> For the complete documentation index, see [llms.txt](https://help.bioinsight.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://help.bioinsight.illumina.com/dragen-single-cell-rna/reference/release-notes.md).
# Release Notes
**Release Notes for DRAGEN v4.6**
Standalone cell filtering mode
* Enabled with option “--single-cell-enable-cell-filter=true”
* Bypasses mapping, dedup, counting steps (only performs cell filtering and guide calling if enabled)
* Takes raw matrix files as input
* “--single-cell-filter-input-directory=\”
* “--single-cell-filter-input-prefix=\”
* Input files must match the following:
* \/\.scRNA.barcodes.tsv.gz
* \/\.scRNA.features.tsv.gz
* \/\.scRNA.matrix.mtx.gz
* Existing cell-filtering options can be used
* “--single-cell-threshold=\”
* “--single-cell-number-cells=\”
* “--scrna-guide-calling-mode=gmm” (for guide calling using a Gaussian mixture model)
Splice junction counts added to the counts matrix with option “--scrna-count-splice-junctions=true”
* Only annotated splice junctions present in the input GTF file are included
* Each splice junction appears as a feature with their corresponding deduplicated counts for each cell
* Splice junction feature entries have the same coordinates appearing as the feature ID and the feature name with the feature type "Splice Junction"
SW trimming added when the STAR aligner is used
* \--trim-adapter-r1-5prime=star\_adapters.fasta – trims the following:
* TSO adapters (AGAGTGAATGGG)
* Smart PCR adapters (TCAACGCAGAGT)
* –trim-adapter-read1=star\_homopolymer\_trim.fasta – trims the following:
* PolyA sequences (AAAAAAAAAAAA)
* PolyG sequences (GGGGGGGGGGGG)
Guide calling improvements
* Posterior probability score added to guide assignment output files
* Simple global threshold approach automatically added
* New output file: \.scRNA.simple\_threshold\_guide\_assignments.csv
* Threshold option: “--scrna-guide-calling-global-count-threshold” (default: 10)
* Guide metrics CSV file
* Updated “Total cells” to “Passing cells with features”
* Updated “Total cells with one or more guides detected” to “Total cell with one or more guides detected” and added fraction over total cells
* Updated “Total cells with two or more guides detected” to “Total cell with two or more guides detected” and added fraction over total cells
* Added “Total features in reference” value
* Added “Features in positive cells” count and fraction over total
* The same metrics as above are also reported on a per-feature-type basis for every feature type in the feature/direct capture barcode reference file.
New paired guide calling feature supported
* Enabled using “--scrna-enable-paired-guide-calling=true”
* “guide\_pair\_id” column must be added to the feature/direct capture barcode reference such that every set of guides to be called together have the same guide pair ID
* Additional paired guide assignment files will be output where only guide pairs whose constituent guides are all passing in the cell are reported
* A separate file will be reported for each method (GMM and simple global threshold)
* \.scRNA.positive\_paired\_guide\_assignments.csv
* \.scRNA.simple\_threshold\_paired\_guide\_assignments.csv
Feature counting updates
* The “target” field for the feature barcode reference can now be used in feature counting mode (for CROP-seq)
* “--scrna-pipseq-crispr-mode” deprecated in favor of “--scrna-direct-capture-mode” (scrna-pipseq-crispr-mode will still function for this release)
* Feature counting support has been split into “features” and “direct capture features” using Illumina direct capture kits
* Features (e.g. SEA kit and CROP-seq features) continue to be supported by including the “--scrna-feature-barcode-reference=\” option and setting the feature barcode read groups using “--scrna-feature-barcode-groups=\”
* Direct capture mode requires the following options
* “--scrna-enable-direct-capture-mode=true”
* “--scrna-direct-capture-barcode-reference=\”
* “--scrna-direct-capture-barcode-groups=\”
* If direct capture mode is enabled, then metrics previously reported as feature metrics will now be reported as corresponding direct capture metrics
* Feature and direct capture references and read groups can be provided in a single DRAGEN run. In this case, both feature and direct capture metrics will be reported simultaneously.
