Space Ranger1.1, printed on 09/21/2023
The microscope slides used to generate Visium data have features that Space Ranger users need to know. In order to run Space Ranger, it is necessary to know the serial number associated with the slide that generated the data you intend to analyze, as well as the capture area associated with each sample.
The image below illustrates these key features of the Visium slide and these terms are explained in the glossary below.
Alignment File - A file produced by the Loupe Browser when using manual alignment and manual tissue detection.
Area (or Capture Area) - One of the four active regions where tissue can be placed on a Visium slide. Each area is intended to contain only one tissue sample. Slide areas are named consecutively from top to bottom: A1, B1, C1, D1.
Brighfield Image - A light-microscopy image depicting tissue on a slide. In a Visium experiment, a brightfield image is used as an anatomical reference. These images are usually stained with hematoxylin and eosin in order to highlight tissue structure (see H&E staining below).
Immunohistochemistry (IHC) and Immunofluorescence (IF) - IHC is an imaging technique whereby protein is stained using a matching, antibody-based label. Immunofluorescence (IF) is a subset of this technique that generates a signal using fluorophores attached to antibodies and fluorescence imaging.
Fiducial Frame - A frame of specially patterned spots surrounding each capture area. These spots help the sample microscopist see where to place tissue and are also used by Space Ranger to determine where the capture area is in an image.
H&E Staining - The process of applying hematoxylin and eosin to tissue in order to highlight tissue structure. Hematoxylin colors cell nuclei blue, and eosin colors the cytoplasm and extracellular matrix pink.
Count Matrix (or Feature-Barcode Matrix) - A matrix of counts representing the number of unique observations of each feature (gene) within each spot barcode. Genes defined by the transcriptome reference appear as rows in the matrix. Each barcode is a column of the matrix.
Dual Indexing - A strategy for sequencing multiple samples on the same flowcell by using two oligonucleotide sequences, one attached to either end of each fragment to be sequenced, in order to uniquely identify the sample. The Visium library construction only supports multiplexing samples using this dual-index strategy. See sample index below.
Sample Index - An oligonucleotide sequence used in library construction to differentiate multiple samples that are sequenced on the same flowcell. On the Illumina platform, these sequences are read out as separate "index reads" and reads are sorted into sample-specific files using mkfastq. The Visium library construction supports only "dual-indexing" (see above).
Sequencing Run (or Flowcell) - The output data, including Illumina BCL files, from one sequencing instrument run. The data can be demultiplexed by lane or by sample index. See mkfastq for more information about demultiplexing.