Question: Why does my Fixed RNA Profiling library look unexpected or have low yield?
Answer: After library construction, Fixed RNA Profiling 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 Fixed RNA Profiling library trace will appear as a single discrete peak with an average library size of ~260 bp.
Representative Fixed RNA Profiling 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.
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 Fixed RNA Profiling 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. FRP 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 Fixed RNA Profiling 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. FRP 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 Fixed RNA Profiling 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). The recommended number of cycles are listed below:
If you've generated unexpected library traces, please reach out to 10x Genomics Technical Support (firstname.lastname@example.org) for guidance.
Product: Fixed RNA Profiling, Single Cell Gene Expression Flex