Question: Which Illumina SBS kit should I use for sequencing?
Answer: This depends on the library type, desired sequencing configuration, sequencer, and reagent kit version.
Illumina provides SBS (Sequencing by Synthesis) reagents for all DNA sequencing platforms. Having sufficient volume of sequencing reagents is critical for proper run performance. Each cycle of sequencing generates one base of sequencing information for each cluster on the flow cell, and more information regarding how many cycles of sequencing reagents is contained in the various kits can be found here on the Illumina webpage. For example, the NextSeq 1000/2000 P3 flow cell v3 100 cycle kit can generate ~1.2 billion clusters in the flow cell and contains enough sequencing reagents for 138 cycles of sequencing. This means a maximum of 138 bp could be sequenced with this SBS kit including the index cycles.
In-house, we sequence in the configuration outlined in the sequencing section of each User Guide. This information can also be found on the 10x Support webpage, for example Sequencing recommendations for Single Cell Gene Expression can be found here. And summary of the recommended sequencing configuration of all our assays is shown here:
To determine which flow cell and SBS kit is most appropriate, the following steps can be taken:
- Determine the total number of read pairs required for a library or pool of libraries. This will determine which flow cell is most appropriate. For Single Cell libraries, the 10x Genomics Flowcell Capacity Calculator can assist with flow cell selection. Please note that the desired 10x read pairs per cell correspond to Illumina’s single-end reads (i.e., the number of clusters) when assessing yield specifications.
- Calculate the total number of required cycles of sequencing to identify the appropriate SBS kit. This can be determined by summing all the read lengths from R1 and R2 as well as the index reads. Please be aware that some Illumina sequencers/reagent versions will require an additional 7 cycles of sequencing reagents when performing a dual indexing run and this will also need to be accounted for in the total number of cycles.
- Double-check to make sure the bp required is less than the cycles for each kit found here on the Illumina webpage.
Single Cell 3'/5'
For example, 3’ v3.1 dual index libraries are recommended to be sequenced in the following configuration for optimal assay performance:
Therefore, 138 bp (28 + 10 + 10 + 90) are needed. This would be appropriate for any Illumina 200 cycle kit, as the cycle number (200) exceeds the required number of base pairs (138 bp).
For example, 5’ v2 dual index libraries are recommended to be sequenced in the following configuration for optimal assay performance:
**Length can be longer than listed above, for example if pooled with 3' v3.1 dual index libraries.
Therefore, 136 bp (26 + 10 + 10 + 90) are needed. This would be appropriate for any Illumina 200 cycle kit, as the cycle number (200) exceeds the required number of base pairs (136 bp).
Some 100 cycle kits can be utilized as well. For example, the NovaSeq v1.5 chemistry 100 cycle kit contains 138 cycles of SBS reagents which is equal to the required number of base pairs. Can I use the 100-cycle NovaSeq kit for Single Cell 3' and 5' assays? The NextSeq P2 v3 100 cycle kit contains 138 cycles of SBS reagents which is also equal to the required base pairs. The NextSeq P2 100 cycle kit on the other hand only contains 127 cycles of SBS reagents, which would be insufficient for sequencing 3’ Single Cell Libraries in the recommended configuration (28 + 10 + 10 + 90) or sequencing 5' Immune Profiling Libraries in the recommended configuration (26 + 10 + 10 + 90).
Similar to single cell libraries, the recommended read configuration and sequencing depth should be identified using the Sequencing section in the respective User Guides. Can I use a 100-cycle NovaSeq kit to sequence Visium libraries?
A few additional considerations / resources:
While it is strongly recommended to follow sequencing guidance outlined in the User Guide, it may be possible to modify the sequencing configuration to utilize a SBS kit with fewer cycles. This is not recommended and may compromise assay performance.
Here are a few articles which outline considerations:
- Can I decrease the sequencing length of Read 2 for Single Cell Gene Expression assays?
- Can I change the sequencing read lengths of the i5 and i7 indices?
- Can I change the sequencing read lengths of the i5 and i7 indices for ATAC libraries?
- Can I change the sequencing read length of R1N and R2N?
For ATAC and ATAC Multiome, it is critical not to shorten index reads:
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell Multiome ATAC+GEX, Single Cell ATAC, Fixed RNA Profiling