Hi all,

I'm wondering whether someone knows if it is possible to create something like a NOT gate in Opencyto (preferably in the CSV template that holds the gating strategy) in two dimensions (e.g. a continueing down-stream gating with all events that fall outside of 1 of the 4 quadrants of a quadrant gate).

Any help will be greatly appreciated!

Thanks!

Yes, you can do this via a Boolean gate. Here is a quick example, which I hope that you can adapt to your own data. In this example, I am interested in a population of cells that fall outside the main density of cells, which are identified via a polynomial gate. After I negate it, I then do a rectangle gate on the identified cells - note the very specific notation when using booleanFilter(), which includes the exclamation mark and backticks:

# gate for RFP+GFP+ cloud, then negate with boolean
  message('--------gating for PE-A (RFP) GFP-A cloud')
  gates <- c('PE.A', 'GFP.A')
  gs_add_gating_method(
    gs, alias = 'RFP GFP cloud', 
    pop = '+', parent = 'BV421-A-',
    dims = paste(gates, collapse = ','),
    gating_method = 'flowClust.2d', 
    gating_args = 'K=1,q=0.99')

# negate the gate
  bg <- booleanFilter(`!RFP GFP cloud`, filterId = 'neg RFP GFP cloud')
  gs_pop_add(gs, bg, parent = 'BV421-A-')
  gs_get_pop_paths(gs[[1]])

# make a further specific selection on the cells selected via booleanGate
  gates <- c('PE.A', 'GFP.A')
  rfp_min <- quantile(exprs(ff)[,'PE-A'], 0.05)
  rfp_max <- quantile(exprs(ff)[,'PE-A'], 0.95)
  gfp_min <- quantile(exprs(ff)[,'GFP-A'], 0.60)
  gfp_max <- max(exprs(ff)[,'GFP-A'])
  rg <- rectangleGate(
    'PE-A' = c(rfp_min,rfp_max),
    'GFP-A' = c(gfp_min, gfp_max),
    filterId = 'target RFP+GFP+')
  gs_pop_add(gs, rg, parent = 'neg RFP GFP cloud')
  gs_get_pop_paths(gs[[1]])
  recompute(gs)

Kevin