Single-copy detection of somatic variants from solid and liquid biopsy

Abstract

Accurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

Ultra-sensitive molecular detection of gene fusions from RNA using ASPYRE

“…Herein we describe a method based on pyrophosphorolysis to include detection of gene fusions from RNA, with identical assay steps and conditions to detect somatic mutations in DNA [ 1 ], permitting concurrent assessment of DNA and RNA in a single instrument run.…”

“…The stages were described previously for DNA: 1) PCR, 2) enzymatic cleanup, 3) exonuclease digestion, hybridisation, pyrophosphorolysis (PPL), and ligation; and 4) rolling circle isothermal amplification (RCA; Supplementary Figs. 2 and [ 1 ]). To adapt this method to incorporate RNA as analyte, we added a reverse transcriptase (RT) step to the initial PCR, and created a pool of primers to amplify 37 gene fusions in which the 3’ partners are ALK exon 20, ROS1 exons 32, 34, 35, and 36, RET exons 8, 11 and 12, MET exon 14 skipping, NTRK1 exon 10, NTRK2 exon 14, and NTRK3 exons 14 or 15 (Supplementary Table 1).…”

“…The PCR mastermix from [ 1 ] was modified to perform RT-PCR in 6 µL of mixture per reaction by the addition of 1x Luna WarmStart reverse transcriptase (E3006, NEB) and the reaction performed in 1x Q5U buffer (M0515, NEB) with 2 mM MgSO 4 (NEB), and 6 µL of RNA target. The thermocycling conditions used were 37 ˚C 1 min; 55 ˚C 10 min; 98 ˚C 1 min; 50 cycles of 98 ˚C 10 s, 63 ˚C 15 s, 72 ˚C 15 s; and 72 ˚C 5 min.…”

“…The thermocycling conditions used were 37 ˚C 1 min; 55 ˚C 10 min; 98 ˚C 1 min; 50 cycles of 98 ˚C 10 s, 63 ˚C 15 s, 72 ˚C 15 s; and 72 ˚C 5 min. Proteinase K digestion and pyrophosphorolysis were performed essentially as described previously [ 1 ]. For the RCA, 10 µL of detection mixture per reaction was prepared by mixing 1x RCA buffer (51.8 mM Tris-HCl pH 8.8, 27.6 mM NH 4 SO 4 , 3.72 mM KCl, 3.49 mM MgSO 4 , 0.0567% Tween-20), 300 U/mL Bst 3.0 WarmStart (M0374, NEB), 0.8 mM dNTPs with dUTP (Promega), 0.3% polydimethylsiloxane emulsifier (A5757, Sigma Aldrich), and 0.253 µM primer mix.…”

“…Previously, we demonstrated that detection of a panel of somatic variants in DNA from tumor tissue was possible down to single-molecule levels of sensitivity by using a simple, fast and low-cost method incorporating Allele-Specific PYrophosphorolysis REaction (ASPYRE) [ 1 ]. Herein we show further capabilities of the ASPYRE technology by detecting gene fusions from RNA, showing single-molecule detection limits equivalent to DNA, scaling to over 30 fusion targets from one initial RT-PCR amplification reaction.…”