Cytoprofiling Run Output Files

The following is an example run output directory of a successful cytoprofiling run:

example-storage-location
└── DemoInstrumentName
    └── 20240506_DemoInstrumentName_ExampleRunName
        ├── BaseCalling
        │   ├── BaseCalls
        │   │   └── BXX_CXXX.zip ... (for n cycles)
        │   ├── CellXform
        │   │   └── BXX ... (for n batches)
        │   │       └── LXRXXCXXS1.xform ... (for n tiles)
        │   ├── Location
        │   │   └── BXX ... (for n batches)
        │   │       └── LXRXXCXXS1ZXX.loc ... (for n tiles)
        │   └── Filter
        │       └── BXX ... (for n batches)
        │           └── LXRXXCXXS1ZXX.filter ... (for n tiles)
        ├── Cell Segmentation
        │   └── WellXX ... (for n wells)
        │       ├── LXRXXCXXS1_Cell.tif ... (for n tiles)
        │       └── LXRXXCXXS1_Nuclear.tif ... (for n tiles)
        ├── Thumbnails
        │   └── WellXX_Thumbnail.png ... (for n wells)
        ├── Projection
        │   └── WellXX ... (for n wells)
        │       └──BXX_LXRXXCXXS1_Target.tif ... (for each target and n tiles)
        ├── Cytoprofiling
        │   ├── Instrument
        │   │   ├── AverageNormWellStats.csv
        │   │   ├── RawCellStats.csv
        │   │   ├── RawCellStats.parquet
        │   │   ├── RunStats.json
        │   │   ├── Versions.json
        │   │   └── Wells
        │   │       └── WellXX ... (for n wells)
        │   │           └── BXX ... (for n batches)
        │   │               └── LXRXXCXXS1_barcodes.parquet ... (for n tiles)
        ├── RunManifest.csv
        ├── RunManifest.json
        ├── RunParameters.json
        ├── Panel.json
        ├── RunStats.bin
        ├── RunAnalysisFilesUploaded.json
        └── RunUploaded.json

Run Output Files

The following table defines the key cytoprofiling run output files from an AVIT24 System. Parquet files are column-based files that efficiently store data. For more information, see the Apache Parquet Documentation.

Directory and File Name	File Format	Description	Quantity
{root}/BaseCalling/BaseCalls/{batch}_C{cycle}.zip	Binary	Reports the raw cytoprofiling base call data	One per batch per cycle
{root}/BaseCalling/Filter/{batch}/{tile}.filter	Binary	For use with future applications	One per tile per batch
{root}/BaseCalling/CellXform/{batch}/{tile}.xform	Binary	Transforms polony locations onto cells	One per tile per batch
{root}/BaseCalling/Location/{batch}/{tile}.loc	Binary	Identifies polony locations on the flow cell	One per tile per batch
{root}/BaseCalling/RunStats.bin	Log	Internal troubleshooting log of offline runs	One per run
{root}/CellSegmentation/{well}/{tile}_Cell.tif	TIF	Cell segmentation masks for a well, where the value for a pixel in a cell is the cell ID	One per tile per well
{root}/CellSegmentation/{well}/{tile}_Nuclear.tif	TIF	Nuclear segmentation masks for a well, where the value for a pixel in a nucleus is 1	One per tile per well
{root}/Cytoprofiling/Instrument/AverageNormWellStats.csv	CSV	Reports filtered and average metrics for each well in the run	One per run
{root}/Cytoprofiling/Instrument/Versions.json	JSON	Reports the version number for CSV output files and bundled software programs	One per run
{root}/Cytoprofiling/Instrument/RawCellStats.csv	CSV	Reports values per cell for all morphology features and raw target counts in a run	One per run
{root}/Cytoprofiling/Instrument/RawCellStats.parquet	Parquet	R values per cell for all morphology features and raw target counts in a run	One per run
{root}/Cytoprofiling/Instrument/RunStats.json	JSON	Reports run metrics	One per run
{root}/Cytoprofiling/Instrument/Wells/ {well}/{batch}/{tile}_barcodes.parquet	Parquet	Barcoding information for each polony in a tile	One per tile per batch per well
{root}/Panel.json	JSON	Records target detection information for the run	One per run
{root}/Projection/{well}/{batch}_{tile}_{target}.tif	TIF	Z-projected images of cell paint targets	One per target per tile
{root}/QC_S12_Avid/B02/C001/L1RXXCXXSXZ00_GRN_F4.tif	TIF	Internal support and run diagnostics files	One per tile per run
{root}/RunManifest.csv.	CSV	Manifest that records biological sample information and well mapping	One per run
{root}/RunManifest.json	JSON	Version of the run manifest that is reserved for Element processes	One per run
{root}/RunParameters.json	JSON	Records information about the run configuration	One per run
{root}/RunAnalysisFilesUploaded.json	JSON	Written after the last analysis file is transferred and post-run analysis may begin	One per run
{root}/RunUploaded.json	JSON	The last file transferred and marks run completion	One per run
{root}/Thumbnails/{well}_Thumbnail.png	PNG	Thumbnail image for a well	One per well

