Output files

The following table lists the files and folders that Cells2Stats outputs. Parquet files are column-based files that efficiently store data. For more information, see the Apache Parquet Documentation.

File	Directory and File Name	Description	Quantity
Spatial Data Object	{root}/SpatialData/RunID.zarr.zip	Zarr-based structured object containing all modalities from a run for downstream analysis and visualization. For more information, see Spatial Data Object.	One per run
CytoCanvas Visualization File	{root}/SpatialData/cyto.viz	File for use by the Element CytoCanvas™ tool	One per run
Run Manifest, JSON	{root}/RunManifest.json	JSON file that is reserved for Element Biosciences™ processes	One per run
Run Parameters	{root}/RunParameters.json	JSON file that records information about the run configuration	One per run
Panel	{root}/Panel.json	JSON file that records information about the targets for the run	One per run
Run Stats	{root}/RunStats.json	JSON file that records overall statistics about the run	One per run
Average Normalized Well Statistics	{root}/AverageNormWellStats.csv	Filtered and average metrics for each well in the run	One per run
Raw Cell Statistics, CSV	{root}/RawCellStats.csv	CSV file that reports values per cell for all morphology features and raw target counts in a run	One per run
Versions, JSON	{root}/Versions.json	File that reports the version number for CSV output files and bundled software programs	One per run
Barcodes	{root}/Wells/{well}/{batch}/{tile}_barcodes.parquet	Parquet files that provide barcoding information for each polony in a tile	One per tile per batch per well
MultiQC Report, HTML	{root}/multiqc_report.html	Interactive HTML quality control (QC) summary that is generated from the RunStats.json summary. Not present if --skip-html-report is used	One per run
Run Manifest, CSV	{root}/RunManifest.csv	Run manifest for the Cells2Stats execution	One per run
Cell Segmentation Mask	{run}/CellSegmentation/{well}/{tile}_Cell.tif	Cell segmentation masks for a well, where the Cell ID is the value for a pixel in a cell	One per tile per well
Nuclear Segmentation Mask	{run}/CellSegmentation/{well}/{tile}_Nuclear.tif	Nuclear segmentation masks for a well, where 1 is the value for a pixel in a nucleus	One per tile per well
Target Counts, JSON	{root}/TargetCounts.json	(For Teton Atlas™ runs with Target Cell Assignment enabled) JSON file that records read counts per spacer target across the run. Use this to assess library representation and identify low-count or absent spacers.	One per run
Target Cell Assignment Manifest, CSV	{root}/TargetCellAssignmentManifest.csv	Copy of the target cell assignment manifest used in this run. Confirms which spacer sequences and settings were applied.	One per run
Target Cell Assignment Manifest, JSON	{root}/TargetCellAssignmentManifest.json	JSON file that is reserved for Element processes	One per run
Antibody Screen Kit Report, CSV	{root}/AntibodyScreenKitReport.csv	(For Teton™ Custom Screen runs) Report with cell, quality control, and custom protein information	One per run

Target cell assignment output details

When target cell assignment is enabled for at least one target site in a run, Cells2Stats populates target cell assignment-specific fields in RunStats.json and generates the following additional output files. For more information on how cell assignment works, see Cell assignment logic.

Cells are classified as pure (single spacer detected), mixed (multiple spacers, one dominant), or unassigned based on a confidence score. For a full explanation of these categories and how cell assignment is calculated, see Pure cells vs. mixed cells.

Key target cell assignment output files

File	What to use it for
TargetCounts.json	See total reads detected per spacer. High dropout (many spacers with zero reads) may indicate a manifest or TargetMask issue.
TargetCellAssignmentManifest.csv	Confirm which sequences and settings were applied. Compare against your source library file to catch discrepancies.
Panel.json	Contains the TargetMask that was applied per target site. Check here if assignment rates are unexpectedly low.
RunStats.json	Contains all target cell assignment-specific quality metrics. See Target cell assignment metrics.
RawCellStats.csv	Per-cell data including raw target read counts at each target site. Use for downstream per-cell analysis.

