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 Reference Correlation

A flag in a blank sample may indicate plasma or serum contamination. In internal studies, uncontaminated blanks generally have low RefCorr values (~0.4) and blanks with greater than 2% plasma or serum contamination were observed to have a RefCorr values of at least 0.6.

Flag at Hybridization Normalization

A flag in a sample indicates that the hybridization controls in that sample had elevated or decreased signal compared to the plate reference. Potential reasons for a high or low hybridization control signal include:

• Issues with the sample itself. These include 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.

• High plate-wide plasma / serum variability. Because Hyb Norm is performed using an internal reference, the composition of the plate can impact the scale factors of other samples. Therefore, a flag in one sample may reflect the quality of the other samples on the plate rather than issues with the sample itself (for example, many hemolyzed samples or blanks). In this case, other error modes for that sample are not expected to be flagged.

Flag at Internal Reference Median Normalization

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 External Reference Median Normalization

A flag in a sample indicates that it had a large discrepancy in signal when compared to the external plasma or serum 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.

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

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

Run Quality

There are two additional metrics on run quality that evaluates blank samples in a plate (see below). The general recommendation is that, if warnings are observed in one of these metrics, 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.

Warning at Reference Correlation

A warning on a plate may indicate plate-wide plasma or serum contamination.