QC your panel

Step 5 of the Getting started workflow.

Every target succeeding individually does not mean the panel is good. A target that scraped through with 12 probes will be dim and unreliable on the microscope, and you want to know that before you pay for oligos, not after.

Count what each target actually got

mkprobes filter-genes panel_a/output --genes panel_a/genes.converted.tss.txt \
    --min-probes 48 --out panel_a/genes.pass.txt

--genes is required — the command needs to know which targets were supposed to exist, not just which files happen to be in the directory.

The count comes from each target's _final_ file: the constructed, encoded probes, which is exactly what that target would contribute to an oligo order. (It is not the _screened_ count, which is larger — screening selects probe pairs, and construction then caps and encodes them.)

Three kinds of result:

  • At or above --min-probes — fine. The comparison is >=, so a target with exactly 48 passes at --min-probes 48.

  • Below the threshold — warned about individually, by name and count. These are the ones to rework.

  • No constructed probes at all — reported separately as an error, with the count and the first several names. These are not thin targets; they are run-panel failures that never completed. Go back to Design probes and check codebook.failed.txt before treating them as a QC problem.

--out writes the passing targets, one per line, so the next step has an explicit list rather than an implicit one.

What threshold to use

48 is the usual floor for a bright, reliably detected target. Below roughly 30 you should expect the target to be unreliable rather than merely dim. The right number depends on your expression levels and imaging setup; pick one for the panel and apply it consistently.

Fixing the thin ones

For each target below threshold, in rough order of what to try:

  1. Check the transcript. A short isoform simply has less room for probes. Try -m longest in Choose your targets and regenerate.

  2. Look at what it was losing probes to. Run mkprobes run-panel ... --list-failed-all, or read the target's _crawled.stats.json. One dominant cross-reactive binder is a different problem from diffuse loss.

  3. Accept verified off-targets. If the dominant binder is a homolog you do not mind labelling, --allow it — or use the interactive triage in Order your oligos, which records your decisions in codebook.acceptable.json and lets run-panel apply them automatically.

  4. Loosen screening. Raise --maxoverlap so probes may overlap slightly to reach the count.

  5. Drop the target. Sometimes the honest answer. Remove it from the target list, regenerate the codebook, and re-run — do not simply delete it from the codebook by hand.

Then re-run filter-genes and check again.

Where this leaves you

The per-target _final_ parquet files are the design deliverable:

panel_a/output/<target>_final_BamHIKpnI_<bits>.parquet

They are not orderable yet — turning them into oligos is Order your oligos.

To see exactly how one was made:

mkprobes provenance panel_a/output/Sox2_final_BamHIKpnI_1,2,3.parquet

Column meanings: Output columns, stage by stage.

On a cluster

  • QC is lightweight; run it as a post-job step, not its own allocation.

  • Archive the codebook, target list, genes.pass.txt and the final parquet files together as one panel release bundle.

  • Keep rework jobs scoped to the specific failing targets.

Next: Order your oligos.