Target cell assignment metrics in RunStats.json

Field	Description	What to watch for
PercentAssignedPureCells	Cells assigned to a single dominant spacer (confidence >0.5, meets minimum read count)	Primary metric — aim as high as possible for your MOI
PercentAssignedMixedCells	Cells with multiple spacers detected, but one is dominant (confidence >0.5)	Expected to increase with higher MOI; still valid data
PercentUnassignedMixedCells	Cells with multiple spacers; no spacer exceeds 50% confidence	High values suggest MOI may be too high for reliable single-guide assignment
PercentUnassignedLowCountCells	Spacer was matched but read count is below the minimum threshold	High values suggest low expression or insufficient sequencing depth
PercentTargetDropout	Percentage of spacers in the manifest not assigned to any cell	High dropout may indicate library representation problems
MedianAbundantTargetCount	Median read count of dominant spacer in assigned cells	Indicator of per-cell sequencing depth at target site
MeanUniqueTargetsPerCell	Mean number of unique spacers detected per cell	Values substantially above 1 suggest high MOI
UnassignedSequences	List of sequences detected at target site but not matching any manifest entry	Primary troubleshooting resource — see Using UnassignedSequences for troubleshooting

Using UnassignedSequences for troubleshooting

The UnassignedSequences field lists actual nucleotide sequences of reads detected at the target locus that did not match any spacer in the manifest. Inspect this field when assignment rates are low.

What you see	Likely cause	Resolution
Sequences that look like your spacers but reversed	Orientation error in manifest	Re-enter spacers in 5' to 3' direction
Spacer-like sequences of a different length	TargetMask or sequence length mismatch	Confirm the Y cycle count in TargetMask equals spacer length
Recognizable spacers from your library	Spacers missing from manifest	Add missing entries to [TARGET INFORMATION]
Unrecognizable sequences	Off-target reads or artifacts	Contact Element Technical Support

For more information on assignment thresholds and additional troubleshooting, see Cell assignment logic.

MultiQC Reports

MultiQC reports, which are generated by Seqera and designed by Element Biosciences, are available for use with Bases2Fastq™ and Cells2Stats. MultiQC reports analyze results and statistics from bioinformatics tool outputs, such as log files and console outputs. These reports help summarize experiments that contain multiple samples and multiple analysis steps and are designed to be placed at the end of pipelines or to be run manually when you finish running your tools. Furthermore, MultiQC reports contain the parsed data in a nice friendly format, ready for any further downstream analysis. For more information, see MultiQC Documentation.

Metrics

The output files contain a variety of metrics such as tile-specific and average metrics.

• The RawCellStats.csv and RawCellStats.parquet contain a full set of morphology and quantification metrics for each target and batch.
• The AverageNormWellStats.csv file provides the averages of these metrics for each well. Metrics that end with .std provide the standard deviation for the metric.

Metrics files report metrics from the following CellProfiler modules, unless the user runs the --skip-cellprofiler argument:

• MeasureObjectSizeShape
• MeasureGranularity
• MeasureObjectIntensity
• MeasureObjectIntensityDistribution
• MeasureTexture

Certain metrics from these modules are not available in these files. For example, the output files do not report Zernike metrics. In some files, columns for Z-axis metrics appear with values of 0. Z-axis metrics are not available in the RawCellStats.csv and RawCellStats.parquet files because they are not relevant to the analysis output.

For more information on cytoprofiling metrics, see the measurement information in the CellProfiler Manuals.

