Question: What are the bait design criteria for 10x pre-designed and custom panels?
Answer: Panels used in the Targeted Gene Expression assay consist of baits that hybridize to library fragments corresponding to genes of interest. The following criteria are used to design baits for 10x pre-designed panels and for custom panels designed using the 10x Custom Panel Designer:
- Baits are 120 nt single stranded DNA oligonucleotides with 5' biotin modifications
- Baits are designed with end-to-end tiling (ie., there is no overlap between adjacent baits)
- Baits are tiled across the entire non-redundant mature transcript space:
- this includes exons (coding regions & UTRs) from all annotated isoforms
- does not include introns
- A small subset of baits are trimmed or removed to avoid:
- Repetitive sequences
- Baits outside reasonable range of melting temperature
Example of bait tiling across exons from all annotated isoforms:
For more information, please refer to the "Design of Baits Sequences for Targeted Gene Expression" section on the Targeted Gene Expression Algorithms Overview page.
Note on full-length tiling:
- Baits are tiled across the entire mature transcript space rather than only the 3' or 5' ends of transcripts.
- Through in-house experiments to compare different bait design strategies, we found that the full-length tiling approach is more robust as it enables the capture of transcript isoforms that have poorly annotated 3' or 5' ends.
- For many genes, multiple isoforms with different 3' or 5' sequences are expressed in tissue-specific patterns. These isoforms may not be fully documented in the gene annotation files (eg. GENCODE) that are used for bait design. These poorly annotated isoforms may not be detected by bait design strategies that target only the 3' or 5' end of annotated isoforms.
Given that baits are tiled across the entire mature transcript, the same bait set can be used for Single Cell 3' and 5' Gene Expression libraries and Visium Spatial Gene Expression libraries for fresh-frozen tissues.
Products: Targeted Gene Expression