I designed a NGS PANEL for human lung cancer related genes using multiplex PCR strategy. This PANEL contains ~6000 PCR primers that have highly specificity across the whole human genome. Now I need to do the last step: Pairwisely detecting secondary structures between any of the two primers and then exclude primers that would form duplex by themselves.
I use the
ntthal program from the
primer3 package. The running speed for
ntthal is relatively fast, only ~0.1s per
sequence. However, there are too many pairwise groups to detect. For 6000 primer sequences, there should be
6000*5999/2 = 18 million group pairs. My script runs for 3days and still not finished.
Besides, it seems difficult to split them and run parallelly, as every two primers should be paired and tested.