A pipeline for sensitive, simple, low-cost, cell free DNA based, liquid biopsy

Early detection of cancer relapse is important for guiding adjuvant treatment decisions. Liquid biopsy (LB) of cell free DNA (cfDNA) can detect relapse months before in appears in imaging studies. This can be based on detection the small fraction of tumor somatic DNA variant within the cfDNA. The main challenge in cfDNA-LB is increasing sensitivity and lowering the detection limit. Some recent works showed that testing large panel of mutations (instead of just one) increases sensitivity and enables lower the detection limit.

Here we present a pipeline for designing tumor specific multiplex for liquid biopsy. This pipeline is based on tumor mutational profile driven from commercial, ~500 genes panel that is part of the standard of care in Israel for a number of malignancies. It includes the selection of variants, primer design, Insilco PCR testing against the human genome and reduction of potential primer dimers. We used this pipeline on 25 patients with 2- 25 variations each and present here the method`s applicability to clinical practice. This pipeline is based on ion-torrent platform, it has a relatively lower costs and user-friendly bioinformatics tools can be applied for the analysis. The library preparation process is simple and short which make it feasible in many pathology institute.