Target site settings
Target site settings are defined per-target site and control how cell assignment is performed during Cells2Stats processing and Bases2Fastq™ FASTQ generation when at least one target has completed cell assignment. The most important setting is the TargetMask, which defines which sequencing cycles contain your spacer sequence.
If you omit the [TARGET SITE SETTINGS] section entirely, the system uses the default TargetMask from the panel.json file generated by your published Teton Atlas™ custom workflow design.
A target will undergo target to cell assignment when you selected that option during custom design creation. If unsure, consult the panel.json from the run output and look for the CellAssignment keys to see if it is true or false for the target site of interest.
Columns
The Target site settings section includes the TargetSiteName, SettingName, and Value columns. All columns are required when the section is populated. When the section is omitted, the default settings from the custom workflow design are applied.
| Column | Constraints | Definition | Allowed Values |
|---|---|---|---|
TargetSiteName | Required | The name of the target site that is sequenced in the cytoprofiling run and is present in the associated Teton Atlas custom design. In the case of gRNA sequencing, this refers to the constant scaffold region shared by all gRNAs in a library. | 1-64 case-sensitive alphanumeric characters, hyphens (-), and underscores (_) |
SettingName | Required | The name of the setting | One of allowed settings |
Value | Required | The value that is applied to the setting | The specific value allowed depends on the defined SettingName. See Settings. |
Settings
| Setting | Description | Default | Example usage |
|---|---|---|---|
TargetMask | A base mask that defines which sequencing cycles contain the cell assignment target (spacer) sequence. Used by Cells2Stats for spacer matching during cell assignment. | The target mask from the panel.json file that is supplied when a Teton Atlas custom workflow is created | Y20N* |
FastqMask | A base mask that defines which cycles to include in the per-target FASTQ output file. Does not affect cell assignment. | Same as TargetMask | Y20N* |
TargetMismatchThreshold | Override the system maximum number of allowed mismatch bases when aligning reads to spacer sequences during cell assignment. Allowed values: 0, 1, 2, or 3. The CLI argument --target-mismatch-threshold, if set when executing Cells2Stats, takes precedence over this per-target value. | 1 per 16 bp in spacer, capped at 3:
| 2 |
Understanding the target mask
The TargetMask is the most critical setting for cell assignment. It tells the system which sequencing cycles to search when matching reads against your spacer library. A misaligned mask — one that is too short, starts at the wrong cycle, or includes non-spacer cycles — is the most common cause of near-zero cell assignment rates.
Target mask format
The TargetMask uses base mask notation. For guide RNA cell assignment, the relevant tokens are:
| Token | Meaning |
|---|---|
Y[5] | Include the next 5 cycles — these contain the spacer sequence |
N[5] | Skip the next 5 cycles |
N* | Skip all remaining cycles (wildcard) |
Common examples:
| Spacer design | TargetMask |
|---|---|
| 20 bp spacer, starts at cycle 1 | Y20N* |
| 23 bp spacer, starts at cycle 1 | Y23N* |
| 20 bp spacer, 4 non-spacer cycles precede it | N4Y20N* |
The TargetMask must be at least 4 cycles. It cannot exceed 36 cycles for guide RNA sequencing applications, or 100 cycles for advanced non-guide RNA sequencing applications.
TargetMask must match sequence lengths in the manifest
The number of Y cycles in the TargetMask must equal the length of every spacer sequence for that target site in the [TARGET INFORMATION] section. Different target sites can use different TargetMask values. This is validated per site when the manifest is attached to a design in Custom Designer — mismatches prevent the manifest from being accepted.
For example, a TargetMask of Y20N* requires all sequences in [TARGET INFORMATION] to be exactly 20 bases.
When to override the default mask
The default TargetMask from your custom workflow design is correct for most runs. Specify a TargetMask in the manifest only when:
- You are using the same custom design with a spacer library of a different length.
- Your construct has changed since the design was published.
- You are troubleshooting unexpectedly low assignment rates and want to confirm cycle alignment.
To check your design's default mask, open the downloaded design package and inspect the panel.json file, or view the TargetMask field in the Custom Designer guide RNA setup.
FastqMask
The FastqMask controls which cycles appear in the per-target FASTQ output generated by Bases2Fastq. By default it mirrors the TargetMask. Set it separately only when:
- You want FASTQ output to include additional flanking cycles beyond the spacer.
- You want to trim FASTQ output to fewer cycles than the spacer length.
The FastqMask has no effect on cell assignment. Only the TargetMask determines which cycles are used when matching reads against the spacer library.