TY - JOUR
T1 - Clinical validation of a next-generation sequencing screen for mutational hotspots in 46 cancer-related genes
AU - Singh, Rajesh R.
AU - Patel, Keyur P.
AU - Routbort, Mark J.
AU - Reddy, Neelima G.
AU - Barkoh, Bedia A.
AU - Handal, Brian
AU - Kanagal-Shamanna, Rashmi
AU - Greaves, Wesley O.
AU - Medeiros, L. Jeffrey
AU - Aldape, Kenneth D.
AU - Luthra, Rajyalakshmi
PY - 2013/9
Y1 - 2013/9
N2 - Transfer of next-generation sequencing technology to a Clinical Laboratory Improvement Amendments-certified laboratory requires vigorous validation. Herein, we validated a next-generation sequencing screen interrogating 740 mutational hotspots in 46 cancer-related genes using the Ion Torrent AmpliSeq cancer panel and Ion Torrent Personal Genome Machine (IT-PGM). Ten nanograms of FFPE DNA was used as template to amplify mutation hotspot regions of 46 genes in 70 solid tumor samples, including 22 archival specimens with known mutations and 48 specimens sequenced in parallel with alternate sequencing platforms. In the archival specimens, the IT-PGM detected expected nucleotide substitutions (n = 29) and four of six insertions/deletions; in parallel, 66 variants were detected. These variants, except a single nucleotide substitution, were confirmed by alternate platforms. Repeated sequencing of progressively diluted DNA from two cancer cell lines with known mutations demonstrated reliable sensitivity at 10% variant frequency for single nucleotide variants with high intrarun and inter-run reproducibility. Manual library preparation yielded relatively superior sequencing performance compared with the automated Ion Torrent OneTouch system. Overall, the IT-PGM platform with the ability to multiplex and simultaneously sequence multiple patient samples using low amounts of FFPE DNA was specific and sensitive for single nucleotide variant mutation analysis and can be incorporated easily into the clinical laboratory for routine testing.
AB - Transfer of next-generation sequencing technology to a Clinical Laboratory Improvement Amendments-certified laboratory requires vigorous validation. Herein, we validated a next-generation sequencing screen interrogating 740 mutational hotspots in 46 cancer-related genes using the Ion Torrent AmpliSeq cancer panel and Ion Torrent Personal Genome Machine (IT-PGM). Ten nanograms of FFPE DNA was used as template to amplify mutation hotspot regions of 46 genes in 70 solid tumor samples, including 22 archival specimens with known mutations and 48 specimens sequenced in parallel with alternate sequencing platforms. In the archival specimens, the IT-PGM detected expected nucleotide substitutions (n = 29) and four of six insertions/deletions; in parallel, 66 variants were detected. These variants, except a single nucleotide substitution, were confirmed by alternate platforms. Repeated sequencing of progressively diluted DNA from two cancer cell lines with known mutations demonstrated reliable sensitivity at 10% variant frequency for single nucleotide variants with high intrarun and inter-run reproducibility. Manual library preparation yielded relatively superior sequencing performance compared with the automated Ion Torrent OneTouch system. Overall, the IT-PGM platform with the ability to multiplex and simultaneously sequence multiple patient samples using low amounts of FFPE DNA was specific and sensitive for single nucleotide variant mutation analysis and can be incorporated easily into the clinical laboratory for routine testing.
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U2 - 10.1016/j.jmoldx.2013.05.003
DO - 10.1016/j.jmoldx.2013.05.003
M3 - Article
C2 - 23810757
AN - SCOPUS:84882289495
SN - 1525-1578
VL - 15
SP - 607
EP - 622
JO - Journal of Molecular Diagnostics
JF - Journal of Molecular Diagnostics
IS - 5
ER -