Troubleshooting Guides


Assay Performance

User Guide – Ion AmpliSeq Library Preparation is available on Ion Community.

For use with:

  • Ion AmpliSeq Library Kits 2.0
  • Ion AmpliSeq Custom Primer Pools
  • Ion AmpliSeq Ready-to-Use Panels
  • Ion AmpliSeq Sample ID Panel

Below is a table that can help determine probable cause and recommended action for performance observation.

Bias in amplicon representation
Observation Possible cause Recommended action
Loss of short amplicons Poor purification Vortex AMPure® XP Reagent thoroughly before use, and be sure to dispense the full volume
In unamplified library purification (“Purify the unamplified library” on page 17), increase AMPure® XP Reagent volume from 1.5X to 1.7X
In amplified library purification (“Purify the amplified library” on page 20), increase AMPure® XP Reagent volume in second round from 1.2X to 1.4X
Denaturation of digested amplicon Use the 60°C/20 minute temperature incubation during the primer digestion step (“Partially digest primer sequences” on page 15)
Loss of long amplicons Inappropriate primer design Use an FFPE assay design for degraded or low quality samples
Inefficient PCR Use the 8-minute anneal and extend step for target amplification (“Amplify targets” on page 14)
Too few nucleotide flows Use an appropriate number of flows on the Ion PGM Sequencer
Loss of AT-rich amplicons Denaturation of digested amplicon Use the 60°C/20 minute temperature incubation during the primer digestion step
Unknown Amplicons with >80% AT often exhibit low representation
Loss of GC-rich amplicons Inadequate denaturation Use a calibrated thermal cycler
Inefficient library amplification Do not amplify the library (not required for qPCR quantification)
Unknown Amplicons with >80% GC often exhibit low representation