10x Genomics
Visium Spatial Gene Expression

Space Ranger2.0, printed on 12/26/2024

Pipestance Structure

Table of Contents

• Pipeline structure
• Directory structure
• Commonly generated metadata
• Navigating pipestances

The pipeline output directory, described in Understanding Output, contains all of the data produced by one invocation of a pipeline (a pipestance) as well as rich metadata describing the characteristics of each stage. This directory contains a specific structure used by the Martian pipeline framework to track the state of the pipeline as execution proceeds.

Pipeline structure

Space Ranger's notion of a pipeline is very flexible in that a pipeline can be composed of stages that run stage code or sub-pipelines that may themselves contain stages or sub-pipelines.

Space Ranger pipelines follow the convention that stages are named with verbs (such as, ALIGN_READS, MARK_DUPLICATES, FILTER_BARCODES) and sub-pipelines are named with nouns and prefixed with an underscore (e.g., _BCSORTER). Each stage runs in its own directory bearing its name, and each stage's directory is contained within its parent pipeline's directory.

For example, the spaceranger mkfastq pipeline has the following process graph:

where

• MAKE_FASTQS_CS is the top-level pipeline stage
• MAKE_FASTQS is a sub-pipeline contained in MAKE_FASTQS_CS
• PREPARE_SAMPLESHEET, BCL2FASTQ_WITH_SAMPLESHEET, MAKE_QC_SUMMARY, and MERGE_FASTQS_BY_LANE_SAMPLE are stages contained in the MAKE_FASTQS sub-pipeline.
• MAKE_FASTQS_PREFLIGHT and MAKE_FASTQS_PREFLIGHT_LOCAL are preflight stages, which validate inputs prior to running the other stages. These also belong to MAKE_FASTQS, but have no connections to other stages because they don't produce any outputs.

Directory structure

Every pipestance operates wholly inside of its pipeline output directory. When the pipestance completes, this pipestance output directory contains three outputs: metadata files, the pipestance output file directory, and the top-level pipeline stage directory.

• Metadata files are files prefixed with an underscore (_) and usually contain unstructured text or JSON-encoded arrays and hashes.
• The pipestance output file directory is a directory called outs/ that contains the pipestance's output files.
• The top-level pipeline stage directory is a directory named according to the top-level pipeline stage that contains the child stage directories that compose this pipestance.

The top-level pipeline stage directory is a stage directory that contains any number of child stage directories as well as one stage output directory for each fork run by that stage. There are two possible top-level pipeline stages:

• MAKE_FASTQS_CS for spaceranger mkfastq
• SPATIAL_RNA_COUNTER_CS for spaceranger count

All Space Ranger pipelines contain only single-fork stages, so there is only one fork0 stage output directory within each stage directory. Chunk output directories are a subset of stage output directories that additionally contain runtime information specific to the job or process being run by that chunk such as a process ID or cluster job ID.

For example, the spaceranger mkfastq pipeline's pipeline output directory contains the following directory structure:

Commonly generated metadata

The metadata contained in the pipeline output directory includes:

Stage directories contain stage output directories, stage output files, and the stage directories of any child stages or pipelines.

Stage output directories typically contain:

Chunk output directories are a subset of stage output directories that, in addition to the aforementioned stage output, may contain:

Metadata files should be treated as read-only. Altering the contents of metadata files is not recommended.

Navigating pipestances

Pipestance output directories can demonstrate very complicated structures, and re-attaching the Space Ranger UI is the easiest way to quickly navigate to a pipeline stage of interest and examine its metadata. In the absence of being able to access the UI, the standard find command can quickly return high-level information about a pipestance.

For example, to find the stages that resulted in the overall failure of a pipestance whose output directory is sample345/,

$ find sample345/ -name _errors
sample345/SPATIAL_RNA_COUNTER_CS/SPATIAL_RNA_COUNTER/SUMMARIZE_REPORTS/fork0/chnk0/_errors

This indicates that the failed stage was SUMMARIZE_REPORTS.

It is helpful to view all _errors files' contents at once by piping to xargs cat:

$ find sample345/ -name _errors | xargs cat
 
Traceback (most recent call last):
  File "/home/jdoe/spaceranger-2.0.1/mro/stages/counter/summarize_reports/__init__.py", line 62, in main
    filtered_gene_bc_matrices=args.filtered_gene_bc_matrices)
  File "/home/jdoe/spaceranger-2.0.1/lib/python/cellranger/webshim/common.py", line 625, in build_web_summary_html
    filtered_gene_bc_matrices=filtered_gene_bc_matrices)
  File "/home/jdoe/spaceranger-2.0.1/lib/python/cellranger/webshim/common.py", line 614, in build_web_summary_json
    filtered_gene_bc_matrices=filtered_gene_bc_matrices)
  File "/home/jdoe/spaceranger-2.0.1/lib/python/cellranger/webshim/common.py", line 579, in build_charts
    plot_data = plot_preprocess_func(sample_properties, filtered_matrices, gene_index)
  File "/home/jdoe/spaceranger-2.0.1/lib/python/cellranger/webshim/common.py", line 319, in plot_preprocess
    silhouette_score = metrics.silhouette_score(kmeans_matrix, clusters, metric='cosine')
  File "/home/jdoe/spaceranger-2.0.1/external/anaconda/lib/python2.7/site-packages/sklearn/metrics/cluster/unsupervised.py", line 84, in silhouette_score
    "and less than n_samples - 1" % n_labels)
ValueError: Number of labels is 5 but should be more than 2 and less than n_samples - 1

In the above case, the error is an unhandled exception without obvious cause. Failures such as this should be reported to the 10x Genomics software support team for assistance with diagnosis.

Stages whose stage code run external binaries (for example, the ALIGN_READS stage which runs STAR) often generate output to their stdout and stderr streams. These messages are captured in the _stdout and _stderr metadata files within the chunk output directories, and combining find and xargs cat to examine their contents can also assist with troubleshooting.