Metrics

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

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

RunStats File

The following table defines the fields that are listed in the RunStats.json file:

Field	Description
AnalysisID	The identifier for the analysis that is generated by Cells2Parquet and assigned by the analysis software
AnalysisVersion	The Cells2Stats or Molecule software version
AssignedCountPerMM2	The target count per cell area mm² that is reported for each control target in the panel
AssignedCountsPerMM2	The target counts per cell area mm² of all barcoding targets
AverageAssignedCountsPerMM2	The target counts per cell area mm² of all barcoding targets, averaged across wells
Batches	Identifies a set of information for each batch
BatchName	The batch name (string)
CellCount	The number of cells that are identifid by cell segmentation (integer)
ControlType	The type of control target (for example, Negative Control 1)
Count	The number of polonies that had a specific sequence basecalled that did not match any target's expected sequence within the allowable mismatch threshold (integer)
DemuxStats	The statistics that are related to the demultiplexing process
ExpectedSequence	The expected barcode sequence for a target (string)
FileVersion	The file format version such as 1.2.0 (string)
FlowCellID	The flow cell identifier (string)
MeanAssignedCountPerCell	The mean number of polonies that are assigned to a target sequence within a cell, across all cells (float)
MedianCellDiameter	The median diameter of cells after cell segmentation (float)
NumPolonies	The number of polonies (integer)
PercentAssignedReads	The percentage of reads that are assigned to a target (float)
PercentConfluency	The percent of the flow cell culture area that is covered by cells (float)
PercentMismatch	The percentage of assigned reads that have a mismatch of 1 or 2 bases in their base calls relative to the reference (float)
PercentNucleatedCells	The percentage of cells that have an identified nucleus
RunID	The run ID (string)
RunName	The run name (string)
Targets	Identifies a set of information for each target
TargetName	The target name (string)
Sequence	The sequence that was basecalled but remained unassigned for a polony because it did not match an expected sequence for any target in the panel (string)
UnassignedSequences	Identifies a set of information for sequences that were not assigned
WellLocation	The location ID of a well. A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1, and F2 are valid values
Wells	Identifies a set of information for each well that was used

RawCellStats Files

The following table defines the fixed fields that are listed in the RawCellStats.csv and RawCellStats.parquet files and are the same for every run:

Field	Description
Area	The area of the cell in pixels
AreaUm	The area of the cell in microns (μm²), which is converted from pixels
Cell	The unique ID of the cell within the run. The cell ID is equal to the sum of (tile_index * $2^{16}$) and the tile-specific local cell ID. This allows cell IDs to be computed independently per tile and ensures that the tile-specific cell ID can be retrieved from the cell ID
NuclearArea	The area of the nucleus in pixels
NuclearAreaUm	The area of the nucleus in microns (μm²), which is converted from pixels
Tile	The specific imaging tile or subregion that the cell was imaged from
Well	The position of the well in the plate. This is calculated from the row and column number (for example, A1)
WellLabel	The label of the well, based on user input from the run manifest. If there is no user input, then this is a duplicate of the Well column
X	The X-coordinate of the cell in pixels, within the tile or image. This is the distance from the tile origin
Xum	The X-coordinate of the cell in microns (μm), which is converted from pixels. This is the distance from the tile origin
Y	The Y-coordinate of the cell in pixels, within the tile or image. This is the distance from the tile origin
Yum	The Y-coordinate of the cell in microns (μm), which is converted from pixels. This is the distance from the tile origin

The following table defines the variable fields that are listed in the RawCellStats.csv and RawCellStats.parquet files and are based on the barcoding and cell paint targets within a run:

