Evaluation of droplet digital PCR and next generation sequencing for characterizing DNA reference material for KRAS mutation detection

Lianhua Dong,Jing Wang,Shangjun Wang,Boqiang Fu

doi:10.1038/s41598-018-27368-3

Abstract

KRAS gene mutations are predictive markers of non-response to anti-epidermal growth factor receptor. An increasing number of techniques are being developed to detect KRAS mutations. To obtain consistent and comparable results, a traceable reference material (RM) is necessary for validation the routinely used method. However, a lack of reference methods is a main impediment for deriving traceability and measurement comparability. In this study, droplet digital PCR (ddPCR) and next generation sequencing (NGS) were evaluated. No cross- reactivity was detected with any of the probe by ddPCR. The measured fraction of KRAS mutant allele by ddPCR and NGS agreed with the prepared value by gravimetrical dilution (concordance (k) >0.95 and >0.93 for ddPCR and NGS, respectively). The reliable limit of quantification (LOQ) was 0.1% and 1% for ddPCR and NGS, respectively. In conclusion, the validated ddPCR and NGS are suitable to characterize the KRAS RM due to the high specificity and accuracy. Verification of the LOD of three commercial kits by using the NIM-KRAS-8 RM showed that the LOD was inconsistent with the claimed LOD of the kits (1%) for some assays. This indicates a traceable RM was important for setting up the criteria regarding the LOD for the commercial kit.

Highlights

KRAS (v-Ki-ras[2] Kirsten rat sarcoma viral oncogene homolog) gene mutations are predictive markers of non response to anti-epidermal growth factor receptor[1,2,3] and valuable for prognosis and treatment of pancreatic cancer and colorectal cancer[4,5]
KRAS mutations are routinely detected by Sanger sequencing, generation sequencing (NGS), ARMS-PCR, mutant-enriched PCR, COLD-PCR and digital PCR
A Quantifiler Human DNA Quantification kit (Thermo Scientific) was used on the QX100 to quantify a single copy target of the human telomerase reverse transcriptase gene[26] which can serve as a reference single copy gene

Summary

Introduction

KRAS (v-Ki-ras[2] Kirsten rat sarcoma viral oncogene homolog) gene mutations are predictive markers of non response to anti-epidermal growth factor receptor (anti-EGFR)[1,2,3] and valuable for prognosis and treatment of pancreatic cancer and colorectal cancer[4,5]. The present study was conducted (1) to establish a highly accurate KRAS allele frequency measurement; (2) develop a traceable KRAS mutant reference material characterized with the established method to validate KRAS mutation detection kits. The results of Sanger sequencing confirmed that each cell line carries its specific target KRAS mutation (Table S3 in the supplemental material).

Results

Conclusion