User Guide – Ion AmpliSeq Library Preparation is available in Ion AmpliSeq Library Kit 2.0 User Guide and Ion AmpliSeq Library Plus Kit User Guide.
For use with:
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 | |