Field	Description
Cellular Counts for Barcoding Targets	The total cellular counts, with nuclear counts included, of each barcoding target. The column name is based on the target and batch and is shown in the format of {target.batch} (for example, ATF2KT1.BO1).
Cellular Intensity for Cell Paint Targets	The background-subtracted sum of cellular intensity for each cell paint target (for example, Mitochondria.CP02).
Morphology Metrics for Cell Paint Targets	The morphology metric output for each cell paint target. The column name is based on the metric, target, and batch and is shown in the format of metric_target.batch (for example, Intensity_MeanIntensityEdge_Mitochondria.CP02). Metrics come from the following CellProfiler modules: MeasureObjectSizeShape MeasureGranularity MeasureObjectIntensity MeasureObjectIntensityDistribution MeasureTexture Certain metrics 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 morphology metrics, see the CellProfiler Manual measurement information.
Nuclear Counts for Barcoding Targets	The nuclear counts of each barcoding target. The column name is based on the target and batch and is shown in the format of {target_Nuclear.batch} (for example, AKT1_Nuclear.BO1).
Nuclear Intensity for Cell Paint Targets	The background-subtracted sum of nuclear intensity for each cell paint target (for example, Mitochondria_Nuclear.CP02).

Barcodes Parquet Files

The {tile}_barcodes.parquet files provide the raw barcode data for each tile in a batch. The files indicate the location of targets in different cells.

The following table defines the columns in the parquet files.

Field	Description	Data Type
BarcodeIndex	A barcode ID number that corresponds to the order of targets for a batch as listed in the Panel.json file. A value of 0 indicates an unassigned barcode.	Int16
Cell	A tile-specific ID that is associated with a cell. The Cell ID in barcode parquet files differs from the Cell ID for the run in other parquet files.	Int16
IsNuclear	Indicates whether the barcode is in the nucleus of the cell	Boolean
X	The position of the barcode on the X-axis of the tile	UInt16
X μm	The X position in microns	Float
Y	The position of the barcode on the Y-axis of the tile	UInt16
Y μm	The Y position in microns	Float
Z	The position of the barcode on the Z-axis of the tile	String
Z μm	The Z position in microns	Float

Panel File

The Panel.json file contains target information for each batch in the run. Each section of the file defines information for batches and targets, such as ImagingPrimerTubes, BarcodingPrimerTubes, ImagingTargets, and BarcodingTargets.

The following tables describe the Panel.json file information:

Field	Description
AnalysisSettings	Identifies an array of settings for the run analysis
Application	The application type for the run, such as Counting
CellBoundaryTarget	Identifies the target that is used to generate cell segmentation masks
FileVersion	The Panel.json file version for the run
ID	A unique ID that is assigned to the panel
KitType	Specifies the type of reagent or assay kit that is used for the run.
Name	The name of the cytoprofiling kit that is associated with the panel
NuclearTarget	Identifies the target that is used to generate nuclear segmentation masks
PanelCartridgePartNumbers	Identifies a set of information that lists the cartridge part numbers for the panel
SpikeInId	The ID of your custom add-on protein panel JSON
SpikeInName	The name of your custom add-on protein panel JSON
SupplementaryCellBoundaryTarget	Identifies an optional additional target that is used to generate cell segmentation masks

Primer Tube Sections

The following table defines information in the ImagingPrimerTubes and BarcodingPrimerTubes sections of the Panel.json file. These sections provide information about the settings for different batches.

Field	Description
BarcodeMask	The mask that is used to support barcoding for target analysis
BarcodingPrimerTubes	Identifies a set of information about the settings for batches with targets for analysis
BatchName	The name of a specific batch
DefaultMismatch	The number of base mismatches that are permitted to assign a barcode to a target, which is typically 2
ImagingPrimerTubes	Identifies a set of information about the settings for batches related to cell paint
MinCycles	The minimum number of cycles for a specific batch
PMGMask	A base mask that is used to generate the map of polonies
RunOrder	Identifies the ordinal position for a batch in the run
Type	The type of batch relative to amplification, PreAmp or PostAmp

Target Sections

The following table defines information in the ImagingTargets and BarcodingTargets sections of the Panel.json file. The sections provide information about the targets in each batch.

Field	Description
Analyses	Identifies an array with the types of analysis for a batch
Barcode	The barcode of bases for a specific target in a batch
BarcodingTargets	Identifies the set of information for targets in each batch for analysis
Base	The base for a particular cycle in a batch
BatchName	The name for a specific batch
ControlType	Identifies the type of control for a target in a batch, if applicable
Cycle	The cycle that a particular base uses in a batch
CycleBases	Identifies a set of information that defines the base in a specific batch cycle
ImagingTargets	Identifies the set of information for targets in each batch that is related to amplification
ProbeConcentration	The concentration of the probe for a specific target in a batch
Target	The name of a target for a batch
TargetType	The type of target for a batch as CellPaint, Protein or Transcript

Run Parameters

The RunParameters.json file contains a record of the input information for a run. When you evaluate run performance or troubleshoot, review this file to confirm that the correct parameters were used for the run.

