Sample Type 2: Various percentages (20%, 10%, 5%, 2%, 1%) of KRAS mutant cells mixed with wild-type cells as well as 100% wild-type and mutant BRAF cells were formaldehyde fixed and paraffin embedded (FFPE) to create a cell block and sections (AcroMetrix, Benicia, CA, USA) from which gDNA was extracted.

Sample preparation

BRAF FFPE sample gDNA was extracted from each section using the MagMAX FFPE DNA Isolation Kit (Ambion, Austin, TX, USA). All other total gDNA was extracted using RecoverAll (Ambion) according to the manufacturer's instructions. The gDNA then was quantified by OD 260 nm using the NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and the Quantifiler Human DNA Quantification Kit (Applied Biosystems, Foster City, CA, USA) using the 7500 Real-Time PCR System (Applied Biosystems).

Type 1 samples were prepared by diluting gDNA from KRAS mutant cell-lines into wild-type KRAS cell-line (HT-29) to a final concentration of 100 ng/µL.

Fluorescent dye terminator Sanger sequencing

PCR primer pairs (5′-tgtaaaacgacggccagtTATTTGATAGTGTATTAACCTTATGTGTG-3′ and 5′-caggaaacagctatgaccGAAACCTTTATCTGTATCAAAGAATG-3′) and (5′-tgtaaaacgacggccagtGCTTGCTCTGATAGGAAAATGAGATC-3′ and 5′-caggaaacagctatgaccATCCAGACAACTGTTCAAACTGATG-3′) with M13 tails (lower case letters) were used respectively to amplify exon 2 of the KRAS gene and exon 15 of the BRAF gene. Each PCR reaction contained 1× AmpliTaq Gold Fast PCR Master Mix UP (Applied Biosystems, Foster City, CA, USA), 10 µM primers, and 100 ng/µL of template gDNA in 30 µL final volume. Cycling conditions performed on the Veriti Thermal Cycler (Applied Biosystems, Foster City, CA, USA) were: 95°C 10 min, 35× (96°C-3 s, 62°C-3 s, 68°C-15 s), 72°C 10 s. Following amplification, the reaction was treated with ExoSAP-IT enzyme (USB Corporation, Cleveland, OH, USA) to remove unincorporated primers and dNTPs. Cycle sequencing was performed on the Veriti Thermal Cycler with reaction containing 1× BigDye Terminator Cycle Sequencing Kit v1.1 (Applied Biosystems, Foster City, CA, USA), 3.2 µM M13 primer, and 2 µL of PCR product in a 10 µL final volume. Cycling conditions were: 96°C-1 min, 25× (96°C-10 s, 50°C-5 s, 60°C-75 s), 72°C-10 s. Cycle sequencing reaction products were purified using Centri-Sep spin columns (Applied Biosystems). Purified reactions were vacuum concentrated and the samples reconstituted with 10 µL of Hi-Di Formamide (Applied Biosystems). Capillary electrophoresis was performed using the 3500xL Genetic Analyzer capillary electrophoresis system (Applied Biosystems) with POP-7 polymer and a 50cm array length. The instrument protocol used was the RapidSeq50_POP7 run module in combination with the E dye set. Sequences were aligned to a reference sequence using Variant Reporter Software v1.1 (Applied Biosystems) and variants assessed by visual inspection.

Fragment analysis of Sanger sequencing (FASS) reactions

For FASS reactions, 0.5 µL of GeneScan-600LIZ v2.0 size standard (Applied Biosystems) was added to purified cycle sequencing reaction products following collection of sequencing sample files (.ab1). Capillary electrophoresis was performed using the 3500xL Genetic Analyzer capillary electrophoresis system with POP-7 polymer and a 50cm array length. The instrument protocol used was the FragmentAnalysis50_POP7 run module in combination with the E5 dye set. GeneMapper v4.1 Software (Applied Biosystems) was used for sizing of fragments and genotyping.

Shifted termination assay

PCR amplification and STA reactions were performed on the 9700 Thermal Cycler (Applied Biosystems). Samples were assessed using the KRAS Mutational Analysis Reagents (Applied Biosystems) according to the manufacturer's protocol. Capillary electrophoresis was performed using the 3500xL Genetic Analyzer capillary electrophoresis system with POP-7 polymer and a 50cm array length. The electrophoresis conditions for c.35G>T (p.Gly12Val) variants used POP-6 polymer and a 50cm array length on the 3500xL Genetic Analyzer. GeneMapper v4.1 Software was used for sizing of fragments and genotyping.

Single-base extension

Single-base extension reaction contained: 1X SNaPshot kit (Applied Biosystems), 0.2 µM of multiplexed primers (KRASc34-5′-AACTTGTGGTAGTTGGAGCT-3′, KRASc35-MM-5′-ACTTGTG-GTAGTTGGAGCTG-3′, KRASc37-MM-5′-GTGGTAGTT-GGAGCTGGT-3′, KRASc38-MM-5′-GTGGTAGTTGGAGCTGGTG-3′ (MM = mobility modified)), and 3 µL of PCR product (from FTSS reaction) in a 10 µL final volume. After treatment with 2 U of shrimp alkaline phosphatase (USB Corporation), 0.5 µL of SBE products was combined with 9 µL of Hi-Di Formamide and 0.5 µL of GeneScan-120LIZ size standard (Applied Biosystems). Following denaturation at 95°C for 2 min, capillary electrophoresis was performed using the 3500xL Genetic Analyzer capillary electrophoresis system with POP-7 polymer and a 50cm array length. GeneMapper v4.1 Software was used for sizing of fragments and genotyping.