Introduction: Tumor mutation detection experiments are increasingly moving from the
characterization of solid primary tumor biopsies to fluid samples like blood and urine and the tracking of dynamic changes of the mutational profile over time. These sample types require the addition of tumor DNA enrichment steps and modifications of the molecular and data analysis protocols be able to detect rare variants. For circulating tumor cell samples, the processing must also function for low copy inputs, as sometimes only a few cells (~5 cells) are recovered from a blood draw. Here we present validation of the IsoFlux workflow, a method to detect somatic mutations from a blood draw. Typical tumor cell enrichment above 10% of total cell numbers allows the use of standard amplicon libraries typically employed in the analysis of tissue-based biopsies.
Results: Multisite analytical validation data, based on spiking of cells into whole blood,
and a matched molecular and bioinformatics approach demonstrates a detection limit
down to 10 cells from a blood draw with a very low false positive rate. This translates into robust detection of variants down to 0.5% mutant allele frequency, without the use of nonstandard NGS techniques like single molecule sequencing. Results are shown for a number of analytical validation experiments, detecting variants down to 2 mutated copies in a background of wild type cells.
Exemplary clinical data is presented from a few patients, including biological replicates,
and analysis replicates using different amplicon sets and sequencing technologies to
validate the results.