Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing

Abstract

Whole exome sequencing (WES) is used to identify mutations in a patient's tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤ 5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. Here we performed ~ 1000 × WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5 and 5% VAF. Each variant was then tested using NuProbe NGSure, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤ 5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.

Figure 1

Somatic variant detection by whole exome sequencing of non-small cell lung cancer tumor samples. (a) Different depths of WES have different costs and LoDs. By incorporating BDA methodology, WES can achieve 0.1% VAF LoD at lower cost. (b) Categorical breakdown of WES detected somatic variants in the fresh-frozen (FF) sample (n = 1719) and the FFPE sample (n = 8201). (c) Number of somatic variants called by WES. All 66 variants with < 5% VAF and in cancer-related genes underwent confirmatory analysis by BDA. Another 160 variants with < 5% VAF but not in cancer-related genes also underwent confirmatory analysis by BDA. (d) VAF histogram and categorical breakdown of somatic variants detected in cancer-related genes.

Figure 2

WES variant confirmation using Blocker Displacement Amplification (BDA). (a) Workflow for BDA confirmation of WES variants. (b) Mechanism for BDA variant enrichment. The blocker preferentially binds to wildtype (WT) DNA sequences and suppresses their PCR amplification, resulting in selective amplification of variants. FP, forward primer. (c) Example 1: WES-called variant confirmed by BDA. (d) Example 2: Locus of interest was identified as wildtype by BDA, indicating that the variant called by WES was absent in the sample. (e) Example 3: Locus of interest showing a peak with an insertion rather than the WES-called substitution, indicating that the variant was misidentified by WES. (f) The amplicon-based NGS result (Integrative Genomics Viewer) for variants in (e) matches the BDA result.

Figure 3

Characterization of WES variant confirmation. (a) The confirmation profile was determined by plotting the WES depths against the VAFs. We tested 94 variants called from the fresh-frozen sample and 132 variants called from the FFPE sample by BDA qPCR/Sanger analysis. Variants were either confirmed (blue) or disconfirmed (red). Variants called in WES but not tested by BDA are shown in grey. (b) BDA confirmation results by VAF distribution. (c) BDA confirmation results by variant functional categories. (d) BDA confirmation results by alteration types.

Figure 4

WES false-negative analysis. BDA qPCR/Sanger analysis was performed for 50 variants on the FFPE sample. By WES, those variants were detected with < 5% VAF in the fresh-frozen sample but not detected in the FFPE sample.