Validation of Candidate Markers in Sézary Syndrome Malignant T-cells using scRNA-seq and TCR Data (Validation Borcherding data)

---

1 Introduction

Dataset: GSE146586 (Borcherding et al., 2023). [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE146586] Includes scRNA-seq and paired TCR-seq from Sézary syndrome (SS) patients and one healthy control. TCR is used to identify the dominant malignant clone.

• Healthy controls: 1
• Sézary syndrome patients: 6

---

2 TCR Analysis

2.1 load libraries

2.2 Define File Paths

# Base directory
base_dir <- "/home/bioinfo/1-Thesis_Final_Year_2025/2025-Final_Year_Results/2025-Year3_Analysis/Biomarkers_Validation_with_Public_Data/Borch_Combined/data/"

# Seurat object
seurat_file <- file.path(base_dir, "ss_Borcherding_Malignant_6_Normal_1_Integrated_object.rds")

# TCR data directory
tcr_dir <- file.path(base_dir, "TCR_Data")

# Output directory
output_dir <- "Borcherding_output"
dir.create(output_dir, showWarnings = FALSE)

2.3 Load Seurat Object

cat("Loading Seurat object...\n")

Loading Seurat object...

merged_seurat <- readRDS(seurat_file)

cat("Dataset dimensions:\n")

Dataset dimensions:

cat(sprintf("  - Cells: %d\n", ncol(merged_seurat)))

  - Cells: 51095

cat(sprintf("  - Genes: %d\n", nrow(merged_seurat)))

  - Genes: 19184

cat(sprintf("  - Samples: %s\n", paste(unique(merged_seurat$Sample_ID), collapse = ", ")))

  - Samples: SS, CTRL

2.4 Load TCR Data

library(tidyr)
library(dplyr)
library(readr)
library(stringr)

# Define your directory path here
# tcr_dir <- "path/to/tcr_files"

# Define gzipped TCR file paths with sample names
tcr_files <- list(
  SS2 = file.path(tcr_dir, "GSM4396048_CTCL2_filtered_contig_annotations.csv.gz"),
  SS3 = file.path(tcr_dir, "GSM4396049_CTCL3_filtered_contig_annotations.csv.gz"),
  SS4 = file.path(tcr_dir, "GSM4396050_CTCL4_filtered_contig_annotations.csv.gz"),
  SS5 = file.path(tcr_dir, "GSM4396051_CTCL5_filtered_contig_annotations.csv.gz"),
  SS6 = file.path(tcr_dir, "GSM4396052_CTCL6_filtered_contig_annotations.csv.gz")
)

cat("Loading gzipped TCR clonotype files:\n")

Loading gzipped TCR clonotype files:

# Load and filter productive TCR chains, clean barcode suffix
tcr_list <- lapply(names(tcr_files), function(sample_name) {
  cat("Reading sample:", sample_name, "\n")
  
  df <- read_csv(tcr_files[[sample_name]], show_col_types = FALSE)
  
  df_filtered <- df %>%
    filter(productive == "True") %>%
    mutate(sample_id = sample_name) %>%
    mutate(barcode = gsub("-1$", "", barcode))  # Remove "-1" suffix if present
  
  return(df_filtered)
})

Reading sample: SS2 
Reading sample: SS3 
Reading sample: SS4 
Reading sample: SS5 
Reading sample: SS6 

# Combine all samples into one dataframe
combined_tcr <- bind_rows(tcr_list)

cat("Columns in combined TCR data:\n")

Columns in combined TCR data:

print(colnames(combined_tcr))

 [1] "barcode"          "is_cell"          "contig_id"        "high_confidence"  "length"          
 [6] "chain"            "v_gene"           "d_gene"           "j_gene"           "c_gene"          
[11] "full_length"      "productive"       "cdr3"             "cdr3_nt"          "reads"           
[16] "umis"             "raw_clonotype_id" "raw_consensus_id" "sample_id"       

cat("Total productive TCR chains across all samples:", nrow(combined_tcr), "\n")

Total productive TCR chains across all samples: 74426 

cat("Sample barcodes (first 10):\n")

Sample barcodes (first 10):

print(head(combined_tcr$barcode, 10))

