How do I use Cell Ranger aggr to aggregate Fixed RNA Profiling datasets?

Question: How do I use Cell Ranger aggr to aggregate Fixed RNA Profiling datasets?

Answer: The cellranger aggr pipeline supports aggregation of Fixed RNA Profiling (FRP) with FRP samples as long as they have the same feature reference (+) and/or probe-set (^). It is also possible to run cellranger aggr on FRP with 3' or 5' gene expression data, but the combination of different chemistries is not officially supported. 

Note:

• Supported (validated by 10x Genomics)
• Possible (will run, but not the validated intended usage)

Example 1: aggregating singleplex FRP and singleplex FRP with Antibody Capture samples.In these cases, you may need to re-run an analysis to make sure it is using the same feature reference CSV and/or probe-set CSV file. 

You will need to re-run the singleplex FRP analysis with a feature reference in the [features] section. The [libraries] section will still specify the Gene Expression library; do not specify a Feature Barcode library in this re-analysis. The multi config CSV file should look similar to:

[gene-expression]
reference,
probe-set,/path/to/probe/set

[libraries]
fastq_id,fastqs,feature_types
gex1,/path/to/fastqs,Gene Expression

[features]
reference,/path/to/feature_reference.csv

The paths in bold must match file paths used in the singleplex FRP with Antibody Capture analysis, e.g.,:

[gene-expression]
reference,
probe-set,/path/to/probe/set

[libraries]
fastq_id,fastqs,feature_types
gex1,/path/to/fastqs,Gene Expression
ab1,/path/to/fastqs,Antibody Capture

[features]
reference,/path/to/feature_reference.csv

Similarly, to aggregate singleplex FRP with other singleplex FRP samples or with multiplex FRP samples, the probe-set file must be the same. For example, if your experiment used the human probe set, all Cell Ranger analyses should specify the human probe set file in `probe-set`.

Note that a similar approach is possible for aggregating 3’ or 5’ Gene Expression samples with singleplex Fixed RNA Profiling with Antibody Capture samples, however these combinations are not officially supported and will require careful validation of results.

Example 2: aggregating 3’ Gene Expression and singleplex FRP with Antibody Capture experiments.

You’ll need to re-run the 3’ Gene Expression analysis using the same feature reference and probe set files as those used for the singleplex FRP with Antibody Capture analysis.

The multi config file for the 3’ Gene Expression re-analysis would look like: 

[gene-expression]
reference,
probe-set,/path/to/probe/set

[libraries]
fastq_id,fastqs,feature_types
gex1,/path/to/fastqs,Gene Expression

[features]
reference,/path/to/feature_reference.csv

Then, run the multi pipeline as cellranger multi --id=<name> --csv=<path to config CSV file>.

To aggregate the datasets, you’ll need the molecule_info.h5 files. See Running cellranger aggr for detail.