Comparison of next-generation sequencing and mutation-specific platforms in clinical practice.
Author(s): Hinrichs JW, Marja van Blokland WT, Moons MJ, Radersma RD, Radersma-van Loon JH, de Voijs CM, Rappel SB, Koudijs MJ, Besselink NJ, Willems SM, de Weger RA
Publication: Am J Clin Pathol, 2015, Vol. 143, Page 573-8
PubMed ID: 25780010 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare mutation analysis results of formalin-fixed paraffin-embedded (FFPE) lung tumors obtained using 2 real-time PCR based methods and 2 next-generation sequencing (NGS) platforms with those obtained by high resolution melting followed by Sanger sequencing.
Conclusion of Paper
All 14 Kirsten rat sarcoma viral oncogene homolog (KRAS) and 7 epidermal growth factor receptor (EGFR) mutations identified using high-resolution melting followed by Sanger sequencing were also identified using next generation sequencing using the Ion Torrent on the Personal Genome Machine (NGS-IonT), but 4 KRAS mutations failed to sequence using the 454 Genome Sequencer junior (NGS-454), 2 EGFR mutations were not included in the real-time PCR based COBAS z 480 (COBAS) and one of these EGFR mutations was not included in the real-time PCR based Rotor-Gene (RotorG). Interestingly, NGS-IonT, RotorG, and COBAS identified 1 specimen with a mutation in EGFR and another with a mutation in KRAS that were not found by high-resolution melting followed by Sanger sequencing. COBAS classified one additional specimen as containing a KRAS mutation, but this was not found using any other platform.
Studies
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Study Purpose
The purpose of this study was to compare mutation analysis results of 25 FFPE lung tumors using 2 real-time PCR based methods (RotorG and COBAS) and 2 NGS platforms (NGS-454 and NGS-IonT) with those obtained using high resolution melting followed by Sanger sequencing. DNA was extracted from sections of twenty-five FFPE non-small cell lung carcinoma (NSCLC) specimens using a modification of the DNeasy tissue kit or the COBAS DNA sample preparation kit (for COBAS analysis only). High-resolution melting combined with Sanger sequencing served as the reference method.
Summary of Findings:
All 14 KRAS mutations identified using high-resolution melting followed by Sanger sequencing were also identified using the real-time PCR based COBAS and RotorG methods and by next generation sequencing using the NGS-IonT, but 4 of these samples failed to sequence using NGS-454. 9 of the 11 specimens identified as KRAS wild-type using high-resolution melting followed by Sanger sequencing were determined to be wild-type on all platforms. One of the 2 specimens with discrepant KRAS results was determined to be pG12X/p.G13X using COBAS, but wild-type using the other 3 platforms and the other was found to be p.G12V using NGS-IonT and RotorG, pG12X/p.G13X using COBAS and failed to sequence using NGS-454. All 7 EGFR mutations identified using high-resolution melting followed by Sanger sequencing were also found using NGS-454 and NGS-IonT, but 1 was not included in the COBAS analysis, and another was not included in the RotorG or COBAS analysis resulting in a wild-type classification. 18 specimens were classified as EGFR wild-type by Sanger sequencing. Of these, 4 failed using NGS-454 and 1 was classified as p.G719A using NGS-IonT, COBAS and RotorG.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA SNP assay DNA Next generation sequencing DNA DNA sequencing DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) SNP assay Specific Technology platform High resolution melting with Sanger sequencing
COBAS z 480 analyzer
Rotor-Gene Q
454 Genome Sequencer junior
Ion Torrent Personal Genome Machine
SNP assay Specific Targeted nucleic acid KRAS exons 2 and 3
EGFR exons 19, 20 and 21