Question: Can I pool libraries prepared from different cell types for use in the Targeted Gene Expression assay?
Answer: For the Targeted Gene Expression assay, up to eight Whole Transcriptome Analysis (WTA) libraries can be pooled prior to hybridization. For optimal performance, we recommend pooling WTA libraries prepared from similar cell types. If WTA libraries from very different cell types are pooled, this can lead to unexpected read balance coming off the sequencer:
- Due to differences in gene expression profiles, WTA libraries prepared from very different cell types are likely to have different panel content (the percentage of library fragments corresponding to genes included in the panel).
- When determining the pooling ratios for WTA input libraries, the pooling calculations make adjustments for cell number and desired sequencing depth. However, these calculations assume that all libraries have the same panel content.
- When a WTA library contains more or less panel content relative to the other WTA libraries in the pool, that WTA library will receive more or less, respectively, of the total sequencing reads in the targeted library (the pool becomes unbalanced).
Generally, a difference in the read distribution does not significantly impact targeting performance. Indeed, having more reads assigned to the library with higher panel content is often desirable, as it ensures that a greater proportion of the complexity from that library is recovered. However, in extreme cases, sequencing read imbalance will result in insufficient read depth for some libraries in the pool.
In the example shown below, Library “A” prepared from one cell type has 5% on-target reads (the percentage of library fragments that correspond to genes included in the panel). Library “B”prepared from a different cell type may have 10% on-target reads, due to differences in gene expression profiles between cell types.
If libraries A and B are pooled at equimolar ratios and used as input in the targeted assay, after sequencing the targeted library, Library A is expected to receive 2-fold fewer sequencing reads than Library B, due to different on-target rates in the parent libraries
Products: Targeted Gene Expression