RNA vs DNA Sequencing in Cancer: Complementary, Not Interchangeable
Tumour molecular profiling can start from DNA or from RNA. They are not two ways of doing the same thing: DNA sequencing reads the genome, RNA sequencing reads what is being expressed and how transcripts are spliced. Each has clear strengths and blind spots.
Quick Answer
Tumour molecular profiling can start from DNA or from RNA. They are not two ways of doing the same thing: DNA sequencing reads the genome, RNA sequencing reads what is being expressed and how transcripts are spliced. Each has clear strengths and blind spots.
What DNA Sequencing Captures
DNA sequencing detects point mutations, small insertions and deletions, copy-number changes and, with adequate intronic coverage, some structural rearrangements. It works from the stable genome, so a variant is present whether or not the gene is expressed.
DNA is also more robust in archival formalin-fixed tissue than RNA, and germline comparison requires DNA.
What RNA Sequencing Adds
RNA sequencing reads transcripts, so it directly shows gene fusions at their spliced junction, splicing changes such as MET exon 14 skipping, and expression levels.
Because it captures the functional consequence, RNA can confirm that a DNA variant of uncertain effect actually alters splicing, and it can catch fusions that a DNA panel missed.
Where Each Falls Short
DNA panels can miss fusions with breakpoints in large introns and cannot confirm a splicing effect. RNA sequencing cannot detect a mutation in a gene that is not expressed, is more sensitive to sample quality, and is less established for copy number and for tumour mutational burden.
Neither replaces germline testing, which is a separate DNA-based analysis on normal tissue.
Running Both
Many comprehensive profiling assays now combine a DNA panel with an RNA fusion panel, using DNA for mutations and copy number and RNA for fusions and splicing events.
Proficiency-testing data show RNA fusion detection performance varies between laboratories, so method and validation still matter even within a combined assay.
Interpretation Notes
If a report is DNA-only and a fusion driver is clinically expected but absent, RNA testing is a reasonable next step.
A splicing-related finding such as MET exon 14 skipping is most confidently established when RNA evidence supports it.
Key Takeaways
- ·DNA sequencing reads the genome: mutations, copy number, some rearrangements, germline comparison.
- ·RNA sequencing reads transcripts: fusions at the junction, splicing changes, expression.
- ·DNA can miss deep-intronic fusions; RNA can miss variants in unexpressed genes and needs good RNA.
- ·Comprehensive assays increasingly pair a DNA panel with an RNA fusion panel.
Put these genes in pathway context
Frequently asked questions
What is the key idea in RNA vs DNA Sequencing in Cancer: Complementary, Not Interchangeable?
Tumour molecular profiling can start from DNA or from RNA. They are not two ways of doing the same thing: DNA sequencing reads the genome, RNA sequencing reads what is being expressed and how transcripts are spliced. Each has clear strengths and blind spots.
What should be kept with the result or mechanism?
RNA sequencing reads transcripts: fusions at the junction, splicing changes, expression. DNA can miss deep-intronic fusions; RNA can miss variants in unexpressed genes and needs good RNA. Comprehensive assays increasingly pair a DNA panel with an RNA fusion panel.
References
- 1RNA Sequencing for Solid Tumor Fusion Gene Detection: Proficiency Testing Practice and Performance. Archives of Pathology & Laboratory Medicine, 2024. PubMed
- 2Guidelines for Validation of Next-Generation Sequencing-Based Oncology Panels. Journal of Molecular Diagnostics, 2017. PubMed
- 3Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors. Archives of Pathology & Laboratory Medicine, 2018. PubMed
- 4Somatic mutation panels: time to clear their names. Cancer Genetics, 2019. PubMed
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