Ambient RNA removal diagnostics

Will Macnair - Neuroscience and Rare Diseases, Roche Innovation Center, Basel, Switzerland

February 28, 2026

Distributions of spliced pct. in empty and cell barcodes

The plots show the relationship between the number of UMIs and the percentage of spliced reads, separated by sample, both before and after ambient RNA removal. Ambient RNA is typically characterized by a high proportion of spliced reads, making the spliced percentage a useful metric for evaluating the effectiveness of an ambient RNA removal method. By examining changes in spliced percentages, we can assess how well the method performed in reducing ambient RNA contamination.

for (ss in s_lvls) {
  cat("### ", ss, "\n")
  print( plot_spliced_vs_umis(ss, usa_dt_ls[[ss]], ok_bcs_ls[[ss]], tot_inc_ls[[ss]]) )
  cat("\n\n")
}

GSM5292143

GSM5292144

GSM5292145

GSM5292146

GSM5292147

GSM5292148

GSM5292149

GSM5292150

GSM5292151

GSM5292152

GSM5292153

GSM5292154

GSM5292155

GSM5292156

GSM5292157

GSM5292158

GSM5292159

GSM5292160

GSM5292161

GSM5292162

GSM5292163

GSM5292164

GSM5292165

GSM5292166

GSM5292167

GSM5292168

GSM5292169

GSM5292170

GSM5292171

GSM5292172

GSM5292173

GSM5292174

GSM5292175

GSM5292176

GSM5292177

GSM5292178

GSM5292179

GSM5292180

GSM5292181

GSM5292182

GSM5292183

GSM5292184

GSM5292185

GSM5292186

GSM5292187

GSM5292188

GSM5292189

GSM5292190

GSM5292191

GSM5292192

GSM5292193

GSM5292194

GSM5292195

GSM5292196

GSM5292197

GSM5292198

GSM5292199

GSM5292200

GSM5292201

GSM5292202

GSM5292203

GSM5292204

GSM5292205

GSM5292206

GSM5292207

GSM5292208

SRR27882073

SRR27882074

SRR27882075

SRR27882077

SRR27882078

SRR27882079

SRR27882080

SRR27882081

SRR27882082

SRR27882084

SRR27882085

SRR27882086

SRR27882088

SRR27882089

SRR27882090

SRR27882091

SRR27882092

SRR27882093

SRR27882094

SRR27882148

SRR27882149

SRR27882150

SRR27882151

SRR27882152

SRR27882153

SRR27882154

SRR27882156

SRR27882157

SRR27882158

SRR27882159

SRR27882160

SRR27882161

SRR27882162

SRR27882163

SRR27882164

SRR27882165

SRR27882166

SRR27882167

SRR27882168

SRR27882169

SRR27882170

SRR27882171

SRR27882172

SRR27882173

SRR27882174

SRR27882175

SRR27882177

SRR27882178

SRR27882179

SRR27882180

SRR27882181

SRR27882182

SRR27882183

SRR27882185

SRR27882186

SRR27882187

SRR27882188

SRR27882189

SRR27882190

SRR27882191

SRR27882192

SRR27882193

SRR27882194

SRR27882196

SRR27882197

SRR27882198

SRR27882199

SRR27882200

SRR27882201

SRR27882202

SRR27882203

SRR27882204

SRR27882205

SRR27882207

SRR27882208

SRR27882209

SRR27882210

SRR27882211

SRR27882212

SRR27882213

SRR27882214

SRR27882215

SRR27882216

How many reads were removed as ambient?

