Routine clinical specimens tested directly
The series includes nine archived FFPE NSCLC specimens and one GIST liquid-biopsy specimen, providing an intended-use feasibility setting rather than synthetic material alone.
↳ Study Design
Abstract
BACKGROUND: Next generation sequencing (NGS) impacted on clinical algorithm of solid tumor patients. A heterogeneous series of NGS platforms have been implemented in clinical practice but challenging handling procedures, high technical costs, and scant affordability on sequencing diagnostic routine specimens can leave behind some patients who could benefit from target drugs. Here, we sought to evaluate technical feasibility of Oxford Nanopore Technologies (ONT) sequencing accurate identification of tumor-associated molecular alterations, in a pilot series of real-world samples.
METHODS: We developed a technical workflow adapting the SiRe® NGS panel, originally designed for Ion semiconductor sequencing, on MinION platform (Oxford nanopore technologies), a portable, cost effective long read sequencer. The SiRe® panel enables detection of ٥٦٨ clinically actionable somatic mutations across six key genes (EGFR, KRAS, NRAS, BRAF, cKIT, PDGFRα) relevant to targeted therapies in several solid tumors. We implemented a multiplexed assay using pooled and barcoded samples, processed on a single MinION flow cell. Performance was benchmarked from a pilot series of nine FFPE samples against Ion Torrent sequencing data. A single liquid biopsy sample was also analyzed testing accuracy of MinION technology.
RESULTS: The adapted ONT workflow demonstrated high concordance ratei in detecting clinically relevant molecular alterations on short-read fragments, achieving comparable accuracy with standardized second generation NGS platforms on tissue and liquid biopsy samples.
CONCLUSIONS: This proof of concept aimed to integrate ONT sequencing into molecular oncology workflows, providing practical, low-cost, and scalable alternative to conventional NGS platforms. The results support the potential of ONT technology to democratize access to precision oncology, particularly in laboratories with limited resources.
Key Claims
Primary findingTentative
The adapted ONT workflow demonstrated high concordance rate in detecting clinically relevant molecular alterations on short-read fragments, achieving comparable accuracy with standardized second generation NGS platforms on tissue and liquid biopsy samples.
Fragile — the claim's weakest requirement is that the effect exists at all. Based on how the study was designed and conducted, not on statistics extracted from the paper. Experimental. Every point below is traceable to a quoted passage, but this forecast has not yet been validated against known replication outcomes.
ten specimens, one liquid biopsy, and no bidirectional accuracy estimates cannot establish comparable diagnostic accuracy
Abstract, Results
SecondaryTentative
A perfect match between MinION workflow and conventional short-read sequencing pipeline was identified. In detail, 14 single-nucleotide variants (SNVs) across all 10 samples, were confirmed by ONT sequencing.
one-directional recovery lacks ONT-only call, indel, precision, and independently verified low-VAF accounting
Results, paragraph 2
SecondaryTentative
SNVs showing a variant allele frequency (VAF) ranging from 0.5% to 5.0% were consistently identified by the GATK MuTect2 pipeline and confirmed visually inspecting BAM files on Genomics Viewer (IGV) tool.
visual confirmation uses the same ONT reads and conflicts with the stated ≥5% filtering threshold
Results, paragraph 3
Derived from the full evaluation — not a separate score.
Strengths · Limitations
Strengths
Specific MinION workflow is documented
The Methods identify the R10.4.1 flow cell, V14 barcoding chemistry, SUP basecalling model, GRCh38 alignment, and GATK calling tools, supporting technical reproducibility at the protocol level.
↳ MinION Protocol
Limitations are explicitly identified
The Discussion recognises that the sample cannot establish robust performance and also identifies the narrow panel, missing clinical outcomes, and cross-platform reference differences.
↳ Discussion, limitations paragraph
Limitations
Low-VAF workflow remains internally inconsistent
The MinION Protocol states a VAF threshold of at least 5%, while Results report calls from 0.1% to 5.0% after visual inspection. The paper does not clearly identify which pipeline generated these calls or provide an independent confirmation strategy.
↳ MinION Protocol; Results, paragraph 3
Analytical accounting cannot be reconstructed
The Ion protocol describes eight pooled libraries although ten samples are reported, and a run used approximately 200 active pores despite an 800-pore minimum in Methods. These inconsistencies bear on the complete-concordance and feasibility claims.
