Quantification of somatic mutation flow across individual cell division events by lineage sequencing.

Mutation data reveal the dynamic equilibrium between DNA damage and repair processes in cells and are indispensable to the understanding of age-related diseases, tumor evolution, and the acquisition of drug resistance. However, available genome-wide methods have a limited ability to resolve rare somatic variants and the relationships between these variants. Here, we present lineage sequencing, a new genome sequencing approach that enables somatic event reconstruction by providing quality somatic mutation call sets with resolution as high as the single-cell level in subject lineages. Lineage sequencing entails sampling single cells from a population and sequencing subclonal sample sets derived from these cells such that knowledge of relationships among the cells can be used to jointly call variants across the sample set. This approach integrates data from multiple sequence libraries to support each variant and precisely assigns mutations to lineage segments. We applied lineage sequencing to a human colon cancer cell line with a DNA polymerase epsilon (POLE) proofreading deficiency (HT115) and a human retinal epithelial cell line immortalized by constitutive telomerase expression (RPE1). Cells were cultured under continuous observation to link observed single-cell phenotypes with single-cell mutation data. The high sensitivity, specificity, and resolution of the data provide a unique opportunity for quantitative analysis of variation in mutation rate, spectrum, and correlations among variants. Our data show that mutations arrive with nonuniform probability across sublineages and that DNA lesion dynamics may cause strong correlations between certain mutations.

LNA-based PCR clamping enrichment assay for the identification of KRAS mutations.

INTRODUCTION: KRAS mutations in colon carcinomas are associated with lack of response to anti-EGFR monoclonal antibody treatment. Therefore, patients must undergo genetic testing to be eligible for treatment. Several methods for KRAS mutation analysis exist, but many are not sensitive enough to detect a mutation in samples with low fraction of malignant cells. In the present study, we developed a KRAS mutations detection method that is both simple and sensitive. METHODS: Using a locked nucleic acid (LNA) containing oligonucleotide, we developed a PCR clamping method that preferentially amplifies the mutated over wild type KRAS. We evaluated the sensitivity of this method using serial dilutions of plasmids containing wild-type and mutated KRAS fragments. Additionally, KRAS mutation status was evaluated on 60 archived tissue samples of colon carcinoma, and compared to direct sequencing and high resolution melting (HRM) methods. RESULTS: The PCR clamping method could detect as little as 1% mutated DNA in the sample analyzed. Of the 29 KRAS mutations identified by the PCR clamping method, only 23 (79%) were identified by standard direct sequencing. The results of PCR clamping correlated with HRM results. CONCLUSIONS: LNA based PCR clamping method is a simple and highly sensitive method for the detection of KRAS mutations.