Observation Possible Cause Recommended Action
Yield of PCR products for RNA samples is low, but is good for Positive Control RNA sample
 
— or —
 
Low yield of PCR products for both experimental and Positive Control RNA samples
Error in quantitation of input RNA, or RNA is degraded Re-analyze the amount and quality of input RNA using Agilent Bioanalyzer, AATI Fragment Analyzer, or Thermo Fisher NanoDrop.
RNA input is too low Add more RNA, 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 RNA amount Re-analyze the amount and quality of input RNA using Agilent Bioanalyzer, AATI Fragment Analyzer, or Thermo Fisher NanoDrop.
RNA input is too high Add less RNA 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 RNA input too low or PCR cycle number too low Add more RNA or increase number of PCR cycles in target amplification step (PCR with Anchor Primers).
RNA is degraded Use highest quality RNA possible. For degraded RNA, 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.
Inaccurate Indexed Library mixing Re-quantify the indexed library samples and mix them in equimolar amounts.
Inconsistent library yields from replicate RNA 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: Aug 08, 2019

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