Adaptable and comprehensive approaches for long-read nanopore sequencing of polyadenylated and non-polyadenylated RNAs
Bibliographic record
Abstract
The advent of long-read (LR) sequencing technologies has provided a direct opportunity to determine the structure of transcripts with potential for end-to-end sequencing of full-length RNAs. LR methods that have been described to date include commercial offerings from Oxford Nanopore Technologies (ONT) and Pacific Biosciences. These kits are based on selection of polyadenylated (polyA+) RNAs and/or oligo-dT priming of reverse transcription. Thus, these approaches do not allow comprehensive interrogation of the transcriptome due to their exclusion of non-polyadenylated (polyA-) RNAs. In addition, polyA + specificity also results in 3'-biased measurements of PolyA+ RNAs especially when the RNA input is partially degraded. To address these limitations of current LR protocols, we modified rRNA depletion protocols that have been used in short-read sequencing: one approach representing a ligation-based method and the other a template-switch cDNA synthesis-based method to append ONT-specific adaptor sequences and by removing any deliberate fragmentation/shearing of RNA/cDNA. Here, we present comparisons with poly+ RNA-specific versions of the two approaches including the ONT PCR-cDNA Barcoding kit. The rRNA depletion protocols displayed higher proportions (30%-50%) of intronic content compared to that of the polyA-specific protocols (5%-8%). In addition, the rRNA depletion protocols enabled ∼20-50% higher detection of expressed genes. Other metrics that were favourable to the rRNA depletion protocols include better coverage of long transcripts, and higher accuracy and reproducibility of expression measurements. Overall, these results indicate that the rRNA depletion-based protocols described here allow the comprehensive characterization of polyadenylated and non-polyadenylated RNAs. While the resulting reads are long enough to help decipher transcript structures, future endeavors are warranted to improve the proportion of individual reads representing end-to-end spanning of transcripts.
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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".