Peptide Nucleic Acid Clamp
The PNA clamp utilised was 6 bases in length and designed to clamp the KRAS wild-type sequence only, preventing amplification while leaving mutant sequences fully able to amplify during PCR. The PNA clamp sequence used was CACCAG.
PCR Primers and Probes
Primers used were those designed for the Ion AmpliSeq™ Cancer Hotspot Panel v2 sequencing panel (Thermo Fisher Scientific, Paisley, UK). The sequences are as follows: KRAS forward primer 5’-CAAAGAATGGTCCTGCACCAGTAATAT-3’ and reverse 5’-AGGCCTGCTGAAAATGACTGAATATAA-3’. For the genomic region under investigation in KRAS, two TaqMan probes were designed to bind to the same genomic location, with one probe specific to the wild-type sequence, 5’-TTGGAGCTGGTGGCGT-3’, and one specific to the G12D mutation, 5’-TGGAGCTGATGGCGT-3’ (Thermo Fisher Scientific, Paisley, UK).
Droplet Digital PCR
All ddPCRs were conducted on a QX200 Droplet Digital PCR System (Bio-Rad, Watford, UK) using the manufacturer’s protocol and reagents. Thermal cycling conditions were as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 30 s, 90°C for 10 min. A no template control and a positive control were included in every assay. Analysis was performed according to manufacturer's instructions on QuantaSoft Software (Bio-Rad, Watford, UK).
Quantitative Real-time PCR was performed on the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific, Paisley, UK). Thermal cycling conditions were as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 30 s. Reactions were conducted in triplicate, including a no template control and positive control. To account for non-detects in the qPCR data, Ct values >35 were replaced with a Ct of 35, which has been previously shown to reduce bias.
Ion Torrent NGS Protocol
Library generation followed the protocol described in the Ion AmpliSeq Library Kit 2.0 User Guide using the Ion AmpliSeq Cancer Hotspot Panel v2 primer pool, comprising 207 amplicons covering approximately 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes. The relevant PNA clamps were added at a concentration of 10 µM per PNA clamp, before the initial multiplex PCR amplification with the primer pool. Two NGS runs were performed on 316v2 chips on an Ion Torrent PGM. These runs consisted of an unmodified run and an NGS run with a KRAS WT PNA clamp. Base calls, PCR duplicate removal and quality control analysis occurred on an Ion Torrent server using tools from the Ion Torrent Suite (v4.0-r76860). Sequencing reads were aligned to the reference human genome 19 (hg19) using the Torrent Mapping Alignment Program (TMAP). Variant calling was performed using the Torrent Server variantCaller plug-in (v18.104.22.168). The experimental protocols and raw data were deposited in ArrayExpress under accession E-MTAB-5718.
DNA Reference Sample
The reference sample used was the “Tru-Q 7 (1.3% Tier) Reference Standard” (Horizon Diagnostics, Cambridge, UK). This pooled DNA contained mutations at a range of frequencies (1.0 to 30.0%), with the majority of ddPCR-verified mutations having an allelic frequency (AF) of 1.3%.
Patient DNA Sample
Blood sample collection from an advanced non-small cell lung cancer patient was conducted in accordance with the Declaration of Helsinki. The study protocol was approved by the relevant Ethics Committee. All patients gave written informed consent prior to participation. Blood was taken by venepuncture into K2-EDTA-containing collection tubes (BD Biosciences, San Jose, CA, USA) and processed for cfDNA isolation as described in Page and colleagues.