Sequencing Requirements for Visium Spatial Gene Expression for FFPE

Specifications, Last Modified on June 9, 2021, Permalink

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:

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.

Read Read 1 i7 Index i5 Index Read 2
Purpose Spatial Barcode, UMI Sample Index Sample Index Insert
Length** 28 10 10 50

** Shorter reads than indicated above can lead to decreased application performance. Sample index reads must not be shorter than indicated. Any read can be longer than recommended. Additional bases in Sample index reads must be trimmed using SpaceRanger mkfastq or Illumina's bcl2fastq prior to further analysis.