Preventing overlaps in output PCR products

[tslaird]

I am trying to design primers for a diverse set of viruses. In the BWT_coverage/ folder I am viewing the core_final_maxprimers_set.out file and noticing a lot of the primers designed for a specific cluster also could amplify other clusters. And, in the case that all primers are used on a particular target, would result in overlapping amplicons that create short products. Do you have any suggestions with how to deal with this issue? Are there specific cd-hit or ani thresholds you would recommend to avoid this?

Thanks.

-Tyler-

[joybio]

Thank you for your in-depth use of multiPrime and for raising this critical issue! Regarding primer cross-amplification and short product issues, the root cause remains excessive sequence similarity. We recommend the following integrated approach:
First, we suggest lowering the cd-hit clustering threshold. The default threshold is 80%, but it can be reduced to as low as 65%. This will result in fewer clusters, and combining this with ANI-based merging (integrating smaller clusters into larger ones) can reduce inter-cluster similarity. However, this approach may significantly reduce primer capture rates.
To address this trade-off, we strongly advise using extract_PCR_product.py to screen the final primer set. If overlapping amplicons are detected, iteratively refine the primer pool by removing problematic primers. For critical cases, manual inspection of cross-cluster primer binding sites is recommended to ensure specificity.