This tutorial describes how to use Olink® Analyze to integrate Limit of Detection (LOD) into Olink® Explore HT and Olink® Explore 384/3072 datasets. Although it is recommended to use all Olink Explore data in downstream analyses, LOD information can be useful when performing technical evaluations of a dataset.
In this tutorial, you will learn how to use olink_lod()
to add LOD information to your Olink Explore dataset. Note that Olink
Analyze does not contain example Olink Explore HT or Olink Explore
384/3072 datasets within the package, so external data will be necessary
for the code below to work. The external data should contain internal
and external controls for proper calculation and normalization. All file
paths should be replaced with a path to your data and fixed LOD
reference file (if applicable).
Limit of Detection (LOD) is a metric that indicates the lowest measurable value of a protein. LOD can be helpful when performing technical evaluations of NPX™ datasets, such as calculating CVs. As a note, LOD is less important in downstream statistical analyses as values under LOD typically converge across groups. As such, including data below LOD is unlikely to increase the risk of false positive discoveries. Furthermore, data below LOD can be instrumental in downstream analyses such as biomarker discovery as a protein may be well expressed in one group and not measured in another group. In this case, this protein can be a strong biomarker candidate for specific groups.
LOD can be added to Olink Explore NPX datasets using
olink_lod()
. This function can calculate LOD from an NPX
dataset using the dataset’s negative controls or a list of predetermined
fixed LOD values (available in the Document Download Center at olink.com). As the default setting,
olink_lod()
will calculate LOD using a dataset’s negative
controls.
Olink Explore data is commonly delivered plate control (PC)
normalized or intensity normalized (the normalization type employed is
indicated in the NPX file column Normalization), where the latter is
dependent on that the analyzed samples are randomized. These are
reported in the two respective columns PCNormalizedNPX and NPX. Please
notice that for PC normalized datasets the content in these two columns
will be identical, while for intensity normalized datasets the NPX
column will include the intensity normalized values. Similarly, the
olink_lod()
function adds two columns to your dataset;
PCNormalizedLOD and LOD respectively. For a PC normalized dataset the
content in these two columns will be identical, while for an intensity
normalized dataset the LOD column will contain intensity normalized LOD
values. Examples of results for plate control and intensity
normalization are shown in the tables below.
SampleID | SampleType | OlinkID | UniProt | Assay | Count | NPX | PCNormalizedNPX | Normalization | LOD | PCNormalizedLOD |
---|---|---|---|---|---|---|---|---|---|---|
A1 | SAMPLE | OID01216 | O00533 | CHL1 | 1425 | 12.96 | 12.96 | Plate control | 2.37 | 2.37 |
A2 | SAMPLE | OID01216 | O00533 | CHL1 | 1240 | 11.27 | 11.27 | Plate control | 2.37 | 2.37 |
A3 | SAMPLE | OID01216 | O00533 | CHL1 | 2800 | 25.45 | 25.45 | Plate control | 2.37 | 2.37 |
A4 | SAMPLE | OID01216 | O00533 | CHL1 | 1590 | 14.45 | 14.45 | Plate control | 2.37 | 2.37 |
A5 | SAMPLE | OID01216 | O00533 | CHL1 | 839 | 7.63 | 7.63 | Plate control | 2.37 | 2.37 |
A6 | SAMPLE | OID01216 | O00533 | CHL1 | 695 | 6.32 | 6.32 | Plate control | 2.37 | 2.37 |
SampleID | SampleType | OlinkID | UniProt | Assay | Count | NPX | PCNormalizedNPX | Normalization | LOD | PCNormalizedLOD |
---|---|---|---|---|---|---|---|---|---|---|
A1 | SAMPLE | OID01216 | O00533 | CHL1 | 1425 | 17.12 | 12.96 | Intensity | 6.53 | 2.37 |
A2 | SAMPLE | OID01216 | O00533 | CHL1 | 1240 | 15.43 | 11.27 | Intensity | 6.53 | 2.37 |
A3 | SAMPLE | OID01216 | O00533 | CHL1 | 2800 | 29.61 | 25.45 | Intensity | 6.53 | 2.37 |
A4 | SAMPLE | OID01216 | O00533 | CHL1 | 1590 | 18.61 | 14.45 | Intensity | 6.53 | 2.37 |
A5 | SAMPLE | OID01216 | O00533 | CHL1 | 839 | 11.79 | 7.63 | Intensity | 6.53 | 2.37 |
A6 | SAMPLE | OID01216 | O00533 | CHL1 | 695 | 10.48 | 6.32 | Intensity | 6.53 | 2.37 |
Olink Explore datasets are standard Olink Explore HT and Olink
Explore 384/3072 NPX tables. The read_NPX()
function can be
used to import an NPX file in parquet form as generated by Olink® NPX
Explore Software. More information on using read_NPX()
can
be found in the Olink Analyze Overview
tutorial.
