2Greenwood Genetic Center, Greenwood, SC, USA
3Life Technologies, Benicia, CA, USA
4Life Technologies, Austin, TX, USA
Fluorescent dye terminator Sanger sequencing (FTSS), with detection by automated capillary electrophoresis (CE), has long been regarded as the gold standard for variant detection. However, software analysis and base-calling algorithms used to detect mutations were largely optimized for resequencing applications in which different alleles were expected as heterozygous mixtures of 50%. Increasingly, the requirements for variant detection are an analytic sensitivity for minor alleles of <20%, in particular, when assessing the mutational status of heterogeneous tumor samples. Here, we describe a simple modification to the FTSS workflow that improves the limit of detection of cell-line gDNA mixtures from 50%–20% to 5% for G>A transitions and from 50%–5% to 5% for G>C and G>T transversions. In addition, we use two different sample types to compare the limit of detection of sequence variants in codons 12 and 13 of the KRAS gene between Sanger sequencing and other methodologies including shifted termination assay (STA) detection, single-base extension (SBE), pyrosequencing (PS), high- resolution melt (HRM), and real-time PCR (qPCR).
The KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) gene encodes a GTPase binding protein that functions as an intermediary in the RAS-MAPK pathway, one of several signaling cascades downstream of epidermal growth factor receptor (EGFR) activation (1). EGFR signaling pathways are important in the development and progression of several aggressive cancers, with KRAS gene mutations common in pancreatic, lung, and colorectal cancers (2). Activating KRAS mutations, most frequently described in codons 12 and 13, result in a constitutively activated form of KRAS causing EGFR pathway signaling independent of EGFR activation, thus making therapeutic agents that block EGFR, such as cetuximab or panitumumab, ineffective (3, 4). Consequently, anti-EGFR therapies are recommended only for patients with wild-type KRAS status (5, 6). Although not limited to, a typical analysis of KRAS mutational status would be performed on biopsies of tumor tissue that are processed into formaldehyde fixed and paraffin embedded (FFPE) blocks, from which sections are generated and gDNA extracted. As a consequence of the fixation process, the quality of gDNA from these samples is compromised and, additionally, the samples can be highly heterogeneous with a mixture of normal cells as well as both KRAS mutant and non-mutant tumor cells. Therefore, the methods used to determine the mutational status of the KRAS gene must be both sensitive and robust.
A number of platforms and techniques can be used to assess the mutation status of the KRAS gene with fluorescent dye terminator Sanger sequencing (FTSS) being widely acknowledged as the standard for direct detection of sequence variants. However, the software analysis and base-calling algorithms used to detect mixed bases, a heterozygous state at a particular nucleotide, were designed for typical resequencing mutation detection in which different alleles are expected as heterozygous mixtures of 50% and, as a result, do not facilitate sensitive detection of minor alleles from heterogeneous samples.
In this paper, we present a simple method for improving the analytic sensitivity of FTSS reactions. A standard cycle sequencing workflow is followed consisting of PCR amplification, followed by cycle sequencing using the BigDye Terminator v1.1 Cycle Sequencing Kit. The reaction is first analyzed by capillary electrophoresis (CE) and a standard sequencing file (.ab1) is generated. Following collection of the sequencing file, a fluorescently labeled size standard is added to the sequencing reaction. Then, the sequencing reaction is analyzed by CE as a fragment analysis reaction generating a standard fragment file (.fsa). The addition of the size standard allows for the sizing of all the fluorescently labeled sequencing reaction fragments. The sized fragments then can be further analyzed with genotyping software, which allows automated allele calling through the use of bins specifying the expected size and dye of a particular nucleotide fragment. The use of genotyping software allows the user greater control over how the raw data are analyzed including control over the peak detection algorithm, a functionality that is not possible with base-calling analysis of sequencing sample files (.ab1). To benchmark the improvements to CE FTSS, the following detection methods were examined: shifted termination assay (STA) (7), single-base extension (SBE) (8, 9), pyrosequencing (PS) (10), high-resolution melt (HRM) (11), and real-time PCR (qPCR) (12).
This study describes a simple extension of the Sanger sequencing workflow for improving the detection of minor component alleles. The novelty of the approach is the addition of a size standard to the completed sequencing reaction and the collection of sequencing data as a fragment file for analysis using genotyping software. Sizing analysis facilitates detection by separating the minor component alleles in two dimensions (by size and dye color) from fragments corresponding to the major component allele. Further, genotyping software allows user control over peak detection, such as setting a peak height detection threshold so that minor component alleles can be readily distinguished from baseline noise.
Materials and methods
Sample Type 1: Various percentages (50%, 20%, 10%, 5%, 2%, 1%, and 0.5%) of gDNA extracted from KRAS mutant cancer cell-lines were mixed with gDNA extracted from a KRAS wild-type cancer cell line (HT-29). The KRAS mutant cancer cell-lines were: A549 (c.34G>A), MIA-PaCa-2 (c.34G>T), SW1116 (c.35G>C); SW480 (c.35G>T), and HCT116 (c.38G>A).
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.
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 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.
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).
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.
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).