The following table describes the information in the RunParameters.json file:

Field	Description
AdvancedSettings	Includes information about advanced run settings, such as custom recipes
AnalysisLanes	The lanes that the run uses, such as 1,2, or 1+2
ApplicationName	The type of application for the run, such as Counting
BarcodeStr	The barcode number for a consumable, which the instrument scans and identifies during consumable loading
BaseForChannels	Identifies the set of bases for the channels
Batches	Identifies the set of batches for the run
Buffer	Identifies a set of information that describes the buffer for the run
Channels	Identifies the colors to associate with channels
ColorForChannels	Identifies the channels to associate with bases and colors
Consumables	Identifies the set of information for run consumables
CustomRecipeName	The name of the custom recipe file that is uploaded in Advanced Run Settings
Cycles	Identifies the number of cycles in a batch
CycleFormat	Defines the format in which cycles appear
Date	Timestamp for the run
Expiration	The expiration timestamp for a consumable, which the instrument identifies or a user inputs during consumable loading
ExpirationStr	An expiration ID number for a consumable that is associated with the expiration date
FileVersion	The version of the RunParameters.json file for the run
Flowcell	Identifies a set of information that describes the flow cell for the run
ImageHeight	The height of the image in pixels
ImageInfo	Identifies the set of information that defines characteristics of the cytoprofiling image
ImageWidth	The width of the image in pixels
Lanes	Defines an array that lists the lanes of the flow cell for the run
InstrumentName	The name of the instrument for the run
LotNumber	The lot number for a consumable, which the instrument identifies during consumable loading before the run
Name	The name of a tile in a set of Tiles
OperatorName	The name of the person that set up the run on the instrument
PanelCartridge	Identifies a set of information that describes the sequencing cartridge for the run
PanelName	The name of the panel that is used for the run
PartNumber	The part number for a consumable, which the instrument scans or a user inputs during consumable loading
PlatformVersion	The version of AVITI OS for the run
PMGMask	A base mask that is used to generate the map of polonies
RecipeExecutionID	A UUID for the run recipe, which governs the stages of a sequencing run
RecipeValues	Contains additional values for the recipe that the run uses, such as a filterMask value
RunDescription	An optional description for the run
RunFolderName	The name of the output folder that AVITI OS creates for the run
RunID	A UUID assigned to the run
RunName	A text-based run identifier that is entered by the operator
RunOrder	Identifies the ordinal position for a batch in the run
RunType	The type of AVITI24 System run, Cytoprofiling
SerialNumber	The serial number for a consumable, which the instrument scans or a user inputs during consumable loading
Side	The side of the instrument that the run uses, such as SideA or SideB
StorageConnectionID	A UUID for the storage connection that the run uses
Tags	The tags that the Operator applies to the run, as applicable
ThroughputSelection	Identifies the path for the text file that is used to determine throughput selection for the run
Tiles	Identifies a set of tiles on the flow cell for a well
Type	Identifies the type of batch, such as BarcodingBatch , PreAmpImagingBatch, or PostAmpImagingBatch
WellLayout	Identifies the well layout, such as 48-well, 12-well, or 1-well
Wells	Identifies a set of information for the wells in the run
WellLocation	Identifies the well for the following Tiles listed
XMillimeters	The well position in millimeters on the X-axis of the image
YMillimeters	The well position in millimeters on the Y-axis of the image
ZPositions	Identifies the Z-positions that are associated with each batch in the run
Zs	Identifies the order of batches for the run to associate them with Z-positions

Run Uploaded

The RunUploaded.json file indicates the completion of the run. The file contains high-level information about the run and an outcome field that confirms the run outcome. AVITI OS always transfers this file last, which allows its creation to serve as a trigger to start automated downstream analysis.

The following table defines the fields in the RunUploaded.json file:

Field	Description
version	The version of the RunUploaded.json file
instrument	The name of the instrument for the run
instrumentId	A UUID for the instrument
outcome	The final outcome of the run, such as OutcomeCompleted, OutcomeStopped, or OutcomeFailed
runType	The type of AVITI System run, such as Cytoprofiling
recipeExecutionId	A UUID for the run recipe that is sourced from RunParameters.json
runID	A UUID assigned to the run that is sourced from RunParameters.json

Run Analysis Files Uploaded

The RunAnalysisFilesUploaded.json file indicates that the required data to begin downstream analysis was successfully transferred to the output location. This file contains high-level information about the run and an outcome field that confirms the run outcome. AVITI OS always transfers this file only after the required files to begin analysis are confirmed to be transferred. This allows the file creation to trigger the start of automated downstream analysis.