Run statistics

The RunStats.json provides overall run statistics. The following table describes the run statistics fields:

Field	Description	Data Type
AnalysisID	Unique ID for the analysis	String
AnalysisVersion	Version of the analysis software	String
AssignedCountPerMM2	Target counts per mm² of the cell area of a quality control target. Teton and Teton Custom Screen with quality control targets.	Float
AssignedCountsPerMM2	Target counts per mm² of the cell area of all barcoding targets.	Float
AverageAssignedCountsPerMM2	Target counts per mm² of the cell area of all barcoding targets, averaged across wells.	Float
BatchName	Name of the batch	String
Batches	List of batch-specific statistics	Array of Objects
Count	Count of how many times a particular unassigned sequence appears	Integer
DemuxStats	Statistics related to demultiplexing	Object
ExpectedSequence	Expected DNA sequence for the target	String
ExtraCellularRatio	The ratio of extracellular to intracellular assigned target density	Float
FileVersion	Version of the file format	String
FlowCellID	ID of the flow cell that is used	String
ManifestLabel	Label of the custom protein in the manifest when a custom protein is included. Teton Custom Screen with antibody targets.	String
MeanAssignedBarcodingCountPerCell	Mean number of polonies assigned to a target sequence in a cell per well	Float
MeanUniqueTargetsPerCell	The mean number of unique targets found in a cell. Teton Atlas with Target Cell Assignment enabled.	Float
MedianAbundantTargetCount	The median count of the target that is assigned to cells. Teton Atlas with Target Cell Assignment enabled.	Float
MedianCellDiameter	Median diameter of a cell after cell segmentation.	Float
NuclearFraction	Fraction of the cellular assigned reads of the target that are in the nucleus. Teton Custom Screen with antibody targets.	Float
NucleatedRate	The fraction of cells that contain a segmented nucleus	Float
NumPolonies (Total)	Number of polonies that are detected	Integer
NumPolonies (Targets)	Number of polonies that are assigned to this target	Integer
NumPolonies (Wells)	Number of polonies that are in the well	Integer
PercentAssigned	Percentage of reads that are assigned to a target within the target site	Float
PercentAssignedMixedCells	Percentage of cells that are assigned and mixed with a confidence score above 50%	Float
PercentAssignedPureCells	Percentage of cells that are assigned that are pure with a confidence score above 50%	Float
PercentAssignedReads (Targets)	Percentage of reads that are assigned to targets	Float
PercentAssignedReads (Wells)	Percentage of reads that are assigned to targets in the well	Float
PercentCellularAssigned	Percentage of reads that are assigned to cells	Float
PercentMismatch (Total)	Percentage of reads with at least one base pair mismatch	Float
PercentMismatch (Targets)	Percentage of reads that are assigned to a target with at least one base pair mismatch	Float
PercentMismatch (Wells)	Percentage of reads in the well with at least one base pair mismatch	Float
PercentTargetDropout	Percent of targets not assigned to any cell. Teton Atlas with Target Cell Assignment enabled.	Float
PercentUnassignedLowCountCells	Percentage of cells that are unassigned that are pure but are below the confidence threshold of 50%.	Float
PercentUnassignedMixedCells	Percentage of cells that are unassigned and mixed with a confidence score below 50%.	Float
RunID	Unique ID for the run	String
RunName	Name of the sequencing run	String
Sequence	DNA sequence of unassigned reads	String
TargetName	Name of the sequencing target	String
TargetSiteName	Name of the target site as provided in the Panel.json. Teton Atlas with Target Cell Assignment enabled.	String
Targets	List of target-specific statistics	Array of Objects
UnassignedSequences	List of unassigned sequence details	Array of Objects
WellLocation	Location of the well (for example, A1-F2)	String
Wells	List of well-specific statistics	Array of Objects

Versions

The Versions.json reports the version number for CSV output files and bundled software programs. The following table describes the fields:

Field	Description	Data Type
FileVersion	Overall version of the file	String
FileVersions	Versions of individual files in the dataset	Object
AverageNormWellStats.csv	Version of AverageNormWellStats.csv file	String
RawCellStats.csv	Version of RawCellStats.csv file	String
ProgramVersions	Versions of programs that are used in the analysis	Object
CellProfiler	Version of the CellProfiler program	String

Other files

Some files are copied from the input directory, such as RunManifest.json, RunParameters.json, Panel.json, Cell Segmentation Masks, Nuclear Segmentation Masks, and Barcodes. For more information on these files, see Cytoprofiling Run Output Files.