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• Knowing the reaction quality of your samples allows you to make informed decisions on how to confidently utilize the reported counts from the Countable System for downstream analyses.
• The quality of each Countable reaction depends on several factors, most notably, the PCR assay design and the individual DNA sample quality. PCR assays with severe oligo interactions can lead to high fluorescent background. Samples with severe levels of PCR inhibitors can result in low fluorescent signal. In either situation, counting accuracy can be impacted.
• To enable you to quickly assess the reaction quality of each sample, the analysis pipeline of the Countable Control Software provides robust quality control (QC) flags and metrics. In this article, you'll learn where to find this vital QC information and how to interpret it. </aside>

Access the Countable Analysis Summary for each Countable experiment

After each Countable experiment, a Countable Analysis Summary file is produced. It can be accessed from within the Countable Control Software under ‘View Experiment Reports’, or accessed as an .html file from the [CountableLabs] folder on the Countable Computer.

Review the overall reaction quality in the ‘Summary’ tab

In the ‘Summary’ tab within the Countable Analysis Summary, you'll find two key sections providing distinct levels of quality control information:

• QC Summary: This section offers a high-level overview of any issues that were flagged during your experiment.
• Sample Summary: The "Flags" column displays icons indicating sample-specific quality flags. To view the detailed channel and flag-level information, click the '+' icon on the left side of the table to expand the view.

There are two categories of flags - ‘Issues’ and ‘Observations’. For the list of flags and suggested follow-up actions, see the section ‘Reported issues and observations’ below.

• Issues ⛔ : Reported counts for this sample/channel should not be used.
• Observations ⚠️ : Additional investigation of sample/channel quality should be performed before the reported counts are used.

Inspect the detailed reaction quality for each sample in the ‘Quality Check’ tab

The ‘Quality Check’ tab provides an in-depth view of individual reaction quality. Here, you'll see histograms that illustrate the fluorescent distribution of your single-molecule signal (colored) and background (gray). In the upper-left corner of each histogram plot, the Intensity Distribution (ID) score is displayed. When assessing these QC figures, consider the following:

ID Score:

• An ID Score > 90 indicates a high-quality sample, while a score < 90 would indicate a possible quality issue that should be considered in your data analysis.
• The ID score is a unitless metric with a scale from 0–100 that describes the distribution of fluorescence intensity of the detected single molecules per channel. It is reported for reactions with >3000 and <1 M counts. The higher the metric, the better the signal distribution is from the background. The metric is influenced by a combination of assay and sample quality.