Why does my Flex Gene Expression library look unexpected or have low yield?

Question: Why does my Flex Gene Expression library look unexpected or have low yield?

 

Answer: After library construction, Flex Gene Expression libraries are run on the BioAnalyzer, Tapestation, or LabChip at a 1:80 dilution for qualitative analysis. The traces should resemble the overall shape of the electropherograms shown in the User Guide. We expect a good Flex Gene Expression library trace will appear as a single discrete peak with an average library size of ~260 bp. 

Representative Flex Gene Expression Trace

 

Final library phenotypes and yields are influenced by sample quality and workflow steps. Listed below are some potential root causes of unexpected libraries and some recommendations. 

Potential Causes of additional peaks <200 bp:

• Suboptimal Post-Hybridization and/or Post-Ligation Washes
  • Deviation in the temperature, timing, the number of washes, or excess supernatant carryover
• Low RNA content
• Under cycling
  • Incorrect number of SI-PCR cycles
• Inefficient SPRIselect cleanup. 
  • See: What are the best practices for using SPRIselect beads in 10x assays?

Presence of singlet unligated probes (Additional peak ~190 bp):

An additional peak (<190 bp) may indicate the presence of singlet unligated probes. An abundance of this smaller peak will likely take up a portion of the sequencing reads and result in poorer quality libraries (e.g., lower mapping rates). A drop between the Reads Mapped to Probe Set and Reads mapped Confidently to Probe Set is expected, and it will be roughly proportional to size of the ~190bp peak. Despite the decrease in mapping metrics, the data obtained is still likely usable for downstream analysis.

Representative Flex Gene Expression Trace with Additional Peak

 

This additional peak at ~190 bp  is more likely to occur in specimens with low RNA content. Occasionally, it may also be observed if there is high debris present in the sample, or if the washing technique for the  Post-Hybridization Washes was suboptimal.

Repeating the SPRIselect cleanup during library construction is unlikely to remove this additional peak.

 

High proportion of low molecular weight products (< 200 bp):

A high proportion of low molecular weight products may be caused by primer dimers. Flex Gene Expression libraries should fall within the range of 50 - 200 nM.  If the concentration falls below or above this range, sequencing data quality may be impacted.  Library concentration should not fall below 2 nM. 

 

Representative Flex Gene Expression Trace for Under Cycling

High proportion of higher molecular weight products (> 350 bp):

A high proportion of higher molecular weight products may be the result of library overamplification. Flex Gene Expression libraries should fall within the range of 50 - 200 nM. 

If the concentration is > 200 nM and contains a high proportion of higher molecular weight peaks, we do not recommend proceeding with sequencing as sequencing data quality may be impacted.  Library concentration should not exceed 500 nM.  

 

Representative Flex Gene Expression Trace for Over Cycling

 

Recommendations for Under or Over Cycling:

Sample Index PCR can be repeated with a greater or fewer number of PCR cycles, respectively, using the leftover pre-amplified product from the first PCR. Additional Amp Mix can be obtained from the Fixed RNA Feature Barcode Kit (PN-1000419).  

 

If you've generated unexpected library traces, please reach out to 10x Genomics Technical Support (support@10xgenomics.com) for guidance.

 

Product: Flex Gene Expression