The negative control (NC) LOD method requires at least 10 negative controls in a dataset. Negative control data is available in the standard exported Explore HT and Explore 384/3072 NPX parquet files. NCs can be identified through the SampleID and SampleType columns.
A negative control will not contribute to the minimum number of required NCs if the negative control does not pass sample QC criteria (sample QC failure or warning) in all of the data (i.e. all Explore HT blocks, all Explore 3072 panels, or all Explore 384 panels that were measured)
Negative controls are used to calculate LOD from either PC normalized NPX or counts. For assays with more than 150 counts in one of the negative controls, LOD is calculated using the median PC normalized NPX and adding 3 standard deviations, or 0.2 NPX whichever is larger. For assays with fewer than 150 counts in all negative controls, LOD is calculated using the count values which are then converted into PC normalized NPX.
The resulting LOD is the PC normalized negative control LOD. In the event that the Explore dataset is intensity normalized, an intensity normalization adjustment factor is applied and the resulting intensity normalized LOD is reported in the LOD column and the PC normalized LOD is reported in the PCNormalizedLOD column.
The fixed LOD method uses fixed LOD values that have been calculated on negative controls used in Olink reference runs using the method described above for negative control LOD. These values are specific to the Data Analysis Reference ID, which can be found in your dataset. The fixed LOD data is available in an external CSV file which can be downloaded from the Document Download Center at olink.com. The fixed LOD values reported in this CSV file are the PC normalized LODs.
The fixed LOD file is read into the olink_lod()
function
to be integrated into an Explore dataset. In the event that the Explore
dataset is intensity normalized, an intensity normalization adjustment
factor is applied and the resulting intensity normalized LOD is reported
in the LOD column and the PC normalized LOD is reported in the
PCNormalizedLOD column.
For smaller sized studies (<10 NCs) we recommend using fixed LOD to integrate LOD values into your NPX dataset, as LOD calculations on fewer NCs may provide non-accurate values. However, it is important to keep in mind that fixed LOD values are not specific to your project, rather these values are generated by Olink when a new lot of reagents is released.
For larger projects we recommend calculating LOD from NC to obtain LOD values that are specific to your project. However, this requires that the dataset has at least 10 NCs with passing SampleQC.
If an Olink Explore dataset is intensity normalized, a normalization
adjustment factor is applied to the PC normalized LOD within the
olink_lod()
function.
For each assay, this adjustment factor is calculated as the median NPX of all samples (excluding Olink’s external controls) within each plate. For Olink Explore 3072, overlapping assays are assessed separately, within their respective panels. The intensity normalized negative control LOD is calculated by subtracting this adjustment factor from the PC normalized negative control LOD.
The intensity normalization LOD adjustment is applied to both the negative control and fixed LOD methods.
Olink Explore data with LOD data can be exported using arrow::write_parquet to export Olink Explore data as a parquet file in long format.
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support@olink.com for Olink lab product and technical support
info@olink.com for more information
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