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Improving the limit of detection for Sanger sequencing: A comparison of methodologies for KRAS variant detection
 
Colin J. Davidson1, Emily Zeringer4, Kristen J. Champion2, Marie-Pierre Gauthier1, Fawn Wang1, Jerry Boonyaratanakornkit3, Julie R. Jones2, and Edgar Schreiber1
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Supplementary Material


Table 2.  Summary results for FFPE mixtures of KRAS mutant and wild-type cancer cell-lines (sample type 2). (Click to enlarge)


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For FFPE mixtures, the limit of detection for FTSS was improved for G>T transversion mutations from 20%–10% by visual inspection to 10%–5% when analyzed by the FASS approach. The analytic sensitivity for variant c.34G>T (p.G12C) declined from 5% compared with cell-line gDNA mixtures (sample type 1) to 10% for sample type 2 but was unchanged for variant c.35G>T (p.G12V). The limit of detection and influence of the FFPE sample source was identical for the PS and HRM assays (10% sample type 2 versus 5% sample type 1) for variant c.34G>T (p.G12C). More marked declines were observed for variant c.35G>T (p.G12V) using sample type 2 with the limit of detection for both the PS and HRM assays determined to be 20% versus 5% for sample type 1. The influence of the FFPE sample source did not impact the limit of detection for variant c.34G>T (p.G12C) for SBE while the analytic sensitivity declined for both STA and qPCR assays (2% sample type 2 versus 1% sample type 1). The analytic sensitivity of the qPCR assay was 2% for sample type 2 (compared with 0.5% for sample type 1) for variant c.35G>T (p.G12V), while the limit of detection using sample type 2 for SBE and the STA assays was 1% (compared with 0.5% for sample type 1).

To demonstrate that the FASS approach is amenable to the detection of both simple and complex substitution/missense mutations, FFPE treated cell lines (sample type 2) that are wild-type and with mutations in codon 600 of the BRAF gene were investigated (Figure 2). The FASS method can be applied to the detection of single point mutation, c.1799T>A, as well as double point mutation, c.1798_1799GT>AA. The precise sizing possible on CE instruments due to the addition of an internal size standard facilitates the identification of variant fragments and differentiates the mutant alleles from wild fragments since the mutant and wild type fragments are separated in two dimensions — by size and dye color. Precise sizing coupled with analysis by genotyping software allows reproducible identification of variants by strictly defining a bin (specifies the dye color and size range of <1bp for a fragment) for mutant alleles versus bins for the identification of wild-type alleles. The dimension of size separation is lost when sequencing reactions are analyzed by sequencing analysis software since the base calling algorithms compensate for mobility differences of the fragments so that minor component variant peaks are represented under the fragment peak of the wild-type allele. Further, unlike genotyping software, sequencing analysis software does not allow user control over peak detection such as setting a peak height detection threshold so that variant fragments from minor component alleles can be readily distinguished from the baseline noise.




Figure 2.  Comparison of sequencing reaction electropherograms derived from the analysis of BRAF wild type, c.1799T>A (p.V600E) and c.1798_1799GT>AA (p.V600K) mutant FFPE-treated cell-line gDNA. (Click to enlarge)


In summary, it is important to consider the influence of the sample (FFPE versus purified gDNA), copy number, and zygosity when determining the limit of detection for a method or assay. Although the relevance of low levels of KRAS mutation is unknown, potential tumor heterogeneity resulting in samples that may contain a low percentage of mutant cells necessitates the need for analytically sensitive and robust methods such as SBE, STA, and qPCR. However, CE-based FTSS has several advantages over the other assays investigated here, in that the technique is not target specific but with proper primer design all nucleotides within a region of interest can be interrogated. Similarly, the sequencing of longer amplicons allows for the detection of rare monoallelic double and triple mutations of value in the cases of tumor heterogeneity. KRAS double mutants have previously been identified, indicating that tumor heterogeneity is possible in certain cancer types (13, 14). Further, the primary PCR amplification of the region of interest can be used as the template for the subsequent FTSS and FASS reactions as well as SBE reactions. Thus, the robust, simple workflow of SBE (with 1% detection level) can be coupled to CE-based FTSS for the identification of unknown mutations, with the analysis as a fragment file increasing the limit of detection and reducing problematic peak under peak detection that can occur when analyzed as a sequencing file (Figure 1). Further, combining the fragment analysis approach with selective amplification methods such as COLD-PCR and ice-COLD PCR would further improve the limit of detection for FTSS and FASS reactions (15, 16).

Acknowledgments

Thomas Ingalls for assistance in growing cell cultures.

Competing interests

C.D., E.Z., M-P. G., F.W., J.B., and E.S. are employees of Life Technologies. Life Technologies products referred to within this publication are currently For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

Correspondence
Address correspondence to Colin Davidson, Life Technologies, 850 Lincoln Centre Drive, Foster City, CA, USA. Email: [email protected]

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