Number of non-empty drops returned by emptyDrops() depends on value of test.ambience
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Entering edit mode
Peter Hickey ▴ 470
@petehaitch
Last seen 30 minutes ago
Walter and Eliza Hall Institute of Medi…

I was playing around with the test.ambience argument of DropletUtils::emptyDrops() and was initially surprised to find that I was getting a different number of non-empty droplets returned by the function, as illustrated below.

suppressPackageStartupMessages(library(DropletUtils))
set.seed(0)
my.counts <- DropletUtils:::simCounts()

set.seed(666)
out_FALSE <- emptyDrops(my.counts)
length(which(out_FALSE$FDR <= 0.001))
#> [1] 942

set.seed(666)
out_TRUE <- emptyDrops(my.counts, test.ambient = TRUE)
length(which(out_TRUE$FDR <= 0.001))
#> [1] 842

After more carefully reading the documentation, I understand that's because a different number of non-NA P-values are being returned

sum(!is.na(out_FALSE$PValue))
#> [1] 1300
sum(!is.na(out_TRUE$PValue))
#> [1] 10727

and therefore the FDR values are different, so at a given FDR cutoff the results may be different.

But does this mean that the test.ambient = TRUE results should only be used for diagnostic plots of the P-value histogram and not be used for the actual analysis?

Thanks!

Aside

I think this line in the documentation is should be test.ambient=TRUE:

If test.ambient=FALSE, non-NA statistics will be reported for all barcodes.

It might also need clarification, as non-NA statistics will only be reported for all barcodes with non-zero counts:

out_FALSE[is.na(out_FALSE$FDR), ]
#> DataFrame with 9800 rows and 5 columns
#>          Total   LogProb    PValue   Limited       FDR
#>      <integer> <numeric> <numeric> <logical> <numeric>
#> 1            2        NA        NA        NA        NA
#> 2            9        NA        NA        NA        NA
#> 3           20        NA        NA        NA        NA
#> 4           20        NA        NA        NA        NA
#> 5            1        NA        NA        NA        NA
#> ...        ...       ...       ...       ...       ...
#> 9796        10        NA        NA        NA        NA
#> 9797         6        NA        NA        NA        NA
#> 9798        10        NA        NA        NA        NA
#> 9799        15        NA        NA        NA        NA
#> 9800         4        NA        NA        NA        NA
out_FALSE[is.na(out_TRUE$FDR), ]
#> DataFrame with 373 rows and 5 columns
#>         Total   LogProb    PValue   Limited       FDR
#>     <integer> <numeric> <numeric> <logical> <numeric>
#> 1           0        NA        NA        NA        NA
#> 2           0        NA        NA        NA        NA
#> 3           0        NA        NA        NA        NA
#> 4           0        NA        NA        NA        NA
#> 5           0        NA        NA        NA        NA
#> ...       ...       ...       ...       ...       ...
#> 369         0        NA        NA        NA        NA
#> 370         0        NA        NA        NA        NA
#> 371         0        NA        NA        NA        NA
#> 372         0        NA        NA        NA        NA
#> 373         0        NA        NA        NA        NA

Session info

devtools::session_info()
#> ─ Session info ───────────────────────────────────────────────────────────────
#>  setting  value                       
#>  version  R version 4.0.0 (2020-04-24)
#>  os       Ubuntu 18.04.5 LTS          
#>  system   x86_64, linux-gnu           
#>  ui       X11                         
#>  language en_AU:en                    
#>  collate  en_AU.UTF-8                 
#>  ctype    en_AU.UTF-8                 
#>  tz       Australia/Melbourne         
#>  date     2021-01-23                  
#> 
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DropletUtils emptyDrops 10x • 234 views
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1
Entering edit mode
Aaron Lun ★ 27k
@alun
Last seen 14 hours ago
The city by the bay

But does this mean that the test.ambient = TRUE results should only be used for diagnostic plots of the P-value histogram and not be used for the actual analysis?

Yes. But after some reflection, this is a bit confusing, so I modified it in BioC-devel so that test.ambient=TRUE will report p-values but not change the FDRs compared to test.ambient=FALSE. If you want the old results where the low-count barcodes are used in the FDR, you can get them with test.ambient=NA, which provides some back compatibility.

I think this line in the documentation is should be test.ambient=TRUE:

Thanks.

It might also need clarification, as non-NA statistics will only be reported for all barcodes with non-zero counts:

That's correct, thanks.

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