Question: What are the recommended sequencing specifications for Visium HD libraries?
Answer: For a Visium HD capture area fully covered by tissue (100%), the sequencing recommendation is a minimum of 275 million read pairs. Sequencing is highly tissue block and tissue type dependent. For RNA-rich samples, more information can be accessed by increasing sequencing reads, in some cases by 2x or more.
The recommended minimum sequencing depth was chosen because it achieved >50% sequencing saturation for >50% formalin fixed, paraffin embedded (FFPE) tissues tested at 10x Genomics. To achieve >50% sequencing saturation for >50% of the Fresh Frozen (FF) tissues tested by 10x Genomics, 700 million read pairs per fully-covered Capture Area were required. To achieve >50% sequencing saturation for >50% of the Fixed Frozen tissues (FxF) tested by 10x Genomics, 500 million read pairs per fully-covered Capture Area were required. Please refer to the Visium HD Spatial Gene Expression Reagent Kits User Guide (CG000685) “Sequencing Depth” section for additional information.
If a capture area is not fully covered by tissue, we recommend either guesstimating capture area coverage and multiplying that percentage by 275 million read pairs or utilizing Loupe Browser Visium HD Manual Alignment Wizard for a more accurate determination (v8.0 or later).
Additional details on considerations for selecting sequencing depth as well as performing calculations based upon estimation can be found in the Sequencing section of the Visium HD Spatial Gene Expression Reagent Kits User Guide (CG000685).
For guidance utilizing Loupe Browser, see the following article: How do I calculate the percentage of the capture area covered, so that I can determine the recommended sequencing depth for Visium HD Spatial Gene Expression libraries?
Visium HD libraries should be sequenced with a paired-end, dual index configuration utilizing the following cycling conditions:
| Read 1 | i5 index | i7 index | Read 2 |
| 43 cycles | 10 cycles | 10 cycles | 50 cycles |
Visium HD libraries derived from samples prepared using different methods (such as FFPE, FF, FxF) may be pooled for sequencing, taking into account differences in percent of capture Area covered by tissue. Pooling Visium HD libraries with other 10x libraries for sequencing is not recommended.
Product: Visium HD