Plots show what proportion of reads were removed from all barcodes called as cells.

for (ii in seq_along(chunks_ls)) {
  chunk       = chunks_ls[[ii]]
  cat("### samples ", min(chunk), "-", max(chunk), "\n")
  print(plot_reads_removed_as_ambient(usa_dt_ls[chunk], ok_bcs_ls[chunk]))
  cat("\n\n")
}

samples 1 - 10

samples 11 - 20

samples 21 - 30

samples 31 - 40

samples 41 - 50

samples 51 - 60

samples 61 - 70

samples 71 - 80

samples 81 - 90

samples 91 - 100

samples 101 - 110

samples 111 - 120

samples 121 - 130

samples 131 - 140

samples 141 - 149

Samples excluded by ambient removal step

CellBender includes all barcodes in the analysis up to the total_droplets threshold covering both cell-containing droplets and empty droplets. If CellBender calls the majority of these included droplets as cells, it may indicate an underlying issue. This typically occurs in low-quality samples where cell-containing barcodes and empty droplets cannot be clearly distinguished in the barcode rank plot. The table below shows the proportion of included droplets that were classified as cells by CellBender. Samples where this proportion exceeded % were excluded from further analysis.

calc_ambient_exclusions(stats_dt, run_var) %>% knitr::kable()

