10x Genomics
Visium Spatial Gene Expression
Space Ranger1.3, printed on 12/22/2024
Release notes for Space Ranger 1.3.1 (November 1, 2021):
Bug Fixes and Miscellaneous Changes
- Adds an alert if image alignment would have produced a different result if the
--reorient-images option was specified.
- Update warning for low fraction of reads confidently mapped to the probe set to be more informative.
- Unsets additional sysconfig environment variables prior to pipeline execution, which may otherwise interfere with the pipeline conda environment.
- Fixes an issue with mkfastq when bcl2fastq requires LD_LIBRARY_PATH.
Release notes for Space Ranger 1.3.0 (June 9, 2021):
System Requirements
- The recommended operating systems for Space Ranger 1.3.0 are CentOS 7 or Ubuntu 14 Linux variants or newer. CentOS 6 and Ubuntu 12 are still supported but have been deprecated. Support may be dropped in future versions.
New Feature: Gene Expression from FFPE Samples
- Gene Expression analysis from formalin fixed paraffin embedded (FFPE) samples is now available in Space Ranger and is invoked by specifying the
--probe-set option when running the spaceranger count command. Space Ranger 1.3.0 or later is required to analyze FFPE data.
- A new
--no-probe-filter option can be used to disable the default filtering of certain probes. See the documentation for more information.
- A new probe aligner tailored for use with probe-based assays, such as Visium for FFPE, was developed. When the
--probe-set option is invoked, Space Ranger aligns reads to the specified probe set to count oligo ligation events. See the algorithms for more details.
- BAM output from probe-based assays includes a new tag
pr:Z that indicates the probe to which a read aligns and specifies the gene ID, gene name, and a computational hash specific to the probe sequence.
- Visium Spatial Gene Expression for FFPE ships with a new dual index set (Dual Index Kit TS Set A) distinct from the dual index set used for fresh frozen tissues (Dual Index Kit TT Set A). The dual index sets can be detected by
spaceranger mkfastq based on their abbreviations (e.g., SI-TS-A1) in the samplesheet. Space Ranger 1.3.0 or later is necessary to detect Dual Index Kit TS Set A with spaceranger mkfastq.
New Feature: Spatial Autocorrelation (Moran's I)
- Space Ranger now calculates the Moran's I spatial autocorrelation metric on a per-gene basis, highlighting genes with patterns of spatially coherent gene expression for presentation in Loupe Browser and in a new output file
spatial_enrichment.csv.
Bug Fixes and Miscellaneous Changes
- The web summary output from Space Ranger now indicates whether the
--reorient-images option was specified in order to enable automatic rotation of input images.
- The web summary now differentiates probe-based, targeted, and whole genome analyses.
- Space Ranger now uses Python 3 for Python-based stages.
- The molecule_info.h5 and unfiltered feature-barcode matrix files (raw_feature_bc_matrix in H5 and MEX formats) will only contain barcodes with at least one read, rather than all barcodes in the whitelist.
- A bug that prevented error messages from
spaceranger mkref from displaying properly when processing GTF files was fixed.
- A bug has been fixed in the graph-based clustering output: previously, in a sample with K clusters, the first K spot-associated barcodes (ordered as in the filtered feature-barcode matrix) may have been assigned incorrect cluster labels. This change does not affect the number of clusters output.
- The
--qc option has been deprecated from spaceranger mkfastq.
Release notes for Space Ranger 1.2.2 (February 3, 2021):
- Adds support for pyramid TIFFs, or TIFFs with multiple pages of the same image at different resolutions.
- Fixes an issue where targeted samples run against new references would fail preflight.
Release notes for Space Ranger 1.2.1 (December 10, 2020):
Bug Fixes and Miscellaneous Changes
- Fixed an issue in aggr where files would fail to be copied on NFSv4 File Systems.
- Disables multithreading in mkref to address an issue where mkref would fail on hardware without AVX support. This will be fixed in a future release.
- Better error handling if the pipeline fails due to a missing .env.json file due to an incomplete copy of the application folder.
Release notes for Space Ranger 1.2.0 (October 27, 2020):
New Features
- Targeted Gene Expression analysis is now available in Space Ranger 1.2 and is invoked by specifying the
--target-panel option when running the spaceranger count command.
- Space Ranger 1.2 introduces the new
spaceranger targeted-compare pipeline for direct comparative analysis of matched parent Whole Transcriptome Amplification (WTA) and Targeted Gene Expression datasets.
