10x Genomics Visium HD, CytAssist
The Visium HD technology provides single-cell, whole-transcriptome profiling of human or mouse tissues. These slides feature barcoded oligonucleotides arranged in 2 µm-by-2 µm grids within 6.5 mm-by-6.5 mm capture areas. The CytAssist device is used for hybridized probe transfer from the treated tissue to the Visium HD slides. This platform is compatible with formalin-fixed paraffin-embedded (FFPE), fresh frozen and fixed frozen samples and based on sequencing technology.
10x Genomics Visium HD for CytAssist drives spatial discoveries, enhancing topographic, whole-transcriptome spatial analysis with single-cell-scale resolution. Improving on the previous Visium technology, Visium HD can detect 11 million barcoded oligonucleotides arrayed in a continuous grid pattern of 2 µm-by-2 µm squares. This compares to just 5,000 oligonucleotides arranged hexagonally in 55-µm spots in the standard Visium Spatial Gene Expression slides. This 2-μm data output without gaps allows for single-cell-scale spatial resolution.
The Visium HD Spatial Gene Expression slides for CytAssist technology are designed to allow users to comprehensively characterize FFPE, fixed frozen or fresh frozen tissue sections in 6.5 mm-by-6.5 mm capture areas arrayed with oligonucleotides. The oligonucleotides feature a:
- Poly(dT) sequence for capture of the ligation product.
- Unique molecular identifier to identify duplicate molecules that arise during the library preparation and sequencing process.
- Spatial barcode shared by all oligonucleotides within each barcoded area.
- Partial Illumina TruSeq Read 1 sequencing primer for use during the library preparation and sequencing portions of the workflow.
The assay uses probe-based gene expression chemistry and requires the use of the Visium CytAssist to facilitate the transfer of transcriptomic probes from tissues on glass slides to Visium HD slides.
Visium HD is compatible with hematoxylin and eosin or immunofluorescence-stained FFPE tissue sections from human and mouse samples. Tissue slides are generated via tissue sectioning, deparaffinization, staining and imaging on a glass slide prior to using the Visium CytAssist. Two tissue slides and a two-capture-area Visium HD slide are placed in the CytAssist instrument so that the tissue slides align on top of the two Visium capture areas.
The instrument captures a brightfield image to provide spatial orientation for data analysis, followed by permeabilization of the tissue and transfer of transcriptomic probes to the Visium HD slide. After the probes are extended, the sample is eluted and transferred to a new tube to initiate the process of constructing a gene expression library. The final gene expression library is sequenced at a recommended minimum depth of 275 million read pairs per capture area.
10x Genomics provides two software tools to process and visualize these Visium data types: Space Ranger and Loupe Browser. Space Ranger processes the input file types to align the Visium sequencing data with the tissue image. Each spatial barcode with the associated unique molecular identifiers captured during the Visium workflow is assigned a spatial location in the tissue image. Space Ranger (version 3.0) produces various output files that can be used in Loupe Browser (version 8.0) or third-party tools to visualize and apply spatial analysis methods to the data.
While Visium HD data is generated from spatially patterned oligonucleotides labeled in 2 µm-by-2 µm bins, data from this resolution may be sparse. Thus, adjacent bins are pooled together to create 8-µm and 16-µm resolutions. 10x Genomics recommends the use of 8-µm binned data for analysis.