Quantifying ultra-rare pre-leukemic clones via targeted error-corrected sequencing.

The quantification of rare clonal and subclonal populations from a heterogeneous DNA sample has multiple clinical and research applications for the study and treatment of leukemia. Specifically, in the hematopoietic compartment, recent reports demonstrate the presence of subclonal variation in normal and malignant hematopoiesis,1,2 and leukemia is now recognized as an oligoclonal disease.3 Currently, clonal heterogeneity in leukemia is studied using next-generation sequencing (NGS) targeting subclone-specific mutations. With this method, detecting mutations at 2–5% variant allele fraction (VAF) requires costly and time-intensive deep resequencing and identifying lower frequency variants is impractical regardless of sequencing depth. Recently, various methods have been developed to circumvent the error rate of NGS.4, 5 These methods tag individual DNA molecules with unique oligonucleotide indexes, which enable error correction after sequencing.