Support homeCytAssist Spatial Gene ExpressionDocumentationSequencing
Sequencing Requirements for Visium CytAssist Spatial Gene Expression

Sequencing Requirements for Visium CytAssist Spatial Gene Expression

For guidance on CytAssist-enabled Visium HD Spatial Gene Expression, refer to the sequencing requirements provided here.

The Visium CytAssist Spatial Gene Expression solution produces spatially barcoded, Illumina® sequencer-ready libraries.

Please note that Dual Index Kit TS, Set A (PN-1000251) is required for use with the Visium CytAssist Spatial Gene Expression Solution.

Supported Sequencers:

  • Illumina® NovaSeq 6000
  • Illumina® NextSeq 500/550
  • Illumina® NextSeq 2000
  • Illumina® MiSeq*
  • Illumina® iSeq 100*

Dual-Indexed Sequencing Run: Visium CytAssist Spatial Gene Expression libraries are dual-indexed. We do not recommend sequencing these libraries with a single-index configuration.

PhiX Spike-In Recommendations: 1%

* Recommended for library QC only

Visium CytAssist Spatial Gene Expression Libraries

Minimum Sequencing Depth: We recommend a minimum of 25k read pairs per spot covered with tissue. To calculate this, first estimate the % of capture area covered by the tissue section based upon the tissue image. Then use the following formula to calculate the recommended sequencing depth:

• Total sequencing depth = (Coverage Area x total spots on the Capture Area) x 25,000 read pairs/spot. The total number of spots/Capture Area should be adjusted based on the Capture Area size (the number of spots for 6.5 mm slides is 4,992 and the number of 11 mm slides is 14,336).

Example calculation for 60% tissue coverage using 6.5mm slides: (0.60 x 5,000 total spots) x 25,000 read pairs/spot = 75 million total read pairs for that sample

Additional guidelines for estimating the number of spots covered: Determining number of spots covered with tissue may be performed visually or by using the Visium Manual Alignment Wizard in Loupe Browser for a more accurate measurement.

ReadRead 1i7 Indexi5 IndexRead 2
PurposeSpatial Barcode, UMISample IndexSample IndexInsert

** Shorter reads than indicated above can lead to decreased application performance, particularly for read 2 which is aligned to the reference. Sample index reads must not be shorter than indicated. Any read can be longer than recommended.

Document Type

Last Modified
June 7, 2024