 [1] "AAACCTGAGACAAAGG" "AAACCTGAGAGTCTGG" "AAACCTGAGAGTCTGG" "AAACCTGAGAGTGAGA" "AAACCTGAGAGTGAGA"
 [6] "AAACCTGAGCCACCTG" "AAACCTGAGCCACCTG" "AAACCTGAGCCACGTC" "AAACCTGAGCCACGTC" "AAACCTGAGCCATCGC"

# Mapping to match Seurat barcodes
prefix_map <- c(
  "SS2" = "SS_P2",
  "SS3" = "SS_P3",
  "SS4" = "SS_P4",
  "SS5" = "SS_P5",
  "SS6" = "SS_P6"
)

convert_tcr_barcode <- function(barcode, sample) {
  new_prefix <- prefix_map[sample]
  cell_bc <- str_remove(barcode, paste0("^", sample, "_sample_id_"))  # Remove old prefix
  paste0(new_prefix, "_", cell_bc, "-1")  # Add Seurat-style suffix
}

# Apply barcode transformation
combined_tcr <- combined_tcr %>%
  rowwise() %>%
  mutate(barcode_seurat = convert_tcr_barcode(barcode, sample_id)) %>%
  ungroup()

cat("Transformed barcodes (first 10):\n")

Transformed barcodes (first 10):

print(head(combined_tcr$barcode_seurat, 10))

 [1] "SS_P2_AAACCTGAGACAAAGG-1" "SS_P2_AAACCTGAGAGTCTGG-1" "SS_P2_AAACCTGAGAGTCTGG-1"
 [4] "SS_P2_AAACCTGAGAGTGAGA-1" "SS_P2_AAACCTGAGAGTGAGA-1" "SS_P2_AAACCTGAGCCACCTG-1"
 [7] "SS_P2_AAACCTGAGCCACCTG-1" "SS_P2_AAACCTGAGCCACGTC-1" "SS_P2_AAACCTGAGCCACGTC-1"
[10] "SS_P2_AAACCTGAGCCATCGC-1"

---

3 CLIC1 VALIDATION

---

3.1 Integrate TCR with Seurat

library(dplyr)
library(scRepertoire)
library(Seurat)
library(stringr)

# Define mapping from TCR sample ids to Seurat prefixes
sample_prefix_map <- c(
  "SS2" = "SS_P2",
  "SS3" = "SS_P3",
  "SS4" = "SS_P4",
  "SS5" = "SS_P5",
  "SS6" = "SS_P6"
)

# Assume combined_tcr dataframe contains:
# - barcode_seurat: corrected barcode with prefix+cellbarcode+"-1"
# - sample_id: sample identifier like "SS2", "SS3", etc.
# - raw_clonotype_id or CTstrict: clonotype identifier column from TCR

# Prepare TCR metadata with corrected barcodes:
# Fix possible NA_id_ prefix by replacing with mapped Seurat prefix
tcr_metadata <- combined_tcr %>%
  mutate(barcode_seurat = str_replace(barcode_seurat, "^NA_id_", paste0(sample_prefix_map[sample_id], "_"))) %>%
  distinct(barcode_seurat, sample_id, raw_clonotype_id) %>%
  rename(raw_clonotype_id = raw_clonotype_id, barcode = barcode_seurat)

# Check barcode matching
common_barcodes <- intersect(tcr_metadata$barcode, colnames(merged_seurat))
cat("Number of matching barcodes:", length(common_barcodes), "\n")

Number of matching barcodes: 36575 

if (length(common_barcodes) == 0) {
  stop("No matching barcodes found between TCR and Seurat object after barcode correction")
}

# Create a named vector for clonotype metadata
clonotype_vector <- tcr_metadata$raw_clonotype_id
names(clonotype_vector) <- tcr_metadata$barcode

# Add clonotype metadata to Seurat object
merged_seurat <- AddMetaData(object = merged_seurat,
                            metadata = clonotype_vector,
                            col.name = "clonotype")

