We are implementing a 2 step PCR for NGS and I have a problem designing overhangs.
The first PCR is a region-specific amplification. For that we want to design primers with overhangs. 1st PCR: Overhang + Primer (region of interest)
The second PCR we want to have the P7 and P5 adaptors and indexes. Also the complementary sequence for the overhangs from the first step. 2nd PCR: P7/P5 + index + Overhang (complementary)
3rd: pooling --> flow cell
I thought the best option for the overhang sequence is to find a non-human sequence, that way the oligo wont bing to human DNA. However, how can I do this,
Anyone has some experience with a similar situation?, any advice?