Pyrosequencing

Samples were amplified and sequenced in triplicate using the KRAS Pyro Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's recommendation and the reactions were analyzed on the PyroMark Q24 instrument (Qiagen). The percentage of mutant allele was calculated by the PyroMark Q24 software (Qiagen).

High-resolution melt

Samples were amplified using MeltDoctor HRM Master Mix (Applied Biosystems) according to the manufacturer's recommendation and run on Viia 7 Real-Time PCR System (Applied Biosystems) with default HRM setting and an annealing temperature of 67°C.

Real-time PCR

Samples were PCR amplified using the DxS K-RAS Mutation Kit (Qiagen) according to the manufacturer's recommendation and the reactions were analyzed on the 7500 Real-Time PCR System.

Copy number variation

Copy number variation (CNV) was assessed for samples using 19 pre-designed TaqMan CNV assays (Applied Biosystems) distributed across the KRAS gene and the reactions were analyzed along with wild-type calibrator samples and reference assays (RNaseP and TERT) using the TaqMan Genotyping Master Mix (Applied Biosystems) according to manufacturer's protocol with the reactions analyzed on the 7500 Real-time PCR System.

Results and discussion

The analysis of a Sanger sequencing reactions as fragment analysis reactions (FASS) is possible on Applied Biosystems Genetic Analyzers because of the spectral compatibility of the E dye set and the E5 dye set, used to spectrally calibrate the four fluorophores in the BigDye Terminator v1.1 chemistry and the five fluorophores of the dRhodamine chemistry (DS-02 dye set: dR110, dRGG, dTAMRA, dROX, LIZ), respectively. Applied Biosystems Genetic Analyzers require a spectral calibration, a deconvolution matrix that compensates for fluorophore overlap (reduces raw data from the instrument) in the 4 dye or 5 dye data stored in each sample file. The fifth dye (LIZ) of the E5 dye set is used to label the size standard, which comprises labeled DNA fragments of known sizes that are added to the sample prior to electrophoresis. The genotyping software assigns the defined size values to the appropriate peaks of the size standard and, by means of a size calling algorithm, the sizes of all unknown fragments are determined based on the known sizes of the internal size standard. Use of internal size standard results in precise molecular length determination since the internal size standard fragments and the unknown sequencing reaction fragments undergo exactly the same electrophoretic forces thus compensating for injection-to-injection variation on CE instruments. With precise sizing, it is possible to identify alleles based on bin definitions for a particular nucleotide position of interest. A bin is a defined basepair size range and dye color for a particular allele fragment (Supplementary Figure S1).

To define the bin positions for the wild-type and mutant alleles of the KRAS gene, the appropriate fragments must be visually identified in the raw data by extrapolation from the analyzed (base-called) sequencing electropherogram (Figure 1A). This can be accomplished by opening a sequencing trace file using the free software Sequence Scanner v1.0 (www.lifetechnologies.com) and selecting the analyzed and raw view tab. Note that the fragments in the analyzed sequencing electropherogram will be shifted relative to the raw view because of the base-calling algorithm adjusting fragments due to mobility differences of the dye terminators (Figure 1A, top panel). The sequencing raw view electropherogram pattern can then be located in the plot view of the genotyping software (compare raw view of Figure 1A with top panel of Figure 1B). To analyze bi-directional sequencing reactions, two panel and bin sets were created, one for M13 forward reactions and a second for M13 reverse reactions (Supplementary Figure S1).

Figure 1.  Comparison of sequencing reaction electropherograms derived from the analysis of KRAS wild type and c.38G>A (p.G13D) mutant gDNA mixtures of 50%, 20%, 10%, 5% (mutant/wild type). (Click to enlarge)

Analysis of a sequencing reaction as a fragment file (.fsa) also benefits from the greater user control over peak detection that is possible with genotyping software compared with sequencing software. In addition to automated allele detection by fragment size and dye color, the advanced peak detection algorithm in GeneMapper software allows user control over peak amplitude thresholds that can be set individually for each dye color. This allows the user to determine the peak height at which the software detects peaks of interest but eliminates noise with the software reporting to the user only those peaks with heights that are at least the peak amplitude threshold for that dye. Additional parameters, such as polynomial degree and peak window size, can be used to adjust the sensitivity of peak detection. It is advisable that the above parameters be empirically determined to optimize the peak detection sensitivity for a particular sequencing reaction.