Visium HD WT Panel

Created

January 1, 2026

Modified

March 28, 2026

Visium HD WT Panel is the high-definition spatial gene expression assay from 10x Genomics. It achieves 2 µm bin resolution — approximately single-cell scale — by using a new slide architecture with a continuous lawn of capture oligonucleotides instead of discrete 55 µm spots. Bins are computationally defined during analysis at 2 µm, 8 µm, or 16 µm resolution.

Like Visium v2, the HD WT Panel uses probe-based hybridisation and requires the CytAssist instrument. This makes it compatible with FFPE, fresh-frozen, and fixed-frozen tissues.

Feature Visium v2 (WT Panel) Visium HD WT Panel
Resolution 55 µm spots 2 µm bins
Capture architecture 5,000 discrete spots Continuous lawn
Probe chemistry WT Panel probes WT Panel probes
CytAssist Required Required
Slide size Standard 6.5 × 6.5 mm or 11 × 11 mm
Sample compatibility FFPE, FF, Fixed-frozen FFPE, FF, Fixed-frozen

The spatial barcode in Visium HD encodes a 2D position within the continuous lawn. Each 2 µm × 2 µm bin contains oligos with the same spatial barcode, and the barcode coordinate maps to a precise tissue location.

Assay Principle

  1. Tissue is prepared on a standard glass slide (H&E, IF staining, or unstained FFPE).
  2. WT Panel probe pairs (Left Probe + Right Probe) hybridise to target transcripts in situ.
  3. Probes are ligated to form Probe-Ligation Products (PLPs).
  4. CytAssist transfers PLPs to the Visium HD slide capture area.
  5. PLPs are captured by the surface-bound oligos (spatial barcode + UMI + poly-T) at their corresponding 2 µm bin.
  6. Extension through the PLP incorporates the spatial barcode and UMI.
  7. Library is prepared using standard TruSeq-based PCR workflow.

Capture Bin Oligonucleotide

5'- CTACACGACGCTCTTCCGATCT[16-bp spatial BC][12-bp UMI](T)30VN -3'    (surface-bound, 2 µm bin)

The spatial barcode is a 16 bp sequence that uniquely identifies each 2 µm bin position on the capture area. Visium HD uses a new barcode design with higher density than the v1/v2 spot layout.

Adapter and Primer Sequences

WT Panel Left Probe (LP):

5'- [~25 bp target-specific][ligation handle] -3'
    3' end is the ligation junction

WT Panel Right Probe (RP):

5'- [5'-phosphate][~25 bp target-specific][poly-A / capture handle] -3'
    5' end is the ligation junction

Capture bin oligo:

5'- CTACACGACGCTCTTCCGATCT[16-bp spatial BC][12-bp UMI](T)30VN -3'

cDNA amplification primer: 5'- CTACACGACGCTCTTCCGATCT -3'

TruSeq Read 1 primer: 5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT -3'

TruSeq Read 2 primer: 5'- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT -3'

TruSeq adapter (dsDNA, T overhang):

5'-  GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3'
3'- TCTAGCCTTCTCG -5'

Library PCR primer 1: 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC -3'

Library PCR primer 2: 5'- CAAGCAGAAGACGGCATACGAGAT[8-bp sample index]GTGACTGGAGTTCAGACGTGT -3'

Illumina P5: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'

Illumina P7: 5'- CAAGCAGAAGACGGCATACGAGAT -3'

Step-by-Step Library Generation

Step 1 — Tissue section preparation and probe hybridisation

FFPE or fresh-frozen tissue section (5–10 µm) is placed on a standard glass slide and prepared histologically. WT Panel probe pairs hybridise to target mRNAs (hybridisation buffer, 37°C overnight).

mRNA  5'--XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX--3'
                 |||||||||||||||||||   |||||||||||||||||||
            [LP target-specific]  [probe BC][RP target-specific]
                           ↑ ligation junction ↑

Step 2 — Probe ligation

Ligase joins LP and RP at the ligation junction on the mRNA template. Unhybridised and unligated probes are washed away.

5'- [LP target][probe BC][RP target][capture poly-A handle] -3'   (PLP, released)

Step 3 — CytAssist transfer to Visium HD slide

CytAssist instrument transfers PLPs from the glass slide surface to the continuous capture lawn of the Visium HD slide.

Step 4 — PLP capture and spatial barcode incorporation

PLPs are captured at their corresponding 2 µm bin via poly-T/poly-A annealing. The bin oligo is extended through the full PLP.

slide 5'- CTACACGACGCTCTTCCGATCT[spatial BC][UMI](T)30VN -3'
                                                            |||||||||||||||
                                 3'- [capture handle(pA)][RP target][probe BC][LP target] -5'  (PLP)

Extension product:

slide 5'- CTACACGACGCTCTTCCGATCT[spatial BC][UMI](dT)VN[RP target][probe BC][LP target][LP adaptor complement] -3'

Step 5 — cDNA release and PCR amplification

Extended product is released and amplified:

   5'- CTACACGACGCTCTTCCGATCT-------->
5'- CTACACGACGCTCTTCCGATCT[spatial BC][UMI]-PLP-[LP adaptor] -3'
3'- GATGTGCTGCGAGAAGGCTAGA[spatial BC][UMI]-PLP-[LP adaptor] -5'
                                              <---------[LP adaptor complement] -5'

Step 6 — Fragmentase, A-tailing, TruSeq adapter ligation, library PCR

The amplifiable fragment (TruSeq Read 1 + spatial BC + UMI + partial PLP) is processed with TruSeq adapters:

5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-------->
          5'- CTACACGACGCTCTTCCGATCT[spatial BC][UMI]-PLP-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -3'
          3'- GATGTGCTGCGAGAAGGCTAGA[spatial BC][UMI]-PLP-TCTAGCCTTCTCG                         -5'
                                                  <--------TGTGCAGACTTGAGGTCAGTG[8-bp idx]TAGAGCATACGGCAGAAGACGAAC -5'

Final Library Structure

5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNNN-PLP-AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNNN-PLP-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
          Illumina P5                   TruSeq Read 1              16 bp spatial BC  12 bp UMI   probe-ligation product     TruSeq Read 2           8 bp idx       Illumina P7

Library Sequencing

Step 1 — Read 1: spatial barcode + UMI (bottom strand as template)

                         5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT------------------------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNNN-PLP-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

28 cycles (16 bp spatial BC + 12 bp UMI).

Step 2 — Index 1 (i7): sample index (bottom strand as template)

                                                                               5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNNNNNNNNNNNNNNN-PLP-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

8 cycles.

Step 3 — Read 2: PLP sequence (top strand as template)

5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNNN-PLP-AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                        <------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG -5'

50+ cycles. Read 2 sequences the PLP, with the probe barcode used by Space Ranger HD to assign gene identity.

Key Points

  • Read 1 (28 cycles): 16 bp spatial barcode (2 µm bin ID) + 12 bp UMI.
  • Read 2 (50+ cycles): PLP sequence, probe barcode for gene identification.
  • 2 µm bin architecture enables near-single-cell spatial resolution; bins can be aggregated to 8 µm or 16 µm computationally.
  • WT Panel protocol 2.0 improves sensitivity — recommended over protocol 1.0 for new experiments.
  • Space Ranger HD processes the data: bins reads by spatial barcode, assigns gene identity from probe barcodes, and generates the multi-resolution spatial expression matrix.
  • Compatible with FFPE, fresh-frozen, and fixed-frozen tissues; CytAssist is required.
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