Settings

By default the scRNA workflow assumes that the overall barcode/UMI sequence is made up of a single-cell barcode (possibly split into multiple blocks) and a single UMI. Enter the following command to identify the location of the single-cell barcode and single-cell UMI in the barcode read:

--single-cell-barcode-position <blockPos>[+<blockPos2>+<blockPos3>...] --single-cell-umi-position <blockPos>

blockPos describes the offset of the first and last inclusive base of the block and is formatted as <startPos>_<endPos>. For example for a library with a 16 bp cell-barcode followed by a 10 bp UMI, enter: --single-cell-barcode-position 0_15 --single-cell-umi-position 16_25. For a library with the cell-barcode split into three blocks of 9 bp separated by fixed linker sequences and an 8 bp UMI, enter --single-cell-barcode-position=0_8+21_29+43_51 --single-cell-umi-position=52_59.

Feature reads are reads with a sequence tag specific to a feature (eg, cell-surface protein or antibody). If performing an scRNA run that contains feature reads with feature-specific UMIs located on Read 2, you can use --single-cell-feature-barcode-r2umi=0_11 to specify a 12 bp feature UMI at the beginning of each feature read.

You can provide a list of cell barcode sequences to include using the following command:

--single-cell-barcode-sequence-whitelist </path/to/barcodeAllowlist.txt>

The file must contain one possible cell barcode sequence per line. You can compress the file with gzip (*.txt.gz). During cell-barcode error correction any observed barcodes that do not match a sequence specified in the file are considered errors. If possible, the barcodes are corrected to a similar allowed sequence. See Barcode Error Correction for more information. If the barcodes cannot be corrected, they are filtered out.

DRAGEN uses a threshold on the number of unique UMIs per cell barcode to determine which barcodes likely correspond to single-cells in the original sample from background noise. The threshold is determined based on the distribution of UMIs per barcode and an expected number of true cells in the sample. For more information on how cell filtering is performed, see Cell Filtering .

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single-cell-number-cells—[Optional] Set the expected number of cells. The default is 3000. Adjust only if the expected number of cells is so far from the default that DRAGEN does not call the correct cell filtering threshold automatically.

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single-cell-threshold— Specify the method for determining the UMI count threshold value. The available values are fixed, ratio, or inflection.

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If using ratio, DRAGEN estimates the expected number of cells as max(T_e, T_m). T_m is a threshold based on a fraction of the UMIs seen in most abundant cell-barcodes. T_e is a threshold based on a fraction of the least abundant expected cell.

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If using inflection, DRAGEN determines the threshold using an algorithm based on the inflection point analysis of the number of cumulative UMI counts of the most UMI abundant cells.

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If using fixed, the UMI count threshold is set so that the expected number of cells pass, rather than estimated dynamically. The exact number of passing cells might be slightly larger than the number of single-cells because of the connection between the UMI count and the different cell barcodes.

To set a specific number of cells ahead of time, use the following command:

--single-cell-threshold=fixed --single-cell-number-cells=X

The command forces the UMI threshold value to pass the top X cells and any extra cells with the same number of unique UMIs.

The following are additional options you can use to configure the Single-Cell RNA Pipeline settings.

Option	Description
rna-libary-type	Set the orientation of transcript reads relative to the genomes. Enter SF for forward, SR for reverse, or U for unstranded. The default is SF.
single-cell-count-introns	Include intronic reads in gene expression estimation. The default false.
qc-enable-depth-metrics	Set to false to disable depth metrics for faster run times. The default is true.
bypass-anchor-mapping	Set to true to disable RNA anchor (two-pass) mapping for increased performance. The default false.

The following is an example command line to run the DRAGEN Single Cell RNA Pipeline.