- Space Ranger includes the new
spaceranger targeted-depth subcommand to estimate sequencing depths appropriate for Spatial Targeted Gene Expression experiments based on input WTA results and an associated target panel file.
- A new command line option,
--no-bam, instructs the pipeline not to construct BAM files.
- The GTF file parsing methods have been changed and some GTF files with non-standard metadata fields may no longer be accepted by Space Ranger. You may need to reformat the metadata fields into a standard layout.
- Performance improvements in reading FASTQ files, writing BAM files, and memory utilization in certain pipeline stages.
spaceranger aggr now outputs a t-SNE projection colored by the input sample.- Support for Gateway slides containing two standard 6mm capture areas per slide.
Bug Fixes and Miscellaneous Changes
- The
--unknown-slide option for spaceranger testrun has been renamed --no-internet to reflect its intended use.
- Fixed an issue in mkfastq that prevented the
--filter-single-index option from working correctly.
- Fixed an issue where files would fail to be copied on NFSv4 File Systems.
- The
--nuclei option was removed from spaceranger mkref.
- The colors used in the web summary output to indicate fiducial alignment and tissue detection have been changed to avoid confusion with an H&E stained image.
- The maximum number of colors (channels) in a fluorescence imaging sample has been increased to nine.
- Fixed parsing of some non-standard FASTQ files to avoid errors.
- Bug fixes for GTF files output by mkref:
- GTF attributes with duplicate keys (e.g., tag "value1"; tag "value2";) are handled correctly. Previously, only the last such attribute was kept.
- GTF attributes with unquoted integer values (e.g., exon_number 1;) are kept. Previously, they were removed.
- GTF lines end with semicolons.
- Unix line endings are used rather than DOS line endings, consistent with other Space Ranger outputs.
- Fixed problem parsing GTF files with semicolon delimiter inside quotes
- Fixed a numerical stability issue in the PCA method that affected the analysis of some datasets.
- BGI/MGI reads can now be analyzed using Space Ranger.
BAM Output Changes
- The duplicate flag (0x400) is set correctly in the secondary alignments (flag 0x100) of PCR duplicate reads and low-support UMI reads (xf:i:2)
- Low-support UMI reads (xf:i:2) have the corrected barcode in UB:Z. Previously, it contained the raw barcode.
- The duplicate flag (0x400) is set correctly in the secondary alignments (flag 0x100) of PCR duplicate reads and low-support UMI reads (xf:i:2)
- Low-support UMI reads (xf:i:2) have the corrected barcode in UB:Z. Previously, it contained the raw barcode.
- Reads that are excluded from analysis due to high-depth subsampling in targeted gene expression are unmapped and marked with (
xf:i:32) in the BAM.
Known Issues
- The minimum dimension for an input image to Space Ranger is 2000 pixels on a side. Even though the Loupe Manual Aligner may allow smaller images to be used during manual alignment, the pipeline will not process images that are smaller than the minimum. These limits will be made consistent in an upcoming release.
- Loupe and Space Ranger currently do not support BigTIFF files that contain a multi-resolution image pyramid. We’re aware that some systems produce such files and we recommend converting such images externally to a single-resolution image at the highest resolution desired. We hope to address this issue in a future release.
Release Notes for Space Ranger 1.1.0
New Features
- Support for fluorescence imaging input such as IHC/IF.
- Aggregation of multiple datasets.
- Optional automatic rotation of slide images.
Runtime Improvements
- Space Ranger 1.1 runs two to five times faster than Space Ranger 1.0, depending on the sequencing data, and reduces disk I/O by half.
Analysis Improvements
- The fraction of reads mapped confidently to a gene was improved by up to 9% by trimming template switch oligo (TSO) sequence from the 5' end of read 2 and poly-A from the 3' end prior to alignment.
- Space Ranger now provides consistent sensitivity across a wider range of read lengths, from 70 bp reads to 120 bp reads, enabling use of smaller sequencing kits.
- The recommended references for mouse and human have been updated (new version: 2020-A). The new references remove annotations with weak evidence that prevent alignment to legitimate annotations.
Other Changes
- Space Ranger 1.1 uses Orbit to perform transcriptome alignment, which leverages a modified STAR v2.7.2a. These modifications provide compatibility with “versionGenome 20201” references, such as those generated by STAR v2.5.1b. In Space Ranger 1.1 we still provide and use STAR v2.5.1b for other purposes such as spaceranger mkref. In our testing we did not note any differences in transcriptome alignments between the STAR shipped in Space Ranger 1.0 (STAR v2.5.1b), STAR v2.7.2a, or Orbit.
Release Notes for Space Ranger 1.0.0