Question: Can single index and dual index libraries be pooled for sequencing?
Answer: While we recommend sequencing single and dual index libraries separately where possible, it may be feasible to optimize sequencing these libraries together.
Pooling single and dual indexed libraries for sequencing can introduce additional complexity due to varying read configurations and is not supported by Illumina. However, we have not encountered any technical issues when performing in-house sequencing of pooled single index and dual index 3’ v3.1 Single Cell Gene Expression libraries on NovaSeq 6000 and NextSeq 500 instruments. Further details regarding our in-house tests are included below.
That being said, the performance of mixed pools of single and dual index libraries may vary based on the sequencing instrument, loading concentrations, run configuration, and pooling ratios. Sequencer and assay performance cannot be guaranteed.
Please note that we have not tested sequencing mixed single and dual index pools on Illumina instruments other than the NovaSeq 6000 and Next Seq 500. Sequencing mixed pools on other instruments may require additional optimization. In particular, additional optimization may be required for the HiSeq 4000. We are aware of some customer reports of poor quality sequence data after sequencing single index 3’ Gene Expression libraries using dual index parameters on the HiSeq 4000.
Please note that we have not tested sequencing mixed single and dual index pools for library types other than 3' v3.1 Single Cell Gene Expression libraries. Additional optimization may be required when sequencing mixed single and dual index pools of other library types (eg. Feature Barcode libraries, 5’ Single Cell Gene Expression libraries).
Summary of in-house experiments on NovaSeq and NextSeq instruments
To determine whether single index and dual index libraries can be sequenced together, we pooled single index and dual index 3' v3.1 Gene Expression Libraries for sequencing on NovaSeq and NextSeq instruments. We found that sequence quality metrics for pooled single and dual index libraries were comparable to sequencing quality metrics obtained when sequencing these libraries separately. These results suggest that it is feasible to pool single and dual index 3' Gene Expression libraries for sequencing on NovaSeq and NextSeq instruments.
Instruments tested:
- NovaSeq 6000 SP
- NextSeq 500 High Output
Libraries and library pools tested:
- Single index 3’ v3.1 Gene Expression library
- Dual index 3’ v3.1 Gene Expression library
- 1:1 pool of single index and dual index 3’ v3.1 Gene Expression libraries
Run configurations tested:
- Single index run configuration
- R1 = 28bp, i7 = 8bp, R2 = 91bp
- Dual index run configuration
- R1 = 28bp, i7 = 10bp, i5 = 10bp, R2 = 90bp
PhiX spike-in:
- None
Results:
- We did not encounter any technical issues with sequencing 1:1 pools of single and dual index libraries using either single index or dual index run configurations.
- We did not encounter any technical issues with sequencing a single index library (on its own) using a dual index run configuration
- We did not encounter any technical issues with sequencing a dual index library (on its own) using a single index run configuration
- As shown in Figure 1 and 2 below, we observed a high percentage of bases with quality scores above 30 for all indexing strategies and run configurations
Conclusions:
- When using NovaSeq 6000 and NextSeq 500 instruments, it is possible to optimize sequencing of single indexed, dual indexed, or mixed pools of single indexed and dual indexed 3’v3.1 Gene Expression libraries, using either single index or dual index run configurations
Figure 1: NovaSeq 6000 data-by-cycle plots from Illumina SAV software
Figure 2: NextSeq 500 data-by-cycle plots from Illumina SAV software
Note: The Targeted Gene Expression assay has been discontinued. For alternative options, please see: What options are there for performing a targeted enrichment for my gene expression libraries?
Products: Single Cell Gene Expression, Single Cell Immune Profiling