Avalilação PREreview de Analytical Evaluation of Whole Genome Sequencing for Acute Myeloid Leukemia

Short summary of the research and its contribution to the field

This preprint evaluates a high-depth, tumor-only whole genome sequencing workflow (WGS TO) for comprehensive genomic profiling of acute myeloid leukemia (AML). The workflow combines commercially available DNA extraction and PCR-free library preparation with NovaSeq 6000 sequencing and DRAGEN Somatic tumor-only analysis, targeting approximately 220× genome coverage. The analytical evaluation includes 68 clinical AML samples, healthy donor samples, cancer cell lines, and reference material.

The study assesses several clinically important classes of genomic alterations, including single-nucleotide variants and small insertions/deletions, structural variants (SVs), copy-number alterations (CNAs), and loss of heterozygosity. The reported analytical sensitivities were 97.6% for small variants, 89.5% for structural variants, and 92.9% for copy-number alterations. The workflow also detected all seven FLT3 internal tandem duplications included in the clinical comparison.

An additional strength is the evaluation of limit of detection as a function of sequencing depth and variant allele frequency. At 140× coverage, the authors estimate a 95% detection rate at approximately 5% VAF for small variants and 7.3% VAF for structural variants. The study also evaluates detection thresholds for copy-number alterations and loss of heterozygosity. The complete workflow is reported to have a turnaround time of approximately five days from DNA extraction to reporting.

Overall, this work provides useful analytical evidence supporting high-depth WGS as a potentially comprehensive approach to AML genomic profiling and contributes to the continuing discussion about how genome-wide sequencing might be incorporated into routine molecular haematopathology.

General assessment

I found this to be a well-designed and clinically relevant analytical study. AML is particularly suitable for evaluating comprehensive genome sequencing because disease classification, prognostic assessment, and therapeutic decision-making depend on a broad spectrum of genomic abnormalities. In current practice, these abnormalities may require a combination of targeted sequencing, cytogenetics, FISH, and other molecular methods.

An important contribution of this study is therefore the demonstration that a single WGS workflow can interrogate several different classes of genomic alterations simultaneously while maintaining a clinically relevant turnaround time.

The manuscript is generally clear, and the analytical validation strategy is strengthened by the inclusion of different types of clinical and reference material and comparison with several orthogonal methods. I have several suggestions that could further improve the presentation and help readers understand the potential clinical implementation of the approach.

Strengths

One of the strongest aspects of this work is the breadth of the analytical evaluation. The assay is not limited to SNVs and indels but also evaluates structural variants, copy-number changes, and loss of heterozygosity. This is important for AML, where clinically meaningful alterations extend across multiple variant classes.

The high analytical sensitivity for small variants is encouraging. In particular, detection of all seven FLT3-ITDs included in the comparison is noteworthy because FLT3 alterations are clinically important in AML and can present technical challenges for some sequencing approaches.

The use of multiple orthogonal reference methodologies is another valuable feature. Comparison with sequencing-based approaches as well as FISH and karyotyping reflects the heterogeneous diagnostic environment in which a comprehensive WGS assay would ultimately be used.

The analytical precision experiments are also useful. Fifteen clinical AML samples were examined across replicates, and the clinical samples showed very high intra-run and inter-run concordance. The additional evaluation using cell-line and reference materials provides further information about assay behaviour close to the detection limit.

Finally, the approximately five-day turnaround time is potentially important from a translational perspective. The authors report less than one day for wet-laboratory procedures, approximately two days for sequencing, and around 13 hours for bioinformatic analysis. Such a workflow could make comprehensive genomic information available within a timeframe relevant to AML diagnostic work-up and treatment planning.

Comments and suggestions for the authors

1. Clinical positioning of WGS TO

It would be useful to expand slightly on how the authors envisage WGS TO being incorporated into the current AML diagnostic pathway. The manuscript demonstrates that several different classes of variants can be evaluated within one workflow, which raises an important practical question: should WGS TO be viewed primarily as a complement to existing cytogenetic and molecular testing, or could it eventually consolidate some of these assays?

