Fig. 3: NULISAseq performance characterization.

a The dynamic range for the detection of each target is indicated by the dark blue region. Values above the limit of detection (LoD) but below the lower limit of quantitation (LLoQ) are shown in lighter blue. Targets are ordered according to the geometric mean of the LLoQ and upper limit of quantitation (ULoQ). Dark blue lines represent generalized additive model (GAM) cubic regression spline fits to the ULoQ and LLoQ. The overall dynamic range for 200-plex NULISAseq spanned 9.6 log10 values. b Density plots of intraplate CV after internal control normalization and interplate CV after internal control and intensity normalization. CV for each target was calculated using the mean CV across 10 samples with 9 technical replicates each. c Cross reactivity. Two sets of 45 random antigen pools containing 4–5 targets each were analyzed with 200-plex NULISAseq. Each cell of the heatmap represents the percent of normalized read counts for that target (rows) occurring in that pool (columns) (each row adds up to 100); scale ranges from zero (light blue) to 100 (dark red). Targets were ordered according to pool membership such that the cells on the diagonal corresponded to the assigned pools. d Detectability of 204 targets in 151 samples, including 79 from healthy controls and 72 from patients with various diseases. The y-axis represents NULISA Protein Quantification (NPQ) units minus the LoD for the respective target. Boxplots show lines at median; boxes indicate interquartile range; whiskers show values extending from interquartile range to up to 1.5 times the interquartile range; data beyond this are shown as plotted points. Boxplots are shaded using a gradient scale corresponding to detectability, where light pink represents 100% detectability and dark blue represents 0% detectability. Source data are provided as a Source data file. Source data for (a, b) are in Supplementary Data 1. Source data for (d) are provided in the Alamar_NULISAseq_Detectability_NPQ.csv file available under accession GSM7734324.