10x Genomics
Visium Spatial Gene Expression

Space Ranger2.0 (latest), printed on 11/22/2024

Comparing Targeted and Parent Samples with spaceranger targeted-compare

Table of Contents

• Requirements
• Command line interface
• Pipeline outputs

Targeted Gene Expression libraries are derived by performing hybrid capture on a Spatial Gene Expression library. This starting input library, referred to as the parent library, therefore contains the molecules present in the targeted library. While it is not necessary to sequence this parent sample when analyzing Targeted Gene Expression data, if both the targeted and parent samples have been sequenced, the targeted-compare pipeline may be used to directly compare the two datasets. This pipeline can be used to assess targeting performance with greater accuracy than when only the targeted data are known. This comparative analysis provides quality control metrics to verify the extent to which targeted genes were enriched and the parent sample data were recovered.

Requirements

First, run spaceranger count on the parent sample (see Single-Library Analysis with Space Ranger). Then run spaceranger count --target-panel target_panel.csv (see Targeted Gene Expression Analysis). The targeted and parent samples must be run using the same transcriptome reference. The code in red indicates user specific inputs. For example:

$ cd /home/jdoe/runs
$ spaceranger count --id=targeted_sample --target-panel target_panel.csv ...
... wait for pipeline to finish ...
$ spaceranger count --id=parent_sample ...
... wait for pipeline to finish ...

After the above two analyses are complete, run targeted-compare as outlined in the following section.

Command line interface

These are the command line arguments (also accessible by running spaceranger targeted-compare --help):

Resuming the above example, run spaceranger targeted-compare as follows. The code in red indicates user specific inputs.

$ cd /home/jdoe/runs
$ spaceranger targeted-compare --id=TGTCMP123 \
 --targeted=targeted_sample/outs/molecule_info.h5 \
 --parent=parent_sample/outs/molecule_info.h5 \
 --target-panel target_panel.csv

The pipeline will begin to run, creating a new folder named with the ID you specified (e.g. /home/jdoe/runs/TGTCMP123) for its output. If this folder already exists, spaceranger will assume it is from an incomplete targeted-compare run and attempt to resume running it.

Pipeline outputs

A successful run should conclude with a message similar to this:

Outputs:
- Sample comparison summary HTML: /home/jdoe/runs/TGTCMP123/outs/web_summary.html
- Sample comparison summary CSV:  /home/jdoe/runs/TGTCMP123/outs/metrics_summary.csv
- Per barcode metrics CSV:        /home/jdoe/runs/TGTCMP123/outs/barcode_summary.csv
- Per feature metrics CSV:        /home/jdoe/runs/TGTCMP123/outs/feature_summary.csv
 
Pipestance completed successfully!

The output folder (e.g. runs/TGTCMP123/outs) will contain the following files:

See Targeted-Compare Outputs for more information on these output files.