A short schematic showing the conventional AML testing pathway alongside a potential WGS-based pathway could make the translational implications of the study particularly clear.

2. Tumor-only workflow

The tumor-only design is a practical strength because it avoids the requirement for a matched normal sample and may simplify implementation. At the same time, readers would benefit from additional information about how potentially germline findings would be managed in a clinical setting.

For example, the authors could briefly describe when confirmatory germline testing would be recommended and how the bioinformatic workflow distinguishes likely somatic events from variants that may warrant further investigation. This would help connect the analytical validation presented here with future routine clinical reporting.

3. Structural variants and copy-number alterations

The reported performance for structural and copy-number alterations is encouraging. The manuscript would be further strengthened by providing a concise summary of the types of SVs and CNAs that were most successfully detected and those that proved more technically challenging.

Such information could be especially useful for laboratories considering implementation because it would identify the genomic contexts in which WGS performs particularly well and where complementary testing may still be useful.

The authors also report additional variants detected by WGS TO that were not identified using the reference methods, including additional coding small variants, SVs, and CNAs. These observations are interesting and illustrate the potential breadth of high-depth WGS. Future orthogonal confirmation of a representative subset would further clarify the added diagnostic information provided by this workflow.

4. Limit-of-detection analysis

The LoD analysis is an important strength of the paper. The combination of titration, computational downsampling, and modelling provides useful information on the relationship between sequencing coverage and detection of low-frequency variants.

For clinical readers, it might be helpful to summarise these findings in a single table showing, for each variant class, the recommended sequencing depth, estimated LoD, and corresponding analytical sensitivity. This could make the validation results easier to translate into laboratory practice.

The observations close to the detection threshold are also informative. Reporting performance by VAF ranges, for example 2–5%, 5–10%, and above 10%, could provide additional practical information for interpretation of low-frequency subclonal alterations.

5. Turnaround time and implementation

The approximately five-day turnaround time is one of the most clinically attractive findings. It would be useful to provide some additional operational detail regarding whether this timing was consistent across sequencing runs and how much of the five-day period is attributable to laboratory processing, sequencing, bioinformatics, variant review, and final interpretation.

Future prospective implementation studies could also assess turnaround time from receipt of a clinical specimen to a signed diagnostic report. This would complement the analytical workflow reported in the present study and provide an informative benchmark for routine laboratory practice.

6. Future validation

The study provides a strong foundation for further evaluation. A particularly valuable next step would be prospective testing of consecutive newly diagnosed AML cases, with WGS TO performed alongside the complete standard diagnostic work-up.

Such a study could examine not only analytical concordance but also whether WGS provides information affecting WHO/ICC classification, ELN risk stratification, selection of targeted therapies, or other clinical decisions.

Independent validation in additional clinical laboratories would also be informative. Many of the authors are employees of Illumina, which develops several of the technologies evaluated in the study, and this relationship is transparently disclosed in the manuscript. Replication in independent laboratories would provide useful evidence regarding reproducibility and generalisability across different clinical environments.

Overall assessment

This is a valuable analytical validation study of high-depth tumor-only whole genome sequencing for AML. The manuscript demonstrates strong performance across multiple clinically relevant variant classes and provides useful data on sensitivity, precision, sequencing depth, and limit of detection.

The ability to evaluate small variants, structural variants, copy-number alterations, and loss of heterozygosity within a single sequencing workflow is particularly attractive. The approximately five-day turnaround time further supports the potential clinical applicability of this approach.

The work provides an important foundation for moving from analytical validation toward prospective clinical implementation. Further evaluation in independent and prospectively recruited cohorts will help determine how WGS TO can best complement or potentially consolidate existing AML genomic testing strategies.

Overall, the study represents a useful contribution to the development of comprehensive genomic diagnostics for AML and provides encouraging evidence for the feasibility of integrating high-depth WGS into clinically relevant laboratory workflows.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.