Table 1.
Analysis of mutations observed in clinical tumor specimens via Illumina next generation targeted amplicon sequencing, after PCR amplification using conventional or COLD-PCR approaches. Observed mutational abundances are presented for conventional PCR and COLD-PCR approaches for each tumor specimen and their respective matched normal counterparts.
| AMPLICON-BASED ILLUMINA NEXT GENERATION SEQUENCING | ||||||||
|---|---|---|---|---|---|---|---|---|
|
| ||||||||
| Specimen | Gene | Exon | Protein change | Mutation | Observed mutational abundance in tumor | Observed mutational abundance in paired normal tissue | ||
|
| ||||||||
| Conventional PCR | COLD-PCR | Conventional PCR | COLD-PCR | |||||
| CT2 | TP53 | 5 | p.Arg175Ser | c.523C>A | *Not detected | 28% | Not detected | Not detected |
| CT2 | TP53 | 7 | p.Asn247Ile | c.739A>T | 50% | 62% | Not detected | Not detected |
| CT20 | TP53 | 5 | p.Cys176Phe | c.527G>T | 50% | 87% | Not detected | Not detected |
| CT20 | TP53 | 8 | p.Arg273His | c.818G>A | *1% | 53% | Not detected | Not detected |
| CT20 | TP53 | 9 | p.Pro309Ser | c.925C>T | 3% | 76% | *Not detected | 14% |
| TL6 | TP53 | 8 | p.Cys277Phe | c.830G>T | *2% | 22% | Not detected | Not detected |
| TL8 | TP53 | 8 | p.Glu285X | c.853G>T | *1% | 29% | Not detected | Not detected |
| TL22 | TP53 | 5 | p.Val157Phe | c.469G>T | 15% | 54% | *Not detected | 6% |
| TL22 | TP53 | 5 | p.Arg158Leu | c.473G>T | *5% | 25% | *Not detected | 17% |
| TL22 | TP53 | 5 | p.Cys176Phe | c.527G>T | §*3% | 10% | Not detected | Not detected |
| TL64 | TP53 | 8 | p.Arg273His | c.818G>A | 13% | 69% | Not detected | Not detected |
| TL71 | KRAS | 2 | p.Gly12Cys | c.34G>T | 17% | 84% | Not detected | Not detected |
| TL96 | TP53 | 7 | p.Arg249Ser | c.747G>T | 10% | 49% | Not detected | Not detected |
| TL119 | KRAS | 2 | p.Gly12Phe | c.34_35GG>TT | *1 | 67% | Not detected | Not detected |
| TL119 | TP53 | 7 | p.Gly244Cys | c.730G>T | 21% | 61% | Not detected | Not detected |
| TL121 | TP53 | 8 | p.Arg273His | c.818G>A | *Not detected | 18% | Not detected | Not detected |
| TL121 | TP53 | 7 | p.Gly245Ser | c.733G>A | 6% | 58% | Not detected | Not detected |
| TL135 | TP53 | 6 | p.Val216Met | c.646G>A | 29% | 75% | Not detected | Not detected |
Specimens with an asterisk (*) indicates when a mutation could not be detected, or due to noise, could not be reliably scored in the conventional PCR, yet COLD-PCR clearly presents the mutational event.
§
As denoted, a previously undocumented mutation was detected in TL22 with the increased combined sensitivity of COLD-PCR-NGS