Day 1

Panel

Code

library(Luciernaga)
library(dplyr)

TheUnstained <- read.csv("data/Unstained3L.csv", check.names=FALSE)
TheUnstained <- TheUnstained |> select(-Experiment, -Condition, -Count, -Sample) |> slice(1)
Unstained <- TheUnstained

Panel <- file.path("data", "Panel.csv")
Panel <- read.csv(Panel, check.names=FALSE) |> dplyr::filter(Day %in% c(1))
GT <- FluorophoreMatrix(data=Panel, NumberDetectors = 38)

D	WV	Fluorophore	Antigen	D	Fluorophore	Antigen	D	Fluorophore	Antigen
V1	428
V2	443
V3	458
V4	473
V5	508			B1
V6	525			B2
V7	542	BV510	CD8	B3	Spark Blue 550	CD3
V8	581			B4
V9	598			B5
V10	615	BV605	CD4	B6
V11	662			B7	7-AAD	Viability	R1
	679			B8			R2
V12	695			B9			R3
V13	718			B10			R4	Alexa Fluor 700	CD45
	738			B11			R5
V14	750
	760			B12			R6
V15	781			B13			R7
V16	812			B14			R8

Fluorescent Signatures

Code

Fluorophore <- FluorophoreMatrix(data=Panel, NumberDetectors = 38, returnType="data") |> pull(Fluorophore)

Plots <- QC_ReferenceLibrary(FluorNameContains = Fluorophore, NumberDetectors=38,
returnPlots=TRUE, plotname=" ", exact=TRUE, unstained=Unstained)

Unmixing Matrix

Code

TheFluorophores <- FluorophoreMatrix(data=Panel, NumberDetectors = 38,
 returnType="data") |> pull(Fluorophore)

Data <- Luciernaga:::InstrumentReferences(NumberDetectors=38)
Data <- Data |> dplyr::filter(Fluorophore %in% TheFluorophores) |> select(-Instrument) |>
    tidyr::pivot_wider(names_from = "Detector", values_from = "AdjustedY")

colnames(Unstained) <- gsub("-A", "", colnames(Unstained))
Unstained <- Unstained |> select(where(is.numeric)) |>
     AveragedSignature(stats="median", normalize=TRUE) |>
     mutate(Fluorophore="Unstained") |> relocate(Fluorophore, .before=1)

Data <- bind_rows(Data, Unstained)

Fluorophore	V1	V2	V3	V4	V5	V6	V7	V8	V9	V10	V11	V12	V13	V14	V15	V16	B1	B2	B3	B4	B5	B6	B7	B8	B9	B10	B11	B12	B13	B14	R1	R2	R3	R4	R5	R6	R7	R8
7-AAD	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.01	0.14	0.38	0.26	0.24	0.19	0.13	0.05	0.00	0.00	0.00	0.02	0.10	0.37	1.00	0.85	0.89	0.54	0.34	0.32	0.24	0.23	0.03	0.04	0.04	0.03	0.02	0.02	0.02	0.01
Alexa Fluor 700	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.03	0.18	0.13	0.07	0.03	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.01	0.02	0.01	0.01	0.01	0.01	0.00	0.04	0.30	1.00	0.86	0.42	0.40	0.20
BV510	0.00	0.02	0.10	0.25	0.70	0.70	1.00	0.66	0.44	0.42	0.15	0.07	0.06	0.04	0.03	0.01	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
BV605	0.05	0.05	0.05	0.02	0.01	0.00	0.00	0.16	0.69	1.00	0.47	0.19	0.16	0.11	0.07	0.03	0.00	0.00	0.00	0.01	0.04	0.04	0.03	0.02	0.02	0.01	0.01	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Spark Blue 550	0.00	0.00	0.00	0.00	0.00	0.08	0.33	0.19	0.13	0.11	0.04	0.02	0.02	0.01	0.01	0.00	0.04	0.37	1.00	0.40	0.28	0.17	0.10	0.07	0.06	0.03	0.02	0.02	0.02	0.02	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Unstained	0.30	0.28	0.36	0.38	0.62	0.60	1.00	0.58	0.48	0.47	0.21	0.16	0.14	0.15	0.11	0.07	0.34	0.36	0.49	0.26	0.26	0.18	0.15	0.11	0.10	0.07	0.05	0.04	0.06	0.06	0.04	0.05	0.04	0.05	0.04	0.03	0.00	0.03

Cosine (Similarity) Matrix

Code

Cosine <- Luciernaga_Cosine(data=Data, limitlow=0, colorlow="orange")

Kappa (Complexity Index)

[1] "The Complexity Index without the Unstained is 1.84"

[1] "The Complexity Index with the Unstained is 8.48"

Unmixing-dependent Spreading (UDS) Hotspot Matrix

Code

Unstained <- Unstained |> select(-Fluorophore)

Hotspot <- Luciernaga:::MagesCauldron(panelfluors=TheFluorophores, unstained = Unstained)