> For the complete documentation index, see [llms.txt](https://help.connected.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://help.connected.illumina.com/dragen-protein-quantification/counting-and-normalization/interpretation-of-results.md).

# Interpretation of Results

## Sample Quality

The purpose of a flag is to highlight that a sample required a high degree of correction during the normalization process. This means a sample had sufficiently high or low signal, causing the normalization scale factors to be out of specification. The general recommendation is to exercise caution when using that sample in downstream analysis; normalization may not have been able to properly correct for the large changes in signal.

### Flag at SOMAmer Reads

A flag in a non-blank sample indicates low SOMAmer read depth. DRAGEN Protein Quantification has a minimum read depth to ensure measurement precision is achieved for each sample. Flagged samples may have a decrease in measurement precision.

### Flag at SOMAmer Normalized Reads

A flag in a blank sample may indicate increased background or contamination.

### Flag at Hyb QC

A flag in a sample indicates that there is an abnormal distribution of Tm controls. This could be caused by:

* The control signal pattern was too disrupted to characterize
  * This can result in the metric not being computed (NaN will be reported)
* The hybridization temperature is elevated beyond the acceptable threshold, indicating a hybridization issue that may affect protein measurements.

Minor deviations in temperature below recommended operating temperature have not been observed to impact sample quality; as such, there is no lower bound for this specification.

### Flag at Readout Normalization (HybNorm\_PassFlag)

A flag in a sample indicates that the hybridization controls in that sample had elevated or decreased signal compared to the plate plasma or serum controls. Flags here indicate significant differences between the sample and the non-blank controls. Potential reasons for this could be poor sample quality, protein loss during hybridization (e.g., due to elevated temperature) or other issues that arise during library prep. In this case, other failure modes will likely also be flagged, such as low Raw Counts, or high / low MedNormExt scale factors.

### Flag at Plate Normalization (MedNormInt\_PassFlag)

A flag in a calibrator indicates that the flagged calibrator replicate had a large discrepancy in signal when compared to the median of all calibrators within that plate. A single flagged calibrator will have limited impact on the plate due to use of median calibrator values in analysis. Multiple flagged calibrators could impact plate performance; reach out to Illumina Support for additional information.

### Flag at Sample Norm (MedNormExt\_PassFlag, CrossPlateMedNormInt\_PassFlag)

A flag in a sample indicates that it had a large discrepancy in signal when compared to the external plasma, serum, or CSF reference. Low signal may be caused by sample degradation or sample dilution; high signal may be caused by hemolysis. A difference in signal could also be caused by the diseased-state of the sample.

<mark style="color:orange;">**NOTE**</mark>: This step is not applicable to MultiMatrix sample types.

### Plate Fail

If a sample is on a plate that receives a FAIL, the sample receives a PLATE\_FAIL whether it was flagged or not at the sample level. PLATE\_FAIL indicates that the sample is likely not suitable for downstream analyses due to a plate-wide issue. Refer to below for more information on plate failure.

## Plate Quality

QC Percent in Tails and Calibration Percent in Tails are used to determine plate quality by examining individual SOMAmers in each well. A QC Percent in Tails failure will likely require a repeat library preparation or re-sequencing for this plate; contact Illumina Support for additional information.

Review the following table for more details.

### Plate Quality Matrix

<table data-header-hidden data-full-width="true"><thead><tr><th></th><th></th><th></th></tr></thead><tbody><tr><td><strong>Calibration Percent in Tails</strong></td><td><strong>QC Percent in Tails</strong></td><td><strong>Interpretation</strong></td></tr><tr><td><p>WARNING</p><p><em>The median of the calibrator replicates for that plate were sufficiently different from the expected.</em></p></td><td>FAIL<br><em>The median of the QC replicates for that plate were sufficiently different from the expected QC reference.</em></td><td><ul><li>Likely a failed run</li><li>Some samples may individually pass but should not be used for downstream analysis due to the plate failure</li><li>One or more normalization steps after Calibration were unable to rescue the run</li><li>Could be caused by a plate-wide issue, including sequencing run failure, automation failure, or reagent issue</li></ul></td></tr><tr><td><p>WARNING</p><p><em>The median of the calibrator replicates for that plate were sufficiently different from the expected.</em></p></td><td>PASS<br>The median of the <em>QC samples were sufficiently similar to expected values from QC reference.</em></td><td><ul><li>Successful Run</li><li>One or more normalization steps after the Calibration step have successfully removed the differences observed in calibrators</li></ul></td></tr><tr><td><p>PASS</p><p><em>The median of the calibrator samples were sufficiently similar to expected values from calibration reference.</em></p></td><td>FAIL<br><em>The median of the QC replicates for that plate were sufficiently different from the expected QC reference.</em></td><td><ul><li>Technically a failed run, but could be a false positive</li><li>Relatively rare combination</li><li>May indicate problem with only QC samples and not entire plate (e.g., edge effect)</li></ul></td></tr></tbody></table>

## Run Quality

There is an additional metric on run quality that evaluates blank samples in a plate (see below). The general recommendation is that, if a warning are observed, the plate should be used with caution in downstream analysis since there may have been sample contamination or elevated background. If issues with run quality are consistently seen, there may be issues with the assay set up. Contact Illumina Support for additional information or support.

### Warning for SOMAmer Normalized Reads

A warning on a plate may indicate plate-wide increased background or contamination.


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