Pheniqs: Fast and flexible quality-aware sequence demultiplexing
Bibliographic record
Abstract
1 Abstract Motivation Output from high throughput sequencing instruments often exceeds what is necessary to assay a single sample. To better utilize this capacity, multiple samples are independently tagged with a unique “barcode” sequence and are then pooled, or “multiplexed”, and sequenced together. Classifying, or “demultiplexing”, the reads involves decoding the barcode sequence. Although instruments estimate the probability of incorrectly calling each nucleobase, available demultiplexers do not consult those estimates or report classification error probabilities. Results We present Pheniqs, a fast and flexible sequence demultiplexer and quality analyzer. In addition to providing an efficient implementation of the widespread minimum distance decoder , Pheniqs introduces a novel Phred-adjusted maximum likelihood decoder that consults base calling quality scores and estimates the probability of a barcode decoding error. Setting an upper bound on the permissible error provides an intuitive way to control demultiplexing confidence and directly influence precision and recall. Pheniqs supports FASTQ and multiple Sequence Alignment/Map formats and uses auxiliary SAM tags to report both library classification and demultiplexing error probability. Evaluation on both real and semi-synthetic data indicates that Pheniqs is faster than existing demultiplexers, substantially when demultiplexing longer reads, and achieves greater accuracy by correctly reflecting quality measurements. Availability and Implementation Implemented in multithreaded C++ and available under the terms of the AGPL-3.0 license agreement at http://github.com/biosails/pheniqs . Manual and examples are available at http://biosails.github.io/pheniqs .
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.006 | 0.010 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.002 | 0.001 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.002 | 0.002 |
| Open science | 0.002 | 0.003 |
| Research integrity | 0.001 | 0.003 |
| Insufficient payload (model declined to judge) | 0.011 | 0.007 |
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 source (direct Gemma or distilled Codex), 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".