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# Release notes for Space Ranger 2.0.0 (July 18, 2022):

## New feature: CytAssist Spatial Gene Expression

• 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 spatial/ subfolder: 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.

• The --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.

## System requirements

• CentOS/RedHat >= 7.0 or Ubuntu >= 14.04 is required to run Space Ranger v2.0. Space Ranger v1.3 was the last version that supported CentOS/RedHat 6 or Ubuntu 12.04.

## General improvements

• 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.

• New metrics 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 --reorient-images=false.

• The following changes have been made to the outs/ folder in spaceranger:

Space Ranger 1.3 outputs Space Ranger 2.0 outputs
tissue_positions_list.csv tissue_positions.csv
spatial_enrichment.csv spatial/spatial_enrichment.csv
(Moved to spatial/ subfolder)
raw_feature_bc_matrix
raw_feature_bc_matrix.h5
(In spaceranger aggr)
Are not output
• For consistency with cellranger, secondary analysis outputs are prefixed with the library type (ie. gene_expression_*).

└── analysis
└── pca
└──gene_expression_10_components/

• The spaceranger count web_summary.html Analysis tab has been renamed to Gene Expression.

• The BAM file and the molecule_info.h5 now include the slide serial number and the capture area information if provided to the spaceranger count run.

## Bug fixes and miscellaneous changes

• 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 metrics_summary.csv and web_summary.html is replaced by the Median Panel Genes per Spot value from previous versions of metrics_summary.csv. 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.

• The --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.

• Added Visium_Mouse_Transcriptome_Probe_Set_v1.0_mm10-2020-A.csv and 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.

• Fixed 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.

• 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.