Question: Can Multiome Single Cell libraries be pooled with other 10x libraries?
Answer: Multiome Gene Expression libraries can be pooled with other dual-indexed gene expression libraries. Multiome ATAC libraries can be pooled with standalone ATAC libraries only on certain Illumina sequencers like MiSeq, HiSeq 2000/2500, NovaSeq 6000 (v1 reagents) sequencers and using 16bp for the i5 read. Although typically, Multiome ATAC libraries are sequenced at i5=24 bp, but when pooled with standalone ATAC libraries (V1, V1.1) on certain Illumina sequencers listed above, they have to be sequenced at i5=16 bp to be compatible with both library structures. Reading past 16 bp on i5 on standalone ATAC libraries could create issues with Cell Ranger ATAC.
Read more on: Why can I not pool Multiome ATAC and Standalone ATAC libraries on certain sequencers?
There are no disadvantages of sequencing Multiome ATAC libraries with i5=16 bp when sequenced on the sequencers listed above.
Note: We do not recommend pooling Multiome Gene Expression libraries with Multiome ATAC libraries on the same flow cell as they have differing sequencing specifications. Additional information can be found at: Can Multiome ATAC and Multiome GEX libraries be sequenced together?
The following libraries can be pooled together for Illumina sequencing:
- Chromium NextGEM Multiome Gene Expression Libraries
- Chromium GEM-X Single Cell 3’ v4 (Dual Index)
- Gene Expression Libraries
- Cell Surface Protein Libraries
- Chromium GEM-X Single Cell 5’ v3 (Dual Index)
- Gene Expression Libraries
- V(D)J Enriched Libraries
- Cell Surface Protein Libraries
- CRISPR Screening Libraries
- Chromium NextGEM Single Cell 3’ v3.1 (Dual Index)
- Gene Expression Libraries
- Cell Multiplexing Libraries
- Cell Surface Protein Libraries
- CRISPR Screening Libraries
- Chromium NextGEM Single Cell 5’ v2 (Dual Index)*
- Gene Expression Libraries*
- V(D)J Enriched Libraries*
- Cell Surface Protein Libraries*
- CRISPR Screening Libraries*
*If run with 28 cycles for Read 1
- Fixed RNA Profiling Gene Expression Libraries
Please note: 1% PhiX is recommended for Singleplexed libraries, 5% for Multiplexed libraries, and 10% for Multiplexed libraries sequenced on NovaSeq
- Visium Spatial Gene Expression Libraries (Dual Index)
- Visium Spatial Gene Expression for FFPE Libraries and Visium CytAssist Spatial Gene Expression libraries (i.e. Probe-based Visium Libraries)*
*Read 2 should be increased to 90 cycles. When pooling in this fashion, we recommend limiting Probe-based Visium Libraries to <40% of the total sequencing pool to maximize quality.
The following libraries can be pooled only of sequencing Miseq, Hiseq 2000/2500, NovaSeq 6000 (v1 reagents) sequencers and using 16bp for the i5 read:
- Chromium NextGEM Multiome ATAC Libraries
- Chromium NextGEM ATAC v2 Libraries
- Chromium NextGEM ATAC v1.1 Libraries
- Chromium NextGEM ATAC v1 Libraries
Products: Single Cell Multiome ATAC + GEX