title: Introduction of the hybcell-based compact sequencing technology and comparison to state-of-the-art methodologies for KRAS mutation detection.
abstract: The detection of KRAS mutations in codons 12 and 13 is critical for anti-EGFR therapy strategies; however, only those methodologies with high sensitivity, specificity, and accuracy as well as the best cost and turnaround balance are suitable for routine daily testing. Here we compared the performance of compact sequencing using the novel hybcell technology with 454 next-generation sequencing (454-NGS), Sanger sequencing, and pyrosequencing, using an evaluation panel of 35 specimens. A total of 32 mutations and 10 wild-type cases were reported using 454-NGS as the reference method. Specificity ranged from 100% for Sanger sequencing to 80% for pyrosequencing. Sanger sequencing and hybcell-based compact sequencing achieved a sensitivity of 96%, whereas pyrosequencing had a sensitivity of 88%. Accuracy was 97% for Sanger sequencing, 85% for pyrosequencing, and 94% for hybcell-based compact sequencing. Quantitative results were obtained for 454-NGS and hybcell-based compact sequencing data, resulting in a significant correlation (r = 0.914). Whereas pyrosequencing and Sanger sequencing were not able to detect multiple mutated cell clones within one tumor specimen, 454-NGS and the hybcell-based compact sequencing detected multiple mutations in two specimens. Our comparison shows that the hybcell-based compact sequencing is a valuable alternative to state-of-the-art methodologies used for detection of clinically relevant point mutations.
authors.affiliation: Red Cross Transfusion Service for Upper Austria, Linz, Austria.
authors.forename: Agnes
authors.initials: A
authors.lastname: Zopf
authors.affiliation: VelaLabs, Vienna, Austria.
authors.forename: Roman
authors.initials: R
authors.lastname: Raim
authors.affiliation: Red Cross Transfusion Service for Upper Austria, Linz, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Danzer
authors.affiliation: Red Cross Transfusion Service for Upper Austria, Linz, Austria.
authors.forename: Norbert
authors.initials: N
authors.lastname: Niklas
authors.affiliation: Laboratory of Pathology Dr. Obrist & Dr. Brunnhuber OG, Zams, Austria.
authors.forename: Rita
authors.initials: R
authors.lastname: Spilka
authors.affiliation: Red Cross Transfusion Service for Upper Austria, Linz, Austria.
authors.forename: Johannes
authors.initials: J
authors.lastname: Pröll
authors.affiliation: Red Cross Transfusion Service for Upper Austria, Linz, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Gabriel
authors.affiliation: VelaLabs, Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Nechansky
authors.affiliation: VelaLabs, Vienna, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Roucka
mesh_terms: D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins / Q000235:genetics*
chemical_list: D003062:Codon; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
keywords: Hyborg; KRAS; compact sequencing; hybcell technology; next-generation sequencing