Key Clinical Summary:

• Whole transcriptome sequencing (WTS) enables validation and rescue of low–variant allele fraction (VAF) mutations and resolution of sequencing artifacts in solid tumors.
• Integrating DNA and RNA analysis improves detection of clinically relevant variants in genes such as KRAS, TP53, and EP300.
• The combined workflow enhances accuracy, interpretability, and turnaround time for somatic variant analysis.

A new study demonstrates how integrating whole transcriptome sequencing (WTS) with DNA-based sequencing can significantly improve single nucleotide variant (SNV) detection in solid tumors. By leveraging RNA data, researchers achieved more accurate identification of low-frequency and clinically significant mutations, while reducing the need for time-intensive manual review of sequencing artifacts.

In routine clinical review of DNA and RNA sequencing data using a customized workflow (AUGMET), researchers assessed SNVs from tumor samples with low tumor content (<10%). Traditional DNA variant analysis applies a ≥5% VAF threshold, often filtering out potentially actionable mutations in key oncogenes such as KRAS and TP53. The team used WTS, originally validated for gene fusion detection, to cross-verify low-VAF (<5%) and strand-biased (SB) DNA variants, provided that the corresponding genes exhibited baseline wild-type expression in RNA.

Several practical applications of WTS variant rescue were identified:

1. Low-VAF rescue: Variants undetectable or below threshold in DNA were recovered at higher frequencies in RNA. For example, a KRAS G12C variant (2.58% VAF in DNA) appeared at 28% in RNA, and a TP53 R280T variant (1.54% in DNA) reached 8% in RNA.
2. Artifact resolution: Some DNA variants with abnormal SB patterns, such as PTPRT A302V, were absent in RNA, confirming their likely artifact status.
3. Splice variant evaluation: DNA-based splice-site mutations were correlated with intron retention events on RNA—for instance, EP300 c.3591-6C>T led to intron 20 retention in transcripts.
4. Protein-truncating variant confirmation: DNA-truncated variants lacking RNA expression were confirmed to be non-expressed, such as an APC R805* variant detected in DNA but absent in the corresponding transcript.

Integrating WTS with DNA sequencing provides a multi-layered molecular view of tumor biology, improving the sensitivity and interpretability of SNV detection. By rescuing low-frequency variants and confirming biologically relevant alterations at the transcript level, this combined approach enhances diagnostic accuracy and clinical confidence in variant reporting.

The workflow also reduces manual review requirements in tools like Integrative Genomics Viewer, accelerating turnaround time for molecular pathology reports. In clinical practice, this strategy can strengthen personalized oncology efforts, particularly for patients with low-tumor-content biopsies where standard DNA assays may underperform.

This study confirms that combined DNA-RNA sequencing substantially enhances variant detection, artifact resolution, and clinical interpretation in solid tumors. Whole transcriptome data complement DNA-based assays, providing a more comprehensive and reliable molecular diagnostic approach for precision oncology.

Source:

Sukhadia S, Shah P, Green D, Tafe L. Whole-Transcriptome Sequencing Improves Variant Analysis and Interpretation of Whole-Exome Sequencing-Based Variants in Solid Tumors in a Clinical Laboratory Setting. Presented at the 2025 AMP Annual Meeting & Expo. November 11-15, 2025; Boston, Massachusetts. ST143