Why some specified "nrom_cells" were classified as tumor cells?

[Zjianglin]

Hi developer, I'm using SCEVAN to identify tumor cells in a LiverCancer sample (10X scRNA-seq). I supposed that all immune cells (i.e. Neutrophils, Myeloid, T, B cells) are normal cells and their cell barcodes were inputed to norm_cell parameter?

However, the SCEVAN results suggests some immune cells were classified as tumor cells, why this happens? Is the norm_cell parameter does not work?

btw. the running output (found 4451 tumor cells) does not equal to result data.frame class count
(> table(sce8154$class) filtered normal tumor 1 4177 4406 ).

analysis code:

normal_cts <- c('Neutrophils', 'Kupffer', 'Proli. Kupffer',
                'Clec4f-Macro', 'Monocyte', 'pDC', 'cDC1', 'cDC2',
                'Cd4+T','Cd8+T', 'NK',  'Plasmocyte', 'B cell', 'Basophils'
                )

raw_count_li <- list()
norm_cells_li <- list()

for (samplex in sort(unique(sc_sbj$orig.ident))) {
  tmp_sbj = subset(sc_sbj, subset=(!!rlang::sym(sample_key) == samplex), slot = 'counts')
  refmtxx = tmp_sbj@meta.data
  refmtxx = refmtxx[(refmtxx[[celltype_key]] %in% normal_cts), ]
  ref_cellids = rownames(refmtxx) #Cells(nrm_sbj)
  nrm_strx = ''
  cntx = table(refmtxx[[celltype_key]])
  for (x in names(cntx)) {
    nrm_strx = sprintf('%s, %s:%d', nrm_strx, x, cntx[x])
  }

  print(sprintf('%s: %s, Normal cells (%d): %s', samplex, toString(dim(tmp_sbj)), nrow(refmtxx), nrm_strx))

  raw_count_li[[samplex]] = GetAssayData(tmp_sbj, assay='RNA', slot='counts')
  norm_cells_li[[samplex]] = ref_cellids
}

for (samplex in sort(unique(sc_sbj$orig.ident))) {
                cat(sprintf('\n\tSCEVAN: Sample %s (%d nrom_cells) \n', samplex, length(norm_cells_li[[samplex]])))
                print(head(norm_cells_li[[samplex]]))
                scevan_sres <- pipelineCNA(raw_count_li[[samplex]],
                         sample = samplex,
                         par_cores = nthreads,
                         norm_cell = norm_cells_li[[samplex]],
                         SUBCLONES = T,
                         beta_vega = 0.5,
                         ClonalCN = T,
                         plotTree = T,
                         AdditionalGeneSets = NULL,
                         SCEVANsignatures = TRUE,
                         organism = "mouse")

        }

running output:

SCEVAN: Sample 8154 (7179 nrom_cells) 
[1] "AAACCCAGTATCTCGA-8154" "AAACCCAGTCTCACGG-8154" "AAACCCAGTGTTCGTA-8154"
[4] "AAACGAAAGAAGGATG-8154" "AAACGAAAGATTGCGG-8154" "AAACGAAAGCGGACAT-8154"
[1] " raw data - genes: 24413 cells: 8584"
[1] "1) Filter: cells > 200 genes"
[1] "low data quality"
[1] "2) Filter: genes > 5% of cells"
[1] "8898 genes past filtering"
[1] "3) Annotations gene coordinates"
[1] "8062 genes annotated"
[1] "4) Filter: genes involved in the cell cycle"
[1] "7686 genes past filtering "
[1] "5)  Filter: cells > 5genes per chromosome "
[1] "6) Log Freeman Turkey transformation"
[1] "A total of 8583 cells, 7686 genes after preprocessing"
[1] "7) Measuring baselines (confident normal cells)"
[1] "8) Smoothing data"
[1] "9) Segmentation (VegaMC)"
[1] "10) Adjust baseline"
[1] "11) plot heatmap"
[1] "found 4451 tumor cells"
[1] "time classify tumor cells:  6.12251765330633"

results:

> table(sce8154$class)
filtered   normal    tumor 
       1     4177     4406 
> table(sce8154[, c('anno1', 'class')])
                class
anno1            filtered normal tumor
  Hepatocyte            0     45   390
  Cholangiocytes        0      0     0
  Endothelial           0      1   908
  HSC                   0      0    61
  Neutrophils           0      0   366
  Kupffer               0    583    39
  Proli. Kupffer        0    193     2
  Clec4f-Macro          0   1731    41
  Monocyte              1    949   468
  pDC                   0     15   133
  cDC1                  0    177    12
  cDC2                  0    477    93
  Cd4+T                 0      0   145
  Cd8+T                 0      2  1110
  NK                    0      0   111
  Plasmocyte            0      0     4
  B cell                0      4   502
  Basophils             0      0    21

>head(sce8154[, 1:6], n=10)
              cell.names  class confidentNormal subclone        anno1 SCEVANpred
1  AAACCCAGTATCTCGA-8154 normal             yes       NA         cDC2     normal
2  AAACCCAGTCTCACGG-8154  tumor             yes        1       B cell      tumor
3  AAACCCAGTGTTCGTA-8154  tumor             yes        2      Kupffer      tumor
4  AAACGAAAGAAGGATG-8154 normal             yes       NA Clec4f-Macro     normal
5  AAACGAAAGATTGCGG-8154  tumor             yes        3        Cd8+T      tumor
6  AAACGAAAGCGGACAT-8154 normal             yes       NA         cDC1     normal
7  AAACGAAAGTCATGGG-8154 normal             yes       NA     Monocyte     normal
8  AAACGAACAATCTGCA-8154  tumor             yes        1       B cell      tumor
9  AAACGAAGTGTACATC-8154 normal             yes       NA Clec4f-Macro     normal
10 AAACGAAGTTGTTGTG-8154  tumor             yes        3        Cd8+T      tumor