A: An incomplete mixing of the pre-mixes is one of the possible explanations. Make sure the reagents are completely thawed and homogeneous prior to use. 

Some other reasons include:

  • Wrong storage of the kit(s), 
  • Insufficient amount of starting input, 
  • Ethanol used at the wrong step or at the wrong concentration,
  • Equipment issue (thermocycler, multi-channel pipette, …), 
  • AMPure beads not equilibrated at room temperature, 
  • Discarding  the supernatant during the dual size, elution with water or IDTE at the wrong step of the protocol. 

If the yield is too low to proceed to the pooling step for a given sample, we recommend re-preparing the libraries.

Pooling various amounts of libraries together will create a read imbalance between samples and potentially cause the CNV detection to fail for some samples.