massively parallel sequencing. Thus, only one non-enzymatic step is performed before pooling hundreds of barcoded samples for subsequent steps and further analysis. We characterize the quantitative aspects of the assay by applying ApharSeq to more than 500 clinical samples in a robotic workflow. The assay results are linear, and the empirical limit of detection is found to be Ct 33 (roughly 1000 copies/ml). A single ApharSeq test currently costs under 1.2$, and we estimate costs can further go down 3-10 fold. Similarly, we estimate a labor reduction of 10-100 fold, automated liquid handling of 5-10 fold, and reagent requirement reduction of 20-1000 fold compared to existing testing methods.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.003 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".