↳ SiRe Protocol; MinION Protocol; Discussion, flow-cell comparison
Accuracy claims exceed reported evidence
Fourteen recovered SNVs in ten samples are described as a perfect match and comparable accuracy without false-positive accounting, confidence intervals, indel performance, or a reported correlation statistic.
↳ Abstract, Results, paragraphs 2–3; Discussion
Nabu Commentary
The paper demonstrates that adapted SiRe libraries can be sequenced on MinION across a small set of archived clinical specimens, giving the work a legitimate feasibility contribution. Its methodology is limited by the ten-sample spectrum, one liquid-biopsy case, shared amplicon libraries, absent diagnostic-agreement statistics, and unspecified independence of ONT interpretation. Most importantly, the MinION Protocol’s ≥5% VAF statement is not reconciled with Results reporting 0.1–5.0% detections after visual inspection. The Discussion acknowledges that the sample cannot prove robust performance, so conclusions about comparable accuracy, cost, scalability, and access should be narrowed and supported by larger validation data.
Check another paper
Author Note / Field Feedback
Nabu’s assessment, alongside the field’s view.
Are you an author of this paper?
Dimension breakdown
Study Quality
Limited2.7
Confidence mediumContribution
2.8
The study adds a specific adaptation of the six-gene SiRe panel to MinION using routine FFPE material and one liquid-biopsy sample. The advance remains incremental because ONT oncology sequencing is already established and the demonstration comprises only ten specimens.
Abstract, Methods; Study Design; Discussion
“A pilot series of 10 diagnostic samples”
Methodological Rigour
2.7
The study uses intended-use specimens and applies an Ion comparator across the series, but it reports no confusion matrix, precision estimates, or independent validation of low-frequency calls. The unresolved VAF threshold, library count, run-acceptance, blinding, and visual-review issues materially constrain execution.
Study Design; SiRe Protocol; MinION Protocol; Results, paragraphs 2–3
“Filtering chain was able to detect molecular alterations with variant allele fraction (VAF) ≥ 5%”
Reporting
2.6
The workflow and overall argument are followable, and the Discussion identifies several limitations. However, “perfect match,” “significant correlation,” and “comparable accuracy” are not accompanied by the quantitative agreement statistics or per-variant accounting required to support those formulations.
Abstract, Results, paragraphs 2–3; Discussion
“A perfect match between MinION workflow and conventional short-read sequencing pipeline was identified.”
Positioning
2.8
The Discussion identifies the small sample, narrow panel, absent clinical outcomes, and differing reference genomes as limitations. Its broader accuracy and access conclusions nevertheless proceed beyond the stated inability to establish robust technical performance.
Discussion, limitations and concluding paragraphs
“the sample set is inadequate to statistically prove robust technical performance”
Reliability
Concerns4 of 4 checks
Internal CoherenceConcern
Material protocol and accounting inconsistencies prevent a clear reconstruction of the low-frequency variant analysis and whether all runs met the stated acceptance criteria. These issues bear directly on the reported perfect match and comparable-accuracy conclusions.
Research ConductNoted
Consent, anonymisation, conflicts, and funding are declared, but the ethics and consent statements are not fully reconciled for the retrospective use of human samples. The unavailable tables and repository-free data statement are assessability limitations rather than the basis of the reliability flag.
Flags: 3 declared / 5 total
Reference IntegrityNoted
29 references in manuscript 28 of 28 checkable references found in an index 1 are books, websites or datasets — counted, but not index-checkable
Post-Publication RecordClean
No retraction notice found in Retraction Watch.
Last checked: 08 Oct 2026
Sources: Retraction Watch ✓
Readiness
Where this paper’s evidence sits on the path from initial observation to real-world use.
Mechanistic
Routine archived specimens and a portable platform provide an early translational setting, but the study remains a retrospective proof of concept. Larger validation, reproducibility, failure-rate, cost, and turnaround evidence would be needed before implementation.
Study Design; Results; Discussion, limitations
“proof of analytical equivalence study”
AI-generated, human-governed. Something look off? Contact us to request a review.