* **Metric updates**
* Changed SINGLE-CELL RNA → SCRNA for all sections in scRNA\_metrics.csv
* Reads missing barcodes → Reads missing cellular or molecular barcodes
* Filtered non-matching reads (split into 2 metrics) → Filtered intergenic reads AND Filtered unmapped reads
* Feature reads with invalid barcodes → Feature reads with non-matching barcodes
* Feature matching reads → Feature reads matching feature sequence
* Feature non-matching reads (is either renamed or split into 2 metrics depending on if "feature barcode reference" target column is populated) → (if target column not populated) "Feature reads not matching to features" OR (if target column is populated) "Feature reads not matching target sequence" AND "Feature reads matching target but not feature sequence"
* Fraction of feature reads in passing cells → Feature reads in passing cells (now contains both a count and a fraction)
* Total features detected → Total features detected (now contains both a count and a fraction)
* **New feature metrics added**
* Feature reads missing cellular or molecular barcodes
* Feature reads with corrected barcodes
* Feature reads with valid molecular identifier sequences
* Feature reads with corrected molecular identifier sequences
* Feature reads with invalid molecular identifier sequences
* Feature reads matching to features in passing cells (contains both a count and a fraction)
* Total features in reference
* Passing cells with feature matching reads
* Median feature molecules in passing cells with feature matching reads
* Median features in passing cells with feature matching reads
* **New direct capture metrics added (replacing CRISPR metrics) – matches all feature metrics reported except with one extra metric**
* Direct capture reads missing cellular or molecular barcodes
* Direct capture reads with exactly matching barcodes
* Direct capture reads with corrected barcodes
* Direct capture reads with non-matching barcodes
* Total input direct capture reads
* Total barcoded direct capture reads
* Direct capture reads matching feature sequence
* Direct capture reads matching target but not feature sequence
* Direct capture reads not matching target sequence
* Total direct capture reads
* Direct capture tag matching rate (extra metric not in feature metrics)
* Direct capture reads with valid molecular identifier sequences
* Direct capture reads with corrected molecular identifier sequences
* Direct capture reads with invalid molecular identifier sequences
* Total direct capture molecules
* Direct capture sequencing saturation
* Direct capture reads in passing cells
* Direct capture reads matching to features in passing cells
* Mean direct capture reads per cell (Total input direct capture reads / Passing cells)
* Median direct capture molecules per passing cell
* Median direct capture features per passing cell
* Total direct capture features in reference
* Total direct capture features detected
* Passing cells with direct capture matching reads
* Median direct capture molecules in passing cells with direct capture matching reads
* Median direct capture features in passing cells with direct capture matching reads
Metrics per read group have been added
* Additional metrics for each read group have been added to a new \.scRNA.read\_group\_metrics.csv file. These metrics are meant to help with per-read group QA.
* For gene expression read groups, the following metrics are reported (descriptions match the corresponding scRNA\_metrics items):
* scRNA Barcode Read Metrics
* Reads missing cellular or molecular barcodes
* Reads with exactly matching barcodes
* Reads with corrected barcodes
* Reads with non-matching barcodes
* Total input reads
* Total barcoded reads
* scRNA Alignment Metrics
* Unique exon matching reads
* Unique intron matching reads
* Filtered antisense reads
* Filtered ambiguously matching reads
* Filtered low MAPQ reads
* Filtered intergenic reads
* Filtered unmapped reads
* Mitochondrial reads
* Mapped reads
* Reads processed through mapper
* scRNA Molecule Count Metrics
* Total gene reads
* Total counted gene reads
* Total molecules
* Sequencing saturation
* scRNA Cell Metrics
* Passing cells
* Fraction of reads in passing cells
* Fraction of reads in passing cells that are genic
* Mean reads per cell (Total input reads / Passing cells)
* Median molecules per passing cell
* Median genes per passing cell
* For feature read groups, the following metrics are reported (definitions match the corresponding scRNA\_metrics.csv feature metrics items):
* Feature reads missing cellular or molecular barcodes
* Feature reads with exactly matching barcodes
* Feature reads with corrected barcodes
* Feature reads with non-matching barcodes
* Total input feature reads
* Total barcoded feature reads
* Feature reads matching feature sequence
* Total feature reads
* Mean feature reads per cell (Total input feature reads / Passing cells)
* For direct capture read groups, the following metrics are reported (definitions match the corresponding scRNA\_metrics.csv direct capture metrics items):
* Direct capture reads missing cellular or molecular barcodes
* Direct capture reads with exactly matching barcodes
* Direct capture reads with corrected barcodes
* Direct capture reads with non-matching barcodes
* Total input direct capture reads
* Total barcoded direct capture reads
* Direct capture reads matching feature sequence
* Total direct capture reads
* Direct capture tag matching rate
* Mean direct capture reads per cell (Total input direct capture reads / Passing cells)
Metrics per feature type have been added
* Additional metrics for each feature type in the feature / direct capture barcode reference file have been added to a new \.scRNA.feature\_type\_metrics.csv file. These metrics are meant to help with feature-type QA.
* For non-direct capture features, the following metrics are reported:
* Feature reads matching feature sequence
* Total feature reads
* Median feature molecules per passing cell
* Median features per passing cell
* Total features in reference
* Total features detected
* Passing cells with feature matching reads
* Median feature molecules in passing cells with feature matching reads
* Median features in passing cells with feature matching reads
* For direct capture features, the following metrics are reported:
* Direct capture reads matching feature sequence
* Total direct capture reads
* Median direct capture molecules per passing cell
* Median direct capture features per passing cell
* Total direct capture features in reference
* Total direct capture features detected
* Passing cells with direct capture matching reads
* Median direct capture molecules in passing cells with direct capture matching reads
* Median direct capture features in passing cells with direct capture matching reads
---
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