# Inspect to confirm success
head(merged_seurat@meta.data)

# Optional: View clonotype distribution
table(merged_seurat$clonotype, useNA = "ifany")

   clonotype1   clonotype10  clonotype100 clonotype1000 clonotype1001 clonotype1002 clonotype1003 
        24770            28             5             2             2             1             1 
clonotype1004 clonotype1005 clonotype1006 clonotype1007 clonotype1008 clonotype1009  clonotype101 
            2             1             1             2             1             2             5 
clonotype1010 clonotype1011 clonotype1012 clonotype1013 clonotype1014 clonotype1015 clonotype1016 
            2             2             2             2             2             2             2 
clonotype1017 clonotype1018 clonotype1019  clonotype102 clonotype1020 clonotype1021 clonotype1022 
            2             2             1             5             2             2             2 
clonotype1023 clonotype1024 clonotype1025 clonotype1026 clonotype1027 clonotype1028 clonotype1029 
            2             1             2             2             2             2             2 
 clonotype103 clonotype1030 clonotype1031 clonotype1032 clonotype1033 clonotype1034 clonotype1035 
            5             1             2             2             2             2             2 
clonotype1036 clonotype1037 clonotype1038 clonotype1039  clonotype104 clonotype1040 clonotype1041 
            2             2             2             2             5             2             2 
clonotype1042 clonotype1043 clonotype1044 clonotype1045 clonotype1046 clonotype1047 clonotype1048 
            2             2             2             2             2             2             2 
clonotype1049  clonotype105 clonotype1050 clonotype1051 clonotype1052 clonotype1053 clonotype1054 
            2             5             2             1             2             2             2 
clonotype1055 clonotype1056 clonotype1057 clonotype1058 clonotype1059  clonotype106 clonotype1060 
            2             2             2             2             2             5             2 
clonotype1061 clonotype1062 clonotype1064 clonotype1066 clonotype1067 clonotype1068 clonotype1069 
            1             1             1             1             1             1             1 
 clonotype107 clonotype1070 clonotype1071 clonotype1072 clonotype1073 clonotype1074 clonotype1075 
            5             1             1             1             1             1             1 
clonotype1076 clonotype1077 clonotype1078 clonotype1079  clonotype108 clonotype1080 clonotype1081 
            1             1             1             1             4             1             1 
clonotype1082 clonotype1083 clonotype1084 clonotype1085 clonotype1086 clonotype1088  clonotype109 
            1             1             1             1             1             1             4 
clonotype1090 clonotype1091 clonotype1092 clonotype1093 clonotype1094 clonotype1095 clonotype1096 
            1             1             1             1             1             1             1 
clonotype1097 clonotype1098 clonotype1099   clonotype11  clonotype110 clonotype1100 clonotype1101 
            1             1             1            24             5             1             1 
clonotype1102 clonotype1103 clonotype1104 clonotype1105 clonotype1106 clonotype1107  clonotype111 
            1             1             1             1             1             1             5 
clonotype1110 clonotype1111 clonotype1112 clonotype1113 clonotype1114 clonotype1115 clonotype1117 
            1             1             1             1             1             1             1 
clonotype1118 clonotype1119  clonotype112 clonotype1120 clonotype1121 clonotype1122 clonotype1123 
            1             1             4             1             1             1             1 
clonotype1124 clonotype1125 clonotype1126 clonotype1127 clonotype1128 clonotype1129  clonotype113 
            1             1             1             1             1             1             5 
clonotype1130 clonotype1131 clonotype1132 clonotype1133 clonotype1134 clonotype1135 clonotype1136 
            1             1             1             1             1             1             1 
clonotype1137 clonotype1138 clonotype1139  clonotype114 clonotype1140 clonotype1141 clonotype1142 
            1             1             1             5             1             1             1 
clonotype1143 clonotype1144 clonotype1145 clonotype1146 clonotype1147 clonotype1148 clonotype1149 
            1             1             1             1             1             1             1 
 clonotype115 clonotype1150 clonotype1151 clonotype1152 clonotype1153 clonotype1154 clonotype1155 
            5             1             1             1             1             1             1 
clonotype1156 clonotype1158 clonotype1159  clonotype116 clonotype1160 clonotype1161 clonotype1162 
            1             1             1             5             1             1             1 
clonotype1163 clonotype1164 clonotype1165 clonotype1166 clonotype1167 clonotype1168 clonotype1169 
