RNA-based NGS

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Overview

Translocations can occur anywhere in the genome, including coding regions, introns, and other non-coding sequences. As translocations may not be expressed and have limited biological relevance, DNA might not be the ideal substrate to identify oncogenic fusions [1]. As shown in the figure below, RNA-based NGS avoids the technical complications associated with the effect of large intronic spaces in detecting NTRK gene fusions, as the intron regions are not present in the mRNA transcript [2-4].

There of the most common RNA-based NGS platforms include, anchored multiplex PCR, amplicon-based multiplex PCR and hybrid capture-based enrichment method; each having distinct chemistry and mechanisms. All three platforms have been found to be highly concordant in detecting NTRK fusions in patient samples and they all displayed equivalent high-level performance in specificity, indicating that all three platforms can detect NTRK gene fusions in clinical samples with similar performance characteristics [5].

RNA-based NGS Avoids the Technical Complications Associated with Large Intronic Spaces

Although there are many platforms available, the basic steps in performing RNA-based NGS are summarised in the following figure.

Click here for an overview of DNA- and RNA-based NGS.

 References

1. Zheng Z, Liebers M, Zhelyazkova B et al. Anchored multiplex PCR for targeted next-generation sequencing. Nat Med 2014; 20: 1479-1484.
2. Kheder ES, Hong DS. Emerging Targeted Therapy for Tumors with NTRK Fusion Proteins. Clin Cancer Res 2018; 24: 5807-5814.
3. Penault-Llorca F, Rudzinski ER, Sepulveda AR. Testing algorithm for identification of patients with TRK fusion cancer. J Clin Pathol 2019.
4. Pfarr N, Kirchner M, Lehmann U, Leichsenring J, Merkelbach-Bruse S, Glade J, Hummel M, Stögbauer F, Lehmann A, Trautmann M, Kumbrink J, Jung A, Dietmaier W, Endris V, Kazdal D, Evert M, Horst D, Kreipe H, Kirchner T, Wardelmann E, Lassen U, Büttner R, Weichert W, Dietel M, Schirmacher P, Stenzinger A. Testing NTRK testing: Wet-lab and in silico comparison of RNA-based targeted sequencing assays. Genes Chromosomes Cancer. 2020 Mar;59(3):178-188.
5. Park HJ, Baek I, Cheang G, Solomon JP, Song W. Comparison of RNA-Based Next-Generation Sequencing Assays for the Detection of NTRK Gene Fusions. J Mol Diagn. 2021; 23(11):1443-1451.