Question: Can Visium for FFPE libraries be pooled with other dual indexed 10x libraries?
Answer: We have tested pooling Visium for FFPE libraries with dual index 3’ v3.1 libraries and saw no impact in library quality when limiting Visium for FFPE libraries to <40% of the total sequencing pool. It would, therefore, be low risk to pool Visium for FFPE libraries with other dual index libraries that have the same sequencing configuration as dual index 3' v3.1 libraries (e.g. Visium for fresh-frozen).
If pooling with other libraries (that require the following configuration: Read 1 - 28 cycles, i5 index - 10 cycles, i7 index - 10 cycles, and Read 2 - 90 cycles), Read 2S will need to be increased to 90 cycles.
If pooling Fixed RNA Profiling libraries (RFP) with probe-based Visium libraries (i.e., Visium for FFPE or CytAssist-enabled applications), please be sure to utilize the proper amounts of PhiX:
- 1% PhiX should be spiked in for Singleplex FRP and probe-based Visium libraries
- 10% PhiX should be spiked in for Multiplex FRP and probe-based Visium libraries
See our Sequencing Metrics & Base Composition of Visium Spatial Gene Expression for FFPE Libraries Technical Note for additional details.
Products: Visium for FFPE, Visium CytAssist for FFPE, Visium CytAssist for FF, Visium CytAssist for FxF, Visium CytAssist Gene and Protein