            1             1             1             1             1             1             1 
 clonotype117 clonotype1170 clonotype1171 clonotype1172 clonotype1173 clonotype1174 clonotype1175 
            5             1             1             1             1             1             1 
clonotype1176 clonotype1177 clonotype1178 clonotype1179  clonotype118 clonotype1180 clonotype1181 
            1             1             1             1             5             1             1 
clonotype1182 clonotype1183 clonotype1185 clonotype1186 clonotype1187 clonotype1188  clonotype119 
            1             1             1             1             1             1             5 
clonotype1190 clonotype1191 clonotype1192 clonotype1193 clonotype1194 clonotype1195 clonotype1196 
            1             1             1             1             1             1             1 
clonotype1197 clonotype1198 clonotype1199   clonotype12  clonotype120 clonotype1200 clonotype1202 
            1             1             1            22             5             1             1 
clonotype1203 clonotype1204 clonotype1205 clonotype1208 clonotype1209  clonotype121 clonotype1210 
            1             1             1             1             1             4             1 
clonotype1211 clonotype1212 clonotype1213 clonotype1214 clonotype1215 clonotype1216 clonotype1217 
            1             1             1             1             1             1             1 
clonotype1218 clonotype1219  clonotype122 clonotype1220 clonotype1221 clonotype1222 clonotype1223 
            1             1             3             1             1             1             1 
clonotype1224 clonotype1225 clonotype1226 clonotype1227 clonotype1228 clonotype1229  clonotype123 
            1             1             1             1             1             1             3 
clonotype1230 clonotype1232 clonotype1233 clonotype1234 clonotype1235 clonotype1236 clonotype1237 
            1             1             1             1             1             1             1 
clonotype1238 clonotype1239  clonotype124 clonotype1240 clonotype1242 clonotype1243 clonotype1245 
            1             1             4             1             1             1             1 
clonotype1246 clonotype1247 clonotype1248 clonotype1249  clonotype125 clonotype1250 clonotype1251 
            1             1             1             1             4             1             1 
clonotype1252 clonotype1254 clonotype1255 clonotype1256 clonotype1257 clonotype1259  clonotype126 
            1             1             1             1             1             1             4 
clonotype1260 clonotype1261 clonotype1262 clonotype1263 clonotype1264 clonotype1265 clonotype1267 
            1             1             1             1             1             1             1 
clonotype1268 clonotype1269  clonotype127 clonotype1271 clonotype1272 clonotype1273 clonotype1274 
            1             1             4             1             1             1             1 
clonotype1275 clonotype1277 clonotype1278 clonotype1279  clonotype128 clonotype1280 clonotype1281 
            1             1             1             1             4             1             1 
clonotype1282 clonotype1283 clonotype1284 clonotype1285 clonotype1287 clonotype1288 clonotype1289 
            1             1             1             1             1             1             1 
 clonotype129 clonotype1290 clonotype1291 clonotype1292 clonotype1293 clonotype1294 clonotype1295 
            4             1             1             1             1             1             1 
clonotype1296 clonotype1297 clonotype1298 clonotype1299   clonotype13  clonotype130 clonotype1301 
            1             1             1             1            22             3             1 
clonotype1302 clonotype1303 clonotype1304 clonotype1305 clonotype1307 clonotype1308 clonotype1309 
            1             1             1             1             1             1             1 
 clonotype131 clonotype1310 clonotype1311 clonotype1312 clonotype1313 clonotype1314 clonotype1315 
            4             1             1             1             1             1             1 
clonotype1316 clonotype1319  clonotype132 clonotype1321 clonotype1322 clonotype1323 clonotype1324 
            1             1             4             1             1             1             1 
clonotype1325 clonotype1326 clonotype1327 clonotype1328 clonotype1329  clonotype133 clonotype1330 
            1             1             1             1             1             4             1 
clonotype1331 clonotype1332 clonotype1334 clonotype1335 clonotype1336 clonotype1337 clonotype1338 
            1             1             1             1             1             1             1 
clonotype1339  clonotype134 clonotype1340 clonotype1341 clonotype1342 clonotype1343 clonotype1344 
            1             4             1             1             1             1             1 
clonotype1345 clonotype1346 clonotype1347 clonotype1348 clonotype1349  clonotype135 clonotype1350 
