How do I remove high molecular weight products in my library?

Overamplification of the libraries can cause the appearance of high molecular weight (HMW) products. These are likely artifacts caused by hetero duplex bubble products and are more often seen in the protein library compared to the DNA libraries. Repeating the library PCR with fewer cycles (e.g., Protein library: 16 instead of 20 cycles, DNA library: 8 instead of 10 cycles) can reduce the appearance of HMW peaks.

If the bubble product persists, the libraries can be sequenced. qPCR is recommended for quantifying libraries containing bubble products, as intercalating fluorometric quantification may be unreliable (Qubit/BioAnalyzer).

Example: Protein library after 20 cycle Library PCR:

Example: Protein library after 16 cycle Library PCR:

1. Additional information via illumina:

https://support.illumina.com/bulletins/2019/10/bubble-products-in-sequencing-libraries--causes--identification-.html