Reference materials and clinical samples
The following genomic DNA reference materials carrying specific mutations were obtained from ATCC and Horizon Discovery Group plc respectively: CTNNB1 S45 (HCT116), APC E1309 (LS1034), APC Q1367 (C2BBe1), APC R1450 (SW837 ), KRAS G12 (Horizon Cat#: HD272), KRAS G13 (Horizon Cat#: HD290), BRAF V600 (Horizon Cat#: HD238). For target mutations which no commercial reference materials were available, APC, CTNNB1 synthetic DNA templates from Integrated DNA Technologies Inc were used. Reference cfDNA standards APC R1450, CTNNB1 T41, KRAS G12 and BRAF V600E were purchased from SeraCare Inc. For reference cfDNA standards that are not available commercially, genomic DNAs carrying APC E1309/Q1367, CTNNB1 S45 and KRAS G13 mutations were sheared by sonication with M220 Focused-ultrasonicator (Covaris Inc). The sonicated DNAs were analyzed on BioAnalyzer (Agilent) to give an average DNA fragment length of about 150 bp which mimics the size of cfDNA fragments justifying their use as cfDNA references.
Most of FFPE and plasma (cfDNA) clinical samples with CRC used for this study were collected from Chinese patients (Second Affiliated Hospital of Zhejiang University, Hangzhou and Jiangsu Cancer Hospital, Nanjing, China). The ethics approval was awarded by Ethics Committee of the Second Affiliated Hospital of Zhejiang University, Hangzhou, China; and Ethics Committee of Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research, Nanjing, China). All subjects provided written informed consent. 10 mL blood were drawn from each patient and stored in cfDNA BCT Streck tubes.
DNA from FFPE samples was extracted with the QIAamp DSP DNA FFPE Tissue Kit (Catalog, Qiagen, REF 60604. QIAGEN GmbH, Hilden, Germany) following manufacturer’s instructions. For cfDNA isolation, the collected blood was first spun at 1600xg on a table centrifuge (Sorvall ST16R, Thermo Fisher Scientific) for 10 minutes at room temperature. The supernatant above the interface phase was carefully taken and spun at 16,000xg for 10 minutes at room temperature. The final plasma supernatant was stored at -20 °C until use. cfDNAs were isolated from plasma by using QIAamp ® MinElute ccfDNA Midi Kit (QIAGEN, Cat# 55284) following the manufacturer’s instructions. Isolated cfDNA was quantified by using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Cat # Q32851) and also assessed by using the beta-actin qPCR assay (as internal control) to check the quantity and quality.
qPCR Primer and Probe Design
The high sensitivity of this multigene biomarker assay is achieved due to XNA clamp probe technology. XNA oligomers are designed that bind to the selected wild-type sequences at the respective genetic loci in the target genes. For each of the selected mutation sites, primers and TaqMan hydrolysis probes were designed by Primer3 software version 0.4.0. For target gene codons with multiple mutations, e.g. KRAS G12 six mutations, a locus specific probe was designed so that all 6 of KRAS G12 mutations can be detected in one assay using one pair of primers with the same XNA designed for the relevant position in KRAS G12. For target gene codons with single mutations, e.g. APC E1309, APC Q1367 and APC R1450 and APC R876, mutant specific probes or allele specific primers were designed. The human beta actin gene (ACTB) was selected as an internal control for the assay (Supplementary Table 1). The designed primers and probes were analyzed in silico to verify the specificity of the oligos (by GenBank Blast against a whole genome reference DNA), no primer dimers (Auto-Dimer), no amplicon secondary structure (M-fold) before synthesis. All primers were synthesized by IDT (Integrated DNA Technology) and probes were ordered from BioSearch Inc.
XNA oligomers were synthesized in house or ordered from CPC Scientific Inc.
ColoScape TM Assay
The assay consists of 10 µl of reaction volume including 5 ul of 2X buffer (Bioline, Bio-11060), 2 ul of Primer/probe mix in 1xTE with final concentration of 100 nM-600 nM of primers and 50 nM – 500 nM of probes, 1 ul of XNA final concentration from 0.125 µM to 1 µM and 2 µl of template (nuclease free water for non-template control or 5–10 ng DNA). Non-template controls (NTC), Clamping controls (CC, human wildtype gDNA) and positive controls (PC, include each mutant DNA) were included in each run. The thermocycling profile is as follows: 95 °C for 2 minutes followed by 50 cycles of 95 °C for 20 seconds, 74 °C for 40 seconds, 62 °C for 30 seconds and 72 °C for 30 seconds. The assay consists of three multiplex qPCR reactions with XNAs to simultaneously detect all the indicated mutations (Supplementary Table 2).
The mutational status of a sample was determined by calculating the Cq value between amplification reactions for a mutant allele assay and an internal control assay. Cq difference (ΔCq) = Mutation Assay Cq - Internal Control Assay Cq. The cut-off values were experimentally determined as its ΔCq value by testing at least 20 wildtype gDNA and/or cfDNA repeatedly during the verification of assay performance. Cut-off ΔCq is calculated as ΔCq cut-off = ΔCq Cq average – 1.96*SD (at 99% CI). If the sample ΔCq is ≤ cut-off value, the mutation is detected as positive. If the sample ΔCq is > cut-off value, the mutation is not detected.
Performance parameters of the assay
Performance parameters of the assay were established on DNA samples extracted from FFPE and plasma of CRC patients as well as reference materials. Assay performance characteristics were verified with respect to precision, limit of detection, specificity and cross-reactivity as well as clinical sample validation and comparison with Sanger Sequence or NGS.
We calculated the sensitivity, specificity, and Area Under Curve (AUC) of each group by sklearn.metrics 40. ROC curves were then plotted by Matplotlib. Pyplot 41.
ROC curve and the area under the curve (AUC) were used to describe the assay performance.