Question: Why are different index plates required for different library types?
Answer: Different index plates are required because different Illumina adapter sequences are used for different library types.
For example, these images show the adapters used for the different library types associated with our Dual Index Single Cell 3' Gene Expression and Feature Barcode technology product:
Gene Expression library contains Illumina TruSeq Read 1 and TruSeq Read 2 adapter sequences:
3' CRISPR Screening library contains Illumina Nextera Read 1 and TruSeq Read 2 adapter sequences:
Cell Surface Protein library contains Illumina Nextera Read 1 and TruSeq Read 2 adapter sequences:
Cell Multiplexing library contains Illumina Nextera Read 1 and Nextera Read 2 adapter sequences:
We use different adapter sequences so that each library type can be generated from the same starting cell sample using different sets of primers during the Sample Index PCR step. Using different primer sets for each library type ensures that only fragments specific to that library type will be amplified during the Sample Index PCR step.
The index plates contain primers that anneal to the Illumina adapters. Different index plates containing different primer sequences are needed based on which Illumina adapters are present in the library. For example:
- Dual Index Plate TT Set A - contains Illumina TruSeq Read 1 and TruSeq Read 2 sequences.
- Dual Index Plate NT Set A - contains Illumina Nextera Read 1 and TruSeq Read 2 sequences.
- Dual Index Plate NN Set A - contains Illumina Nextera Read 1 and Nextera Read 2 sequences.
If the incorrect index plate is used, library preparation will fail. Please refer to the appropriate User Guide and use only the specified index plate for your library type.
Products: Single Cell Gene Expression