Question: Why does my Visium Spatial Gene Expression for FFPE library look unexpected or have a low yield?
Answer: After library construction, Visium Spatial Gene Expression for FFPE libraries are run on the BioAnalyzer, Tapestation, or LabChip at a 1:5 dilution for qualitative analysis. The traces should resemble the overall shape of the electropherograms shown in the User Guide. We expect a good Visium for FFPE library trace will appear as a single discrete peak with an average library size of ~240 bp.
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.
Causes of additional peaks <200 bp:
- Suboptimal Post-Hybridization and/or Post-Ligation Washes (temperature, timing, or number)
- Tissue detachment before Probe Release. See Q&A articles below:
- Inefficient SPRIselect cleanup.
Causes of low library yields or flat libraries when Cq values were low (<20 cycles):
- Denaturation of ligated, extended probes with the incorrect concentration of KOH
- Failure to neutralize eluted Ligation product with 1M Tris-HCl, pH 7.0
- Use of incorrect Sample Index Plate
- Incorrect number of SI-PCR cycles
- Incorrect thermal cycler program (lid and/or block)
- Thermal cycler incompatible with Thermocycler Adaptor or cannot accommodate full reaction volume
- Failure to use SPRIselect best practices
- Additional peaks (<200 bp) indicate the presence of singlet probes or primer dimers. An abundance of these smaller peaks will likely take up a portion of the sequencing reads and result in poorer quality libraries (e.g., lower mapping rates). If the integrated area beneath the off-target peaks is much smaller than the overall integrated area beneath the desired library fragment (~240 bp) and you can tolerate the presence of some adaptor dimers. In that case, the library can be sequenced after KAPA quantification. If the off-target peaks are significant in comparison to the major peak, it may be possible to repeat the SPRIselect cleanup (Step 4.3), so long as the loss of ~40% of the total library yield can be tolerated and repeated with the understanding that a loss in library complexity may be observed.
- If there is no final library due to an incorrect Sample Index Plate, it may be possible to repeat the SI-PCR (Step 4.2) using the Dual Index TS Set A Plate.
- If the final library yield is low due to under-cycling during SI-PCR, the final library could be re-amplified with the understanding that a loss in library complexity may be observed.
If you've generated unexpected library traces, please reach out to 10x Genomics Technical Support (firstname.lastname@example.org) for guidance.
Product: Visium for FFPE