            1             1             1             1             1             4             1 
clonotype1351 clonotype1352 clonotype1353 clonotype1355 clonotype1356 clonotype1357 clonotype1358 
            1             1             1             1             1             1             1 
clonotype1359  clonotype136 clonotype1360 clonotype1361 clonotype1362 clonotype1363 clonotype1364 
            1             4             1             1             1             1             1 
clonotype1366 clonotype1368 clonotype1369  clonotype137 clonotype1370 clonotype1371 clonotype1372 
            1             1             1             4             1             1             1 
clonotype1373 clonotype1374 clonotype1375 clonotype1376 clonotype1377 clonotype1378 clonotype1379 
            1             1             1             1             1             1             1 
 clonotype138 clonotype1380 clonotype1381 clonotype1382 clonotype1383 clonotype1384 clonotype1385 
            4             1             1             1             1             1             1 
clonotype1386 clonotype1387 clonotype1388  clonotype139 clonotype1391 clonotype1392 clonotype1393 
            1             1             1             4             1             1             1 
clonotype1394 clonotype1395 clonotype1396 clonotype1397 clonotype1398 clonotype1399   clonotype14 
            1             1             1             1             1             1            18 
 clonotype140 clonotype1400 clonotype1401 clonotype1402 clonotype1403 clonotype1404 clonotype1406 
            4             1             1             1             1             1             1 
clonotype1407 clonotype1408 clonotype1409  clonotype141 clonotype1410 clonotype1411 clonotype1412 
            1             1             1             4             1             1             1 
clonotype1413 clonotype1414 clonotype1416 clonotype1418 clonotype1419  clonotype142 clonotype1421 
            1             1             1             1             1             4             1 
clonotype1422 clonotype1423 clonotype1424 clonotype1425 clonotype1426 clonotype1427 clonotype1428 
            1             1             1             1             1             1             1 
clonotype1429  clonotype143 clonotype1431 clonotype1432 clonotype1433 clonotype1434 clonotype1435 
            1             4             1             1             1             1             1 
clonotype1437 clonotype1438 clonotype1439  clonotype144 clonotype1440 clonotype1441 clonotype1442 
            1             1             1             3             1             1             1 
clonotype1443 clonotype1444 clonotype1445 clonotype1446 clonotype1447 clonotype1448  clonotype145 
            1             1             1             1             1             1             4 
clonotype1450 clonotype1451 clonotype1452 clonotype1453 clonotype1454 clonotype1455 clonotype1456 
            1             1             1             1             1             1             1 
clonotype1457 clonotype1458 clonotype1459  clonotype146 clonotype1460 clonotype1461 clonotype1462 
            1             1             1             4             1             1             1 
clonotype1463 clonotype1464 clonotype1465 clonotype1466 clonotype1467 clonotype1468 clonotype1469 
            1             1             1             1             1             1             1 
 clonotype147 clonotype1470 clonotype1471 clonotype1472 clonotype1474 clonotype1475 clonotype1476 
            3             1             1             1             1             1             1 
clonotype1477 clonotype1478 clonotype1479  clonotype148 clonotype1480 clonotype1481 clonotype1482 
            1             1             1             4             1             1             1 
clonotype1483 clonotype1484 clonotype1485 clonotype1486 clonotype1488 clonotype1489  clonotype149 
            1             1             1             1             1             1             4 
clonotype1490 clonotype1492 clonotype1493 clonotype1494 clonotype1495 clonotype1496 clonotype1497 
            1             1             1             1             1             1             1 
clonotype1498   clonotype15  clonotype150 clonotype1500 clonotype1501 clonotype1502 clonotype1504 
            1            18             4             1             1             1             1 
clonotype1505 clonotype1506 clonotype1507 clonotype1508 clonotype1509  clonotype151 clonotype1510 
            1             1             1             1             1             4             1 
clonotype1511 clonotype1512 clonotype1513 clonotype1514 clonotype1515 clonotype1516 clonotype1517 
            1             1             1             1             1             1             1 
clonotype1518 clonotype1519  clonotype152 clonotype1520 clonotype1521 clonotype1522 clonotype1523 
            1             1             4             1             1             1             1 
clonotype1524 clonotype1525 clonotype1526 clonotype1527 clonotype1528 clonotype1529  clonotype153 
            1             1             1             1             1             1             4 
