10x Genomics Support/Spatial Gene Expression for FFPE/Documentation/Sequencing/

Sequencing Requirements for Visium Spatial Gene Expression for FFPE

The Visium Spatial Gene Expression for FFPE 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 Spatial Gene Expression for FFPE Solution.

Supported Sequencers:

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

Dual-Indexed Sequencing Run: Visium Spatial Gene Expression for FFPE 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 Spatial Gene Expression for FFPE 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

Example calculation for 60% tissue coverage: (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
August 2, 2023