Appendix A. Tips and Troubleshooting - DriverMap™ AIR TCR-BCR Profiling Kit (RNA) - v1

Observation	Possible Cause	Recommended Action
Yield of PCR products for DNA samples is low, but is good for Positive Control DNA sample — or — Low yield of PCR products for both experimental and Positive Control DNA samples	Error in quantitation of input DNA, or DNA is degraded	Re-analyze the amount and quality of input DNA using Agilent Bioanalyzer, Agilent Fragment Analyzer, or Thermo Fisher NanoDrop.
	DNA input is too low	Add more DNA, or increase target amplification cycles (Refer to PCR with Anchor Primers for cycle numbers recommended based on input amounts).
	RT step is not optimal	Please check for procedural mistakes. Make master mixes where possible. For individual reaction setups, make sure that the correct volume of RT-EXT Buffer and RT enzyme is added into each reaction.
	PCR cycling conditions are not optimal	Increase PCR amplification cycles. Ensure proper dispensing and mixing of viscous components at each step.

Observation	Possible Cause	Recommended Action
Low Cluster Density.	Inefficient purification from low molecular weight DNA products or low yield of PCR products	Re-purify PCR products using different technology (e.g. QIAGEN QIAquick PCR Purification Kit), and/or increase the concentration of pooled PCR products in the cluster generation step.

Observation	Possible Cause	Recommended Action
Yields of PCR products are significantly higher than Positive Control	Incorrect quantitation of input DNA amount	Re-analyze the amount and quality of input DNA using Agilent Bioanalyzer, Agilent Fragment Analyzer, or Thermo Fisher NanoDrop.
	DNA input is too high	Add less DNA or decrease number of PCR cycles in the PCR with Indexed Primers step.
Low fluorescence readings on Illumina sequencer — or — Lower than expected number of on-target reads — or — High mutation rate in the Index reads	PCR primers from amplified indexed library quantification step (Quantify and Combine Samples for NGS) were not removed	Check that you added Primer Removal Master Mix in the Quantify and Combine Samples for NGS step. If it had been added, use a new vial of Primer Removal Reagent and repeat.
Lower than expected number of on-target reads	DNA input too low or PCR cycle number too low	Add more DNA or increase number of PCR cycles in target amplification step (PCR with Anchor Primers).
Lower than expected number of on-target reads	DNA is degraded	Use highest quality DNA possible. For degraded DNA, increase the number of PCR cycles.
Uneven representation of Indexed Libraries	Inaccurate Indexed Library quantification	Check that you correctly calculate the molar concentration of each indexed library sample.
Uneven representation of Indexed Libraries	Inaccurate Indexed Library mixing	Re-quantify the indexed library samples and mix them in equimolar amounts.
Inconsistent library yields from replicate DNA samples	Sample evaporation in thermal cycler	Seal 96-well plates well with an Adhesive Film Applicator and use a Compression Pad. Fill empty wells with water to minimize evaporation from experimental samples.

Last modified: 14 February 2023

9.2. Appendix B. Structure of Amplified Indexed Library

Need more help with this?
Contact Us