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Evaluating Sequencing Depth for Targeted Gene Expression Libraries with cellranger targeted-depth

Overview

Cell Ranger 4.0 includes targeted-depth, a lightweight program for summarizing a whole transcriptome analysis (WTA) dataset in the context of a hypothetical Targeted Gene Expression experiment. Given an existing WTA dataset and a target panel CSV file, targeted-depth computes the fraction of reads mapped to targeted genes from the panel. This metric can help in two ways when performing a Targeted Gene Expression experiment based on the analyzed whole transcriptome library (or one from a similar sample):

• The targeted fraction computed from the WTA dataset estimates the fraction of library molecules likely to be recovered from the target enrichment step, which can be used to help select appropriate PCR cycling conditions.
• The targeted fraction is helpful in choosing the sequencing depth of the targeted experiment. The targeted-depth tool provides depth recommendations designed to take advantage of the efficiency enabled by targeting, while sequencing enough to match the sensitivity of the WTA dataset.

Usage

The targeted-depth tool requires two inputs:

• A molecule info H5 file produced by cellranger count from a WTA sample. Both 3' and 5' Gene Expression libraries are compatible, as are samples that include Feature Barcode libraries. For more information on using cellranger count, see Single-Library Analysis with Cell Ranger.
• A target panel CSV file. This file can be taken from the target_panel directory included with Cell Ranger or downloaded from the Panel Selection page. For more details on the use and structure of these files, see Count (Targeted GEX).

The cellranger targeted-depth command can be invoked as follows:

cellranger targeted-depth \
--molecule-h5 sample345/outs/molecule_info.h5 \
--target-panel /opt/cellranger-4.0.0/target_panels/pan_cancer_v1.0_GRCh38-2020-A.target_panel.csv


Instructions can also be seen by displaying the help text: cellranger targeted-depth -h.

Output

Input Sample Metrics

The top section of output from cellranger targeted-depth contains metrics relating to the WTA input sample:

Whole Transcriptome Analysis (WTA) Input Sample Metrics:
---------------------------------------------------------
Estimated Number of Cells                           7,742
Sequencing Saturation                               85.2%
Fraction of Reads from Targeted Genes               4.79%
Number of Reads from Targeted Genes            24,045,376
Mean Reads per Cell from Targeted Genes             3,105


The first four metrics are Gene Expression metrics that do not involve the target gene panel, but are shown to give context to the other results.

The next three metrics quantify the sample's target gene content:

• Fraction of Reads from Targeted Genes: Among all reads from the Gene Expression library, these reads contain a valid cell-barcode, are mapped confidently to a targeted gene in the transcriptome, and are not removed during UMI counting due to annotation disagreements.
• Number of Reads from Targeted Genes: Number of reads counted towards targeted gene UMIs, i.e., the numerator of the fraction in the metric above.
• Mean Reads per Cell from Targeted Genes: Equal to Number of Reads from Targeted Genes divided by Estimated Number of Cells. This number is the effective sequencing depth of the targeted portion of the whole transcriptome library.

Recommended Sequencing Depths

The final section of the output indicates recommended sequencing depths for a Targeted Gene Expression library enriched from the analyzed whole transcriptome library (or one from a similar sample):

Targeted GEX Recommended Sequencing Depths:
---------------------------------------------------------
---------------------------------------------------------
Original      6,211                   48,090,752
20k rpc       1,914                   14,822,813
50k rpc       4,786                   37,057,032
The recommended Targeted Gene Expression sequencing depth
is calculated as 2.0 * WTA Depth * Fraction of Reads from
Targeted Genes. The 2.0 depth adjustment factor can help
cannot be mapped confidently to targeted genes. These are
approximate estimates, and final results may vary.


The recommended depths in the two columns above are computed as follows, based on the targeted fraction and depth (Mean Reads per Cell) of the WTA sample:

Recommended Mean Reads per Cell = 2.0 * [WTA Depth] * [WTA Fraction of Reads from Targeted Genes]
Recommended Total Reads = [Recommended Mean Reads per Cell] * [WTA Estimated Number of Cells]


The recommended Mean Reads per Cell is also equal to twice the Mean Reads per Cell from Targeted Genes in the WTA sample. The depth adjustment factor of 2 is used to provide a conservative recommendation. For example, this WTA sample had 64,887 mean reads per cell (rpc) and 4.79% of reads from targeted genes, translating to a recommendation of 6,211 rpc for the Targeted Gene Expression library, or about 48 million reads if the number of recovered cells matches the WTA sample.

Recommendations are also given to approximately match the sensitivity of a whole transcriptome library sequenced to a depth of 20k rpc (the minimum recommended depth for whole transcriptome libraries) or 50k rpc.

Saving Output to a File

Output can be saved to a file instead of output to the console by appending > followed by a filename:

cellranger targeted-depth \
--molecule-h5 sample345/outs/molecule_info.h5 \
--target-panel /opt/cellranger-4.0.0/target_panels/pan_cancer_v1.0_GRCh38-2020-A.target_panel.csv \
> sample345_pan_cancer_depth.txt


Common errors

Incompatible reference and target gene panel: The molecule info H5 file must be created using a reference genome compatible with the target gene panel. For the pre-designed gene panels, the required reference version is GRCh38 2020-A, which can be obtained from the Downloads page. An incompatible reference genome generates an error like this:

error: The gene ENSG00000286522 from the target panel csv is not present
in the reference transcriptome used by the molecule info h5 file.