sample_id	total_droplets	kept_droplets	pct_kept	bad_run
GSM5292143	35173	11679	33.2	FALSE
GSM5292144	37914	16306	43.0	FALSE
GSM5292145	29983	7186	24.0	FALSE
GSM5292146	35361	12222	34.6	FALSE
GSM5292147	37893	13946	36.8	FALSE
GSM5292148	36487	12769	35.0	FALSE
GSM5292149	41396	16483	39.8	FALSE
GSM5292150	40048	14190	35.4	FALSE
GSM5292151	37819	11602	30.7	FALSE
GSM5292152	33970	5836	17.2	FALSE
GSM5292153	35151	8837	25.1	FALSE
GSM5292154	35114	7581	21.6	FALSE
GSM5292155	36153	7526	20.8	FALSE
GSM5292156	34555	9000	26.0	FALSE
GSM5292157	40829	9877	24.2	FALSE
GSM5292158	37305	11266	30.2	FALSE
GSM5292159	28882	8232	28.5	FALSE
GSM5292160	40863	15402	37.7	FALSE
GSM5292161	30376	9463	31.2	FALSE
GSM5292162	33760	11168	33.1	FALSE
GSM5292163	38375	15452	40.3	FALSE
GSM5292164	38757	13156	33.9	FALSE
GSM5292165	35933	14462	40.2	FALSE
GSM5292166	31982	10335	32.3	FALSE
GSM5292167	29697	8779	29.6	FALSE
GSM5292168	27530	4241	15.4	FALSE
GSM5292169	25636	10110	39.4	FALSE
GSM5292170	28106	4603	16.4	FALSE
GSM5292171	26013	3370	13.0	FALSE
GSM5292172	31901	5743	18.0	FALSE
GSM5292173	27815	5683	20.4	FALSE
GSM5292174	32868	8040	24.5	FALSE
GSM5292175	35862	8644	24.1	FALSE
GSM5292176	30626	9818	32.1	FALSE
GSM5292177	27604	5668	20.5	FALSE
GSM5292178	34758	7913	22.8	FALSE
GSM5292179	24487	7138	29.2	FALSE
GSM5292180	35542	8810	24.8	FALSE
GSM5292181	33774	7517	22.3	FALSE
GSM5292182	27916	3716	13.3	FALSE
GSM5292183	13008	1856	14.3	FALSE
GSM5292184	31384	6391	20.4	FALSE
GSM5292185	28720	6241	21.7	FALSE
GSM5292186	36164	9651	26.7	FALSE
GSM5292187	33555	8364	24.9	FALSE
GSM5292188	34637	6880	19.9	FALSE
GSM5292189	37125	14088	37.9	FALSE
GSM5292190	31745	7506	23.6	FALSE
GSM5292191	37785	9860	26.1	FALSE
GSM5292192	30344	8945	29.5	FALSE
GSM5292193	33076	8060	24.4	FALSE
GSM5292194	39819	14566	36.6	FALSE
GSM5292195	28079	6666	23.7	FALSE
GSM5292196	35030	8912	25.4	FALSE
GSM5292197	37197	11653	31.3	FALSE
GSM5292198	26739	9722	36.4	FALSE
GSM5292199	36340	9856	27.1	FALSE
GSM5292200	33537	9522	28.4	FALSE
GSM5292201	34889	7267	20.8	FALSE
GSM5292202	34340	8527	24.8	FALSE
GSM5292203	28656	6256	21.8	FALSE
GSM5292204	28264	5792	20.5	FALSE
GSM5292205	33974	8918	26.2	FALSE
GSM5292206	24306	5001	20.6	FALSE
GSM5292207	26218	4257	16.2	FALSE
GSM5292208	35771	9686	27.1	FALSE
SRR27882073	29280	8385	28.6	FALSE
SRR27882074	33844	10155	30.0	FALSE
SRR27882075	40876	18327	44.8	FALSE
SRR27882077	39344	16662	42.3	FALSE
SRR27882078	36400	11305	31.1	FALSE
SRR27882079	36652	12050	32.9	FALSE
SRR27882080	34560	12622	36.5	FALSE
SRR27882081	33960	11481	33.8	FALSE
SRR27882082	35856	14312	39.9	FALSE
