Please note that the data analysis steps below will be taken care of for you when you send your samples to one of our dedicated, highly skilled Analysis Service teams, who will provide you with a comprehensive Normalized Protein eXpression (NPX) data/quality control report after your study is completed.
NPXis Olink’s relative protein quantification unit on a log2 scale. NPX values from Olink Explore® 1536 are calculated from the number of matched counts (= i.e. the number of sequence reads for each specific combination of sample and assay) from the NovaSeq run.
Running the assay
Detection of the digital PEA signal and sample analysis for Olink® Explore are performed via NGS, using Illumina’s NovaSeq 6000 instrument*. This high-throughput system enables true multiplexing, with simultaneous detection and readout of 384 analytes/controls in 96 samples (36 864 data points) in each lane. The NovaSeq instrument can process four lanes in parallel, generating 147 456 data points from a single run.
Olink Explore workflow, from immunoassay to NGS readout
About sample volumes:
Olink Explore technically requires 2.8 µL of plasma or serum to measure 1536 analytes.
Another 7 µL are needed to compensate for dead-volumes in the automated liquid handling system
Analysis Service requests you actually send 40 µL of each sample in case re-runs are required.
The sample preparation steps take about 24 hours, while the final NGS readout takes less than 12 hours to complete.
Data generation and quality control
Data from the NovaSeq 6000 run is uploaded to the MyData cloud. After this, Olink MyData cloud software processes the data, performs QC-analysis, calculates NPX values and produces a Certificate of Analysis (CoA). The cloud software provides Normalized Protein eXpression (NPX) values for further data analysis. This is an entirely new software specially developed for Olink Explore 1536, although most of the functionality is based on the existing NPX Manager Software that was developed for Olink Target 96 panels.
Olink has developed a built-in QC system using internal controls, enabling full control over the technical performance of assays and samples. Three internal controls are spiked into every sample for each dilution and panel, and are designed to monitor the quality of assay performance, as well as the quality of individual samples:
Immuno control (incubation step)
Extension control (extension and pre-amplification step)
Amplification control (amplification step)
Each sample plate also includes a control strip with the following controls:
Two Sample controls used to estimate the precision (intra- and inter-CVs)
Three Negative controls used to set the background levels for each protein assay and to calculate the limit of detection (LOD)
Three Plate controls used to compensate for potential variation between runs and plates
Due to the huge amount of data generated by NovaSeq 6000 instruments, all data storage and analysis is handled by a secure AWS cloud service.
Our Analysis Service labs provide customers with project reports generated using the MyData Cloud software. Examples of the documents you will receive from our Analysis Service once your samples have been run are available via the links below:
You may also try our new post-assay statistical analysis app, Olink® Insights Stat Analysis, where you can perform basic statistical analyses and generate heatmap, sample distribution or Principal Component Analysis (PCA) plots yourself – Read More
Example of Principle Component Analysis (PCA) plot
*”Illumina” and “NovaSeq” are trademarks and the property of Illumina Inc. or their respective owners.