Managing false positives during detection of pathogen sequences in shotgun metagenomics datasets
Bibliographic record
Abstract
BACKGROUND: Culture-independent diagnostic tests are gaining popularity as tools for detecting pathogens in food. Shotgun sequencing holds substantial promise for food testing as it provides abundant information on microbial communities, but the challenge is in analyzing large and complex sequencing datasets with a high degree of both sensitivity and specificity. Falsely classifying sequencing reads as originating from pathogens can lead to unnecessary food recalls or production shutdowns, while low sensitivity resulting in false negatives could lead to preventable illness. RESULTS: We used simulated and published shotgun sequencing datasets containing Salmonella-derived reads to explore the appearance and mitigation of false positive results using the popular taxonomic annotation softwares Kraken2 and Metaphlan4. Using default parameters, Kraken2 is sensitive but prone to false positives, while Metaphlan4 is more specific but unable to detect Salmonella at low abundance. We then developed a bioinformatic pipeline for identifying and removing reads falsely identified as Salmonella by Kraken2 while retaining high sensitivity. Carefully considering software parameters and database choices is essential to avoiding false positive sample calls. With well-chosen parameters plus additional steps to confirm the taxonomic origin of reads, it is possible to detect pathogens with very high specificity and sensitivity.
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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 | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| 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 | 0.000 | 0.000 |
| 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".