SRR27882084	39543	7860	19.9	FALSE
SRR27882085	40989	8579	20.9	FALSE
SRR27882086	41889	9570	22.8	FALSE
SRR27882088	42788	9514	22.2	FALSE
SRR27882089	41816	8876	21.2	FALSE
SRR27882090	41326	9341	22.6	FALSE
SRR27882091	45593	13498	29.6	FALSE
SRR27882092	44460	14009	31.5	FALSE
SRR27882093	44478	13993	31.5	FALSE
SRR27882094	45151	12893	28.6	FALSE
SRR27882148	40254	8463	21.0	FALSE
SRR27882149	41538	10412	25.1	FALSE
SRR27882150	38367	10916	28.5	FALSE
SRR27882151	40678	10083	24.8	FALSE
SRR27882152	40305	9619	23.9	FALSE
SRR27882153	37919	10088	26.6	FALSE
SRR27882154	40897	12161	29.7	FALSE
SRR27882156	40536	10797	26.6	FALSE
SRR27882157	40451	10470	25.9	FALSE
SRR27882158	37214	8806	23.7	FALSE
SRR27882159	35887	9297	25.9	FALSE
SRR27882160	35410	10892	30.8	FALSE
SRR27882161	29457	7371	25.0	FALSE
SRR27882162	6287	1703	27.1	FALSE
SRR27882163	38995	14087	36.1	FALSE
SRR27882164	44100	16831	38.2	FALSE
SRR27882165	31510	12266	38.9	FALSE
SRR27882166	31296	7292	23.3	FALSE
SRR27882167	35618	10898	30.6	FALSE
SRR27882168	32262	11076	34.3	FALSE
SRR27882169	35378	14293	40.4	FALSE
SRR27882170	29529	12479	42.3	FALSE
SRR27882171	38752	15852	40.9	FALSE
SRR27882172	29158	18812	64.5	FALSE
SRR27882173	35731	11511	32.2	FALSE
SRR27882174	38376	11363	29.6	FALSE
SRR27882175	27856	6665	23.9	FALSE
SRR27882177	34173	13445	39.3	FALSE
SRR27882178	35427	14195	40.1	FALSE
SRR27882179	34644	11646	33.6	FALSE
SRR27882180	41815	19204	45.9	FALSE
SRR27882181	38825	16311	42.0	FALSE
SRR27882182	38542	13042	33.8	FALSE
SRR27882183	33905	22026	65.0	FALSE
SRR27882185	42864	15173	35.4	FALSE
SRR27882186	37651	9973	26.5	FALSE
SRR27882187	40324	10414	25.8	FALSE
SRR27882188	34575	8310	24.0	FALSE
SRR27882189	41612	37137	89.2	FALSE
SRR27882190	85000	69389	81.6	FALSE
SRR27882191	27170	6038	22.2	FALSE
SRR27882192	30063	6396	21.3	FALSE
SRR27882193	30288	6841	22.6	FALSE
SRR27882194	30397	5340	17.6	FALSE
SRR27882196	27661	5464	19.8	FALSE
SRR27882197	27852	5832	20.9	FALSE
SRR27882198	27794	5476	19.7	FALSE
SRR27882199	47047	28539	60.7	FALSE
SRR27882200	41117	11334	27.6	FALSE
SRR27882201	40758	14323	35.1	FALSE
SRR27882202	33113	8251	24.9	FALSE
SRR27882203	21813	5242	24.0	FALSE
SRR27882204	41305	15855	38.4	FALSE
SRR27882205	42521	10151	23.9	FALSE
SRR27882207	33634	8062	24.0	FALSE
SRR27882208	40549	10146	25.0	FALSE
SRR27882209	32569	9768	30.0	FALSE
SRR27882210	32448	6535	20.1	FALSE
SRR27882211	36596	9993	27.3	FALSE
SRR27882212	39104	10227	26.2	FALSE
SRR27882213	36336	9662	26.6	FALSE
SRR27882214	32912	8011	24.3	FALSE
SRR27882215	34203	7496	21.9	FALSE
SRR27882216	40307	10338	25.6	FALSE

