Space Ranger2.0 (latest), printed on 09/24/2022
Space Ranger now supports CytAssist instrument enabled Gene Expression analysis and expands the image processing sub pipeline to include image registration between the CytAssist image, which contains the tissue section and fiducial frame, and the microscope image (brightfield or fluorescence) of the same tissue section on standard glass slide.
The CytAssist generated image is specified using the new option
--cytaimage in the spaceranger count pipeline.
spaceranger count uses the CytAssist generated image to perform automatic fiducial alignment and tissue detection irrespective of the type of microscope image inputs supplied (H&E or immunofluorescence stained tissues). Loupe manual fiducial alignment is optional and not required.
For image registration using a fluorescence microscope image specified using
--darkimage, there is a new option
--dapi-index, which can be used to specify the page index corresponding to a tissue morphology counterstain (e.g., DAPI). When
--dapi-index is specified, Space Ranger will only use the information from this fluorescence channel to perform automatic image registration.
New CytAssist Image Alignment QC tool with an interactive opacity slider in the
web_summary.html to check the image alignment between the CytAssist and microscope image inputs.
Additional CytAssist-related outputs in the
aligned_tissue_image.jpg, which is useful to check the quality of the image registration and
cytassist_image.tiff, which is a copy of the original CytAssist image.
spaceranger count will now quantify fluorescence intensity in the image at every barcoded-spot location. A new output file,
barcode_fluorescence_intensity.csv is produced that contains measurements from every fluorescence channel and can be combined with feature-spot matrices by matching the barcode sequences.
Channel names in the column headers of
barcode_fluorescence_intensity.csv can be set by renaming fluorescence image channels during the manual fiducial alignment workflow in Loupe Browser. The channel names will also persist when opening the
cloupe.cloupe file in Loupe.
--unknown-slide flag is now an option that requires user to specify slide version when the slide serial number is unavailable (e.g.,
--unknown-slide=visium-1 for non-CytAssist slides).
Space Ranger is now compatible with the
QPTIFF image format.
Visium CytAssist Spatial Gene Expression for FFPE introduces a new and improved Human Transcriptome Probe Set with 3-fold coverage of most genes. The
probe set CSV file also contains an additional
region column which indicates whether the probe targets exon-junction-spanning (spliced) or non-exon-junction-spanning (unspliced) regions of the gene.
Estimated UMIs from Genomic DNA and
Estimated per Probe Background UMI Count as well as a new
Segmented Linear Model Plot are added to the Gene Expression tab in the
web_summary.html when using v2 probe sets that include the
region column. These metrics provide insight into the level of background noise in the overall UMI counts.
Images are rotated and mirrored by default to find the optimal fiducial alignment. Users no longer need to specify
--reorient-images in the spaceranger count pipeline when their input image does not have fiducial markers in the correct orientation.
--reorient-images is now a boolean option instead of a flag so rotation and mirroring can be disabled by setting
The following changes have been made to the
outs/ folder in spaceranger:
|Space Ranger 1.3 outputs||Space Ranger 2.0 outputs|
(Added header row)
(In spaceranger aggr)
|Are not output|
For consistency with
cellranger, secondary analysis outputs are prefixed with the library type (ie.
└── analysis └── pca └──gene_expression_10_components/
The spaceranger count
web_summary.html Analysis tab has been renamed to Gene Expression.
Improved error messages and better handling of poorly formatted inputs in spaceranger mkref. Enable users to generate references for analyses with large genomes containing chromosomes longer than 512 Mbp. spaceranger count pipeline will output a
.csi BAM index file instead of
.bai in these cases.
Updated spaceranger mkfastq to autodetect i5 orientation for users who upgraded to Novaseq instrument control software v1.8.
For spaceranger count runs that use a probe set, the value of
Median Genes per Spot in
web_summary.html is replaced by the
Median Panel Genes per Spot value from previous versions of
Median Panel Genes per Spot is now removed from the
metrics_summary.csv. In a nutshell,
Median Genes per Spot used to include all probes in the probe set and now only includes filtered probes.
Read 2 length is trimmed to 50 bp by default when a probe set reference CSV is passed using
--probe-set. User no longer has to remember to set
--r2-length=50 when processing FFPE sequencing libraries.
--no-probe-filter flag is deprecated and superseded by the boolean option
--filter-probes with default set to true for filtering probes with off-traget activity.
Visium_Human_Transcriptome_Probe_Set_v2.0_GRCh38-2020-A.csv to tarball.
Sequencing quality scores (ie., QV) up to Q93 are now supported. Previously supported up to Q41.
Fixed bug where pipeline would crash when only 35 spots under tissue were detected. Now pipeline will run to completion but Moran's I
spatial_enrichment.csv will not be output when fewer than 37 spots.
Fixed bug where
spaceranger count with
--no-bam flag still generated a BAM file.
software_version in matrix mtx and h5 headers to reflect the Space Ranger version used to generate the matrices.
Fixed a bug that resulted in a segmentation fault error when mapping to references that contain small contigs, for example, the rabbit genome.
Various error messages were updated to be more informative.
Web summary usability improvements: QC image(s) were moved from the
Spots panel to a new
Image panel. Chemistry descriptions were fixed to reflect slide version and assay used. Help text was updated to improve clarity.
Third-party licensing information added to
THIRD-PARTY-LICENSES.spaceranger.txt in the tarball.