spillover matrix error - Error in .check_sm(sm, isotope_list) :

[MayaCyTOFnewbie]

Hi Helena,

I know this issue was addressed before, but I could not use the solutions there for some reason.

I get an error during the creation of a spillover matrix. I get this value (179.686.157.636.768) for Lu175Di vs Ir191Di.

thank you very much for all your help!!

here is what I did:

read single stained bead fcs file not normalized but with normalization beads

ff =read.FCS(file.path(dir,"20220309_CompBeads_0.FCS"), transformation = FALSE, truncate_max_range = FALSE)

#check colnames
colnames(ff)
[1] "Time" "Event_length" "Pd102Di" "Rh103Di" "Pd104Di" "Pd105Di" "Pd106Di" "Pd108Di" "Pd110Di" "Cd111Di" "Cd112Di"
[12] "In113Di" "Cd114Di" "In115Di" "Cd116Di" "La139Di" "Ce140Di" "Pr141Di" "Nd142Di" "Nd143Di" "Nd144Di" "Nd145Di"
[23] "Nd146Di" "Sm147Di" "Nd148Di" "Sm149Di" "Nd150Di" "Eu151Di" "Sm152Di" "Eu153Di" "Sm154Di" "Gd155Di" "Gd156Di"
[34] "Gd157Di" "Gd158Di" "Tb159Di" "Gd160Di" "Dy161Di" "Dy162Di" "Dy163Di" "Dy164Di" "Ho165Di" "Er166Di" "Er167Di"
[45] "Er168Di" "Tm169Di" "Er170Di" "Yb171Di" "Yb172Di" "Yb173Di" "Yb174Di" "Lu175Di" "Yb176Di" "BCKG190Di" "Ir191Di"
[56] "Ir193Di" "Center" "Offset" "Width" "Residual"

specify mass channels stained for & debarcode

bc_ms <- c(111:116, 141:176)
sce <- prepData(ff)
sce <- assignPrelim(sce, bc_ms, verbose = TRUE)
Debarcoding data...
o ordering
o classifying events
Normalizing...
Computing deltas...
sce <- applyCutoffs(estCutoffs(sce))

compute & extract spillover matrix

sce <- computeSpillmat(sce)
sm <- metadata(sce)$spillover_matrix

#export sm
write.csv(sm, file= "sm_20220309.csv")

do some sanity checks

chs <- channels(sce)
ss_chs <- chs[rowData(sce)$is_bc]
all(diag(sm[ss_chs, ss_chs]) == 1)
[1] TRUE

all(sm >= 0 & sm <= 1)
[1] FALSE

plotSpillmat(sce)
Error in .check_sm(sm, isotope_list) :
The supplied spillover matrix is invalid as it contains entries greater than 1.
Valid spill values are non-negative and mustn't exceed 1.

session_info()

• Session info --------------------------------------------------------------------------------------------------------------------------------------------------------
  setting value
  version R version 4.1.2 (2021-11-01)
  os Windows Server x64 (build 17763)
  system x86_64, mingw32
  ui RStudio
  language (EN)
  collate English_Europe.1252
  ctype English_Europe.1252
  tz Asia/Jerusalem
  date 2022-05-08
  rstudio 1.4.1717 Juliet Rose (desktop)
  pandoc 2.11.4 @ C:/Program Files/RStudio/bin/pandoc/ (via rmarkdown)

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[1] C:/Users/MayaB/Documents/R/R-4.1.2/library

D -- DLL MD5 mismatch, broken installation.

---

[HelenaLC]

Could you try using the prepData function directly to create the SCE? I.e., prepData(fcs_file_name) (by default,
transformation = FALSE and truncate_max_range = FALSE)? You're SCE currently contains non-mass channels (e.g., time, event length). These will be moved to the internal cell metadata (int_colData) by prepData. I am not certain, but my guess is that these cause issues when trying to match masses internally.

[HelenaLC]

Hey, is this still an active issue? If no, please close it (or I will do so soon). Else, any feedback re my comment above?