R session info

Details of the R package versions used are given below.

devtools::session_info()

## ─ Session info ───────────────────────────────────────────────────────────────
##  setting  value
##  version  R version 4.3.3 (2024-02-29)
##  os       Red Hat Enterprise Linux 8.10 (Ootpa)
##  system   x86_64, linux-gnu
##  ui       X11
##  language (EN)
##  collate  en_US.UTF-8
##  ctype    en_US.UTF-8
##  tz       Europe/Zurich
##  date     2026-03-04
##  pandoc   3.8.2.1 @ /home/macnairw/packages/scprocess/.snakemake/conda/fd31be42acf0c42e462db0f4d9adae50_/bin/ (via rmarkdown)
##  quarto   NA
## 
## ─ Packages ───────────────────────────────────────────────────────────────────
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##  parallelly             1.45.1     2025-07-24 [2] CRAN (R 4.3.3)
##  patchwork            * 1.3.2      2025-08-25 [2] CRAN (R 4.3.3)
##  pbapply                1.7-4      2025-07-20 [2] CRAN (R 4.3.3)
##  pillar                 1.11.0     2025-07-04 [2] CRAN (R 4.3.3)
##  pkgbuild               1.4.8      2025-05-26 [2] CRAN (R 4.3.3)
##  pkgconfig              2.0.3      2019-09-22 [2] CRAN (R 4.3.3)
##  pkgload                1.4.0      2024-06-28 [2] CRAN (R 4.3.3)
##  plotly                 4.11.0     2025-06-19 [2] CRAN (R 4.3.3)
##  plyr                   1.8.9      2023-10-02 [2] CRAN (R 4.3.3)
##  png                    0.1-8      2022-11-29 [2] CRAN (R 4.3.3)
##  polyclip               1.10-7     2024-07-23 [2] CRAN (R 4.3.3)
##  processx               3.8.6      2025-02-21 [2] CRAN (R 4.3.3)
##  profvis                0.4.0      2024-09-20 [2] CRAN (R 4.3.3)
##  progressr              0.15.1     2024-11-22 [2] CRAN (R 4.3.3)
##  promises               1.3.3      2025-05-29 [2] CRAN (R 4.3.3)
##  ps                     1.9.1      2025-04-12 [2] CRAN (R 4.3.3)
##  purrr                * 1.1.0      2025-07-10 [2] CRAN (R 4.3.3)
##  QuickJSR               1.8.0      2025-06-09 [2] CRAN (R 4.3.3)
##  R.methodsS3            1.8.2      2022-06-13 [2] CRAN (R 4.3.3)
##  R.oo                   1.27.1     2025-05-02 [2] CRAN (R 4.3.3)
##  R.utils                2.13.0     2025-02-24 [2] CRAN (R 4.3.3)
##  R6                     2.6.1      2025-02-15 [2] CRAN (R 4.3.3)
##  RANN                   2.6.2      2024-08-25 [2] CRAN (R 4.3.3)
##  RColorBrewer         * 1.1-3      2022-04-03 [2] CRAN (R 4.3.3)
##  Rcpp                 * 1.1.0      2025-07-02 [2] CRAN (R 4.3.3)
##  RcppAnnoy              0.0.22     2024-01-23 [2] CRAN (R 4.3.3)
##  RcppHNSW               0.6.0      2024-02-04 [2] CRAN (R 4.3.3)
##  RcppParallel         * 5.1.9      2024-08-19 [2] CRAN (R 4.3.3)
##  RcppZiggurat         * 0.1.8      2025-03-30 [2] CRAN (R 4.3.3)
##  RCurl                  1.98-1.17  2025-03-22 [2] CRAN (R 4.3.3)
##  readr                * 2.1.5      2024-01-10 [2] CRAN (R 4.3.3)
##  readxl               * 1.4.5      2025-03-07 [2] CRAN (R 4.3.3)
##  registry               0.5-1      2019-03-05 [2] CRAN (R 4.3.3)
##  remotes                2.5.0      2024-03-17 [2] CRAN (R 4.3.3)
##  reshape2               1.4.4      2020-04-09 [2] CRAN (R 4.3.3)
##  reticulate             1.43.0     2025-07-21 [2] CRAN (R 4.3.3)
##  Rfast                * 2.1.0      2023-11-09 [2] CRAN (R 4.3.3)
##  rhdf5                * 2.46.1     2023-11-29 [2] Bioconductor 3.18 (R 4.3.3)
##  rhdf5filters           1.14.1     2023-11-06 [2] Bioconductor
##  Rhdf5lib               1.24.0     2023-10-24 [2] Bioconductor
##  rjson                  0.2.23     2024-09-16 [2] CRAN (R 4.3.3)
##  rlang                  1.1.6      2025-04-11 [2] CRAN (R 4.3.3)
##  rmarkdown              2.29       2024-11-04 [2] CRAN (R 4.3.3)
##  rmdformats             1.0.4      2022-05-17 [2] CRAN (R 4.3.3)
##  robustbase           * 0.99-6     2025-09-04 [2] CRAN (R 4.3.3)
##  ROCR                   1.0-11     2020-05-02 [2] CRAN (R 4.3.3)
##  rprojroot              2.1.1      2025-08-26 [2] CRAN (R 4.3.3)
##  RSpectra               0.16-2     2024-07-18 [2] CRAN (R 4.3.3)
##  rstan                  2.32.7     2025-03-10 [2] CRAN (R 4.3.3)
##  rstantools             2.5.0      2025-09-01 [2] CRAN (R 4.3.3)
##  rstudioapi             0.17.1     2024-10-22 [2] CRAN (R 4.3.3)