clonotype1530 clonotype1531 clonotype1532 clonotype1533 clonotype1534 clonotype1535 clonotype1536 
            1             1             1             1             1             1             1 
clonotype1537 clonotype1538 clonotype1539  clonotype154 clonotype1540 clonotype1541 clonotype1542 
            1             1             1             4             1             1             1 
clonotype1543 clonotype1544 clonotype1545 clonotype1546 clonotype1547 clonotype1548 clonotype1549 
            1             1             1             1             1             1             1 
 clonotype155 clonotype1550 clonotype1551 clonotype1552 clonotype1554 clonotype1555 clonotype1556 
            4             1             1             1             1             1             1 
clonotype1557 clonotype1558  clonotype156 clonotype1560 clonotype1561 clonotype1562 clonotype1563 
            1             1             4             1             1             1             1 
clonotype1564 clonotype1565 clonotype1566 clonotype1568 clonotype1569  clonotype157 clonotype1570 
            1             1             1             1             1             4             1 
clonotype1571 clonotype1572 clonotype1573 clonotype1574 clonotype1575 clonotype1576 clonotype1577 
            1             1             1             1             1             1             1 
clonotype1578 clonotype1579  clonotype158 clonotype1580 clonotype1581 clonotype1582 clonotype1583 
            1             1             4             1             1             1             1 
clonotype1584 clonotype1585 clonotype1586 clonotype1587 clonotype1588 clonotype1589  clonotype159 
            1             1             1             1             1             1             4 
clonotype1590 clonotype1591 clonotype1592 clonotype1593 clonotype1594 clonotype1595 clonotype1596 
            1             1             1             1             1             1             1 
clonotype1597 clonotype1598   clonotype16  clonotype160 clonotype1600 clonotype1601  clonotype161 
            1             1            15             3             1             1             4 
 clonotype162  clonotype163  clonotype164  clonotype165  clonotype166  clonotype167  clonotype168 
            3             4             3             4             3             3             4 
 clonotype169   clonotype17  clonotype170  clonotype171  clonotype172  clonotype173  clonotype174 
            4            15             4             4             4             3             4 
 clonotype175  clonotype176  clonotype177  clonotype178  clonotype179   clonotype18  clonotype180 
            3             4             4             4             4            13             4 
 clonotype181  clonotype182  clonotype183  clonotype184  clonotype185  clonotype186  clonotype187 
            4             4             4             4             4             4             4 
 clonotype188  clonotype189   clonotype19  clonotype190  clonotype191  clonotype192  clonotype193 
            4             4            14             4             2             4             4 
 clonotype194  clonotype195  clonotype196  clonotype197  clonotype198  clonotype199    clonotype2 
            4             4             3             4             4             4          7241 
  clonotype20  clonotype200  clonotype201  clonotype202  clonotype203  clonotype204  clonotype205 
           14             4             4             4             4             4             4 
 clonotype206  clonotype207  clonotype208  clonotype209   clonotype21  clonotype210  clonotype211 
            4             3             4             4            14             4             4 
 clonotype212  clonotype213  clonotype214  clonotype215  clonotype216  clonotype217  clonotype218 
            4             4             4             4             4             3             4 
 clonotype219   clonotype22  clonotype220  clonotype221  clonotype222  clonotype223  clonotype224 
            4            11             4             4             4             3             4 
 clonotype225  clonotype226  clonotype227  clonotype228  clonotype229   clonotype23  clonotype230 
            4             4             4             4             3            12             4 
 clonotype231  clonotype232  clonotype233  clonotype234  clonotype235  clonotype236  clonotype237 
            3             3             4             4             3             4             4 
 clonotype238  clonotype239   clonotype24  clonotype240  clonotype241  clonotype242  clonotype243 
            4             4            10             4             4             4             4 
 clonotype244  clonotype245  clonotype246  clonotype247  clonotype248  clonotype249   clonotype25 
            4             4             4             4             4             4            12 
 clonotype250  clonotype251  clonotype252  clonotype253  clonotype254  clonotype255  clonotype256 
            4             4             4             4             4             3             4 
