Illumina Single Cell 3' RNA Preparation library sequencing recommendations and guidelines

Illumina Single Cell 3’ RNA preparation libraries are composed of standard Illumina paired-end constructs that begin with P5 and end with P7.

Read 1 contains barcode information and must be sequenced > 45 bases.

Read 2 contains gene expression information and must be sequenced > 72 bases.

The libraries are dual-indexed with 10-base i5 and i7 indexes.

Together this combines for a total of a minimum of 137 cycles needed to sequence Illumina Single Cell 3’ libraries.

Read Depth Recommendations

The recommended sequencing read depth for the Illumina Single Cell 3’ RNA Kit sizes of T2, T10, and T20 is 20,000 reads per input cell. The required read depth for kits loaded at the recommended cell/nuclei loading is as follows (M = million):

• For T2, 5,000 cells loaded requires 100 M reads per T2 sample

• For T10, 17,000 cells loaded requires 340 M reads per T10 sample

• For T20, 40,000 cells loaded requires 800 M reads per T20 sample

The recommended sequencing read depth for the T100 kit is 10,000 reads per input cell.

• For T100, 200,000 cells loaded requires 2 billion reads per T100 sample

After the users first run with their specific sample type and a specific kit size, users can evaluate capture rate and sequencing saturation metrics to determine if sequencing depth can be adjusted in future experiments based on sample type and experimental needs.

Final Loading Recommendations

The final library loading concentrations listed are general recommendations for sequencing Illumina Single Cell 3’ RNA Prep libraries. The following concentrations may need to be adjusted to optimize performance:

• NextSeq 500/550 recommended final library loading concentration 1.6 pM including ≥ 1% PhiX.

• NextSeq 2000 recommended final library loading concentration: 550 pM including ≥ 1% PhiX.

• NovaSeq 6000 final library loading concentration 210 pM including ≥ 1% PhiX (equivalent to Pooled Loading Concentration of 1.05 nM).

• NovaSeq X Series final library loading concentration 190 - 200 pM including ≥ 2% PhiX.

  

It is recommended to pool libraries together from all experimental conditions before single-cell sequencing with an Illumina sequencing system, as this will minimize batch effects and can help with index color balancing.

Refer to the Supplemental Enrichment and Amplification (SEA) kit user guides for pooling recommendations for the applications that generate a targeted library to be sequenced with Illumina Single Cell 3’ RNA libraries.

A minimum of 1% PhiX is required in the final library loading pool. If a NovaSeq X Series instrument is being used, a minimum of 2% PhiX should be used.

For more information, refer to the Sequencing section of the Illumina Single Cell Training Packet.