##  Rtsne                  0.17       2023-12-07 [2] CRAN (R 4.3.3)
##  S4Arrays               1.2.0      2023-10-24 [2] Bioconductor
##  S4Vectors            * 0.40.2     2023-11-23 [2] Bioconductor 3.18 (R 4.3.3)
##  sass                   0.4.10     2025-04-11 [2] CRAN (R 4.3.3)
##  scales               * 1.4.0      2025-04-24 [2] CRAN (R 4.3.3)
##  scattermore            1.2        2023-06-12 [2] CRAN (R 4.3.3)
##  sctransform            0.4.2      2025-04-30 [2] CRAN (R 4.3.3)
##  scuttle                1.12.0     2023-10-24 [2] Bioconductor
##  seriation            * 1.5.8      2025-08-20 [2] CRAN (R 4.3.3)
##  sessioninfo            1.2.3      2025-02-05 [2] CRAN (R 4.3.3)
##  Seurat                 5.3.0      2025-04-23 [2] CRAN (R 4.3.3)
##  SeuratObject           5.2.0      2025-08-27 [2] CRAN (R 4.3.3)
##  shape                  1.4.6.1    2024-02-23 [2] CRAN (R 4.3.3)
##  shiny                  1.11.1     2025-07-03 [2] CRAN (R 4.3.3)
##  SingleCellExperiment * 1.24.0     2023-10-24 [2] Bioconductor
##  sp                     2.2-0      2025-02-01 [2] CRAN (R 4.3.3)
##  spam                   2.11-1     2025-01-20 [2] CRAN (R 4.3.3)
##  SparseArray            1.2.2      2023-11-07 [2] Bioconductor
##  sparseMatrixStats      1.14.0     2023-10-24 [2] Bioconductor
##  spatstat.data          3.1-8      2025-08-18 [2] CRAN (R 4.3.3)
##  spatstat.explore       3.5-2      2025-07-22 [2] CRAN (R 4.3.3)
##  spatstat.geom          3.5-0      2025-07-20 [2] CRAN (R 4.3.3)
##  spatstat.random        3.4-1      2025-05-20 [2] CRAN (R 4.3.3)
##  spatstat.sparse        3.1-0      2024-06-21 [2] CRAN (R 4.3.3)
##  spatstat.univar        3.1-4      2025-07-13 [2] CRAN (R 4.3.3)
##  spatstat.utils         3.1-5      2025-07-17 [2] CRAN (R 4.3.3)
##  StanHeaders            2.32.10    2024-07-15 [2] CRAN (R 4.3.3)
##  statmod                1.5.0      2023-01-06 [2] CRAN (R 4.3.3)
##  strex                * 2.0.1      2024-10-03 [2] CRAN (R 4.3.3)
##  stringi                1.8.7      2025-03-27 [2] CRAN (R 4.3.3)
##  stringr              * 1.5.2      2025-09-08 [2] CRAN (R 4.3.3)
##  SummarizedExperiment * 1.32.0     2023-10-24 [2] Bioconductor
##  survival               3.8-3      2024-12-17 [2] CRAN (R 4.3.3)
##  tensor                 1.5.1      2025-06-17 [2] CRAN (R 4.3.3)
##  testit               * 0.13       2021-04-14 [2] CRAN (R 4.3.3)
##  tibble               * 3.3.0      2025-06-08 [2] CRAN (R 4.3.3)
##  tidyr                * 1.3.1      2024-01-24 [2] CRAN (R 4.3.3)
##  tidyselect             1.2.1      2024-03-11 [2] CRAN (R 4.3.3)
##  tidyverse            * 2.0.0      2023-02-22 [2] CRAN (R 4.3.3)
##  timechange             0.3.0      2024-01-18 [2] CRAN (R 4.3.3)
##  TSP                    1.2-5      2025-05-27 [2] CRAN (R 4.3.3)
##  tzdb                   0.5.0      2025-03-15 [2] CRAN (R 4.3.3)
##  urlchecker             1.0.1      2021-11-30 [2] CRAN (R 4.3.3)
##  usethis                3.2.1      2025-09-06 [2] CRAN (R 4.3.3)
##  uwot                   0.2.3      2025-02-24 [2] CRAN (R 4.3.3)
##  vctrs                  0.6.5      2023-12-01 [2] CRAN (R 4.3.3)
##  vipor                  0.4.7      2023-12-18 [2] CRAN (R 4.3.3)
##  viridis              * 0.6.5      2024-01-29 [2] CRAN (R 4.3.3)
##  viridisLite          * 0.4.2      2023-05-02 [2] CRAN (R 4.3.3)
##  whisker                0.4.1      2022-12-05 [2] CRAN (R 4.3.3)
##  withr                  3.0.2      2024-10-28 [2] CRAN (R 4.3.3)
##  workflowr            * 1.7.2      2025-08-18 [2] CRAN (R 4.3.3)
##  xfun                   0.53       2025-08-19 [2] CRAN (R 4.3.3)
##  xtable                 1.8-4      2019-04-21 [2] CRAN (R 4.3.3)
##  XVector                0.42.0     2023-10-24 [2] Bioconductor
##  yaml                 * 2.3.10     2024-07-26 [2] CRAN (R 4.3.3)
##  zlibbioc               1.48.0     2023-10-24 [2] Bioconductor
##  zoo                    1.8-14     2025-04-10 [2] CRAN (R 4.3.3)
## 
##  [1] /home/macnairw/R/x86_64-conda-linux-gnu-library/4.3
##  [2] /home/macnairw/packages/scprocess/.snakemake/conda/fd31be42acf0c42e462db0f4d9adae50_/lib/R/library
##  * ── Packages attached to the search path.
## 
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