 clonotype257  clonotype258  clonotype259   clonotype26  clonotype260  clonotype261  clonotype262 
            4             3             3            10             3             2             3 
 clonotype263  clonotype264  clonotype265  clonotype266  clonotype267  clonotype268  clonotype269 
            3             3             3             3             2             3             3 
  clonotype27  clonotype270  clonotype271  clonotype272  clonotype273  clonotype274  clonotype275 
           10             3             2             2             3             3             3 
 clonotype276  clonotype277  clonotype278  clonotype279   clonotype28  clonotype280  clonotype281 
            3             2             2             3             9             3             3 
 clonotype282  clonotype283  clonotype284  clonotype285  clonotype286  clonotype287  clonotype288 
            2             2             3             3             3             2             3 
 clonotype289   clonotype29  clonotype290  clonotype291  clonotype292  clonotype293  clonotype294 
            3             9             3             3             3             3             2 
 clonotype295  clonotype296  clonotype297  clonotype298  clonotype299    clonotype3   clonotype30 
            2             3             3             3             2           221             9 
 clonotype300  clonotype301  clonotype302  clonotype303  clonotype304  clonotype305  clonotype306 
            3             3             3             3             3             3             3 
 clonotype307  clonotype308  clonotype309   clonotype31  clonotype310  clonotype311  clonotype312 
            3             2             3             8             3             3             2 
 clonotype313  clonotype314  clonotype315  clonotype316  clonotype317  clonotype318  clonotype319 
            3             2             3             3             3             3             2 
  clonotype32  clonotype320  clonotype321  clonotype322  clonotype323  clonotype324  clonotype325 
            9             3             3             2             3             3             3 
 clonotype326  clonotype327  clonotype328  clonotype329   clonotype33  clonotype330  clonotype331 
            3             2             3             3             8             3             3 
 clonotype332  clonotype333  clonotype334  clonotype335  clonotype336  clonotype337  clonotype338 
            3             3             2             3             3             3             2 
 clonotype339   clonotype34  clonotype340  clonotype341  clonotype342  clonotype343  clonotype344 
            3             9             3             1             3             2             3 
 clonotype345  clonotype346  clonotype347  clonotype348  clonotype349   clonotype35  clonotype350 
            3             3             2             3             3             8             2 
 clonotype351  clonotype352  clonotype353  clonotype354  clonotype355  clonotype356  clonotype357 
            3             3             2             3             3             3             3 
 clonotype358  clonotype359   clonotype36  clonotype360  clonotype361  clonotype362  clonotype363 
            3             2             8             2             3             3             3 
 clonotype364  clonotype365  clonotype366  clonotype367  clonotype368  clonotype369   clonotype37 
            2             3             2             3             3             3             8 
 clonotype370  clonotype371  clonotype372  clonotype373  clonotype374  clonotype375  clonotype376 
            2             2             2             3             2             3             3 
 clonotype377  clonotype378  clonotype379   clonotype38  clonotype380  clonotype381  clonotype382 
            3             2             3             7             3             3             3 
 clonotype383  clonotype384  clonotype385  clonotype386  clonotype387  clonotype388  clonotype389 
            3             3             3             3             3             2             3 
  clonotype39  clonotype390  clonotype391  clonotype392  clonotype393  clonotype394  clonotype395 
            7             3             3             3             3             3             3 
 clonotype396  clonotype397  clonotype398  clonotype399    clonotype4   clonotype40  clonotype400 
            3             3             2             2            87             6             2 
 clonotype401  clonotype402  clonotype403  clonotype404  clonotype405  clonotype406  clonotype407 
            1             3             3             3             3             2             2 
 clonotype408  clonotype409   clonotype41  clonotype410  clonotype411  clonotype412  clonotype413 
            3             3             7             3             3             1             3 
 clonotype414  clonotype415  clonotype416  clonotype417  clonotype418  clonotype419   clonotype42 
            3             3             2             3             3             2             7 
 clonotype420  clonotype421  clonotype422  clonotype423  clonotype424  clonotype425  clonