Space Ranger1.2, printed on 04/01/2023
Space Ranger is a set of analysis pipelines that process Visium spatial RNA-seq output and brightfield and fluorescence microscope images in order to detect tissue, align reads, generate feature-spot matrices, perform clustering and gene expression analysis, and place spots in spatial context on the slide image. Space Ranger includes two pipelines relevant to spatial gene expression experiments:
spaceranger mkfastq wraps Illumina's bcl2fastq to correctly demultiplex Visium-prepared sequencing runs and to convert barcode and read data to FASTQ files.
spaceranger count takes a microscope slide
image and FASTQ files from
spaceranger mkfastq and performs alignment, tissue detection, fiducial
detection, and barcode/UMI counting. The pipeline uses the
Visium spatial barcodes to generate feature-spot
matrices, determine clusters, and perform gene expression analysis.
spaceranger aggr takes the output of
multiple runs of
spaceranger count from related samples and aggregates
their output, normalizing those runs to the same sequencing depth, and then
recomputing the feature-barcode matrices and the analysis on the combined data.
The aggr pipeline combines data from multiple samples into an experiment-wide
feature-barcode matrix and analysis.
These pipelines combine Visium-specific algorithms with the widely used RNA-seq aligner STAR. Output is delivered in standard BAM, MEX, CSV, HDF5, TIFF, PNG, JPEG and HTML formats that are augmented with spatial information.
Space Ranger takes a slide image as input to be used as an anatomical map on which gene expression measures are visualized. Space Ranger can take two styles of images: either a brightfield image stained with hematoxylin and eosin (H&E) with dark tissue on a light background or a fluorescence image with bright signal on a dark background. While brightfield input comprises a single image, fluorescence input comprises one or more channels of information generated by separate excitations of the sample.
Space Ranger combines algorithms for processing Visium slide images with the robust gene expression analysis used in Space Ranger.
spaceranger count pipeline performs the following
automated image processing tasks:
These image processing tasks are described further in the Image Processing section. We encourage you to read Input Recommendations for imaging and sequencing suggestions prior to data collection.
The automated alignment and tissue identification processes require images
taken with proper tissue placement and image exposure and currently only
works for brightfield images. Samples with fluorescence images or brightfield
images where these conditions cannot be met can be processed by
using Loupe Browser to manually align the spot pattern and
identify tissue in the slide image. When this "manual workflow" is
spaceranger count pipeline
will still take the full resolution image or images as input in order to
prepare data for visualzation within Loupe Browser. Note that both the
Loupe Manual Aligner and Space Ranger must be given the same set of images for a given