Optimizing the BESTSeq protocol for clinical laboratory testing
When cancer is identified early, patient survival rates are dramatically higher. Current cancer diagnostics such as imaging, biopsy, and general screening tests are powerful, but there’s a clinical need for an early detection test for asymptomatic individuals. TGen developed the BESTSeq protocol for this purpose. BESTSeq uses a minimal amount of blood to detect fragmentation in cell-free DNA (cfDNA). As clinical testing begins, there are still some procedural questions. The first is how many times can plasma be thawed before sacrificing DNA quality. To test this, blood plasma was evaluated at three conditions: thawed once, twice, and three times. DNA quality was highest when the plasma was thawed a minimal amount of times. Next, we tested the amount of magnetic beads to add to plasma to maximize the amount of cfDNA collected. This was done by adding various amounts of beads to five plasma samples. We determined that in order to maximize the amount of cfDNA in the plasma extraction step, at least 7.5 microliters should be added. Finally, the effect of primer storage on BESTSeq was assessed. Using primers pooled at various points, we looked for signs of primer dimerization as a measure of degradation. Primers that were pooled over a year ago showed signs of dimerization which can impact BESTSeq results. These results will give important guidelines when BESTSeq is transitioned into a clinical laboratory environment and are crucial to creating an accurate and reproducible test that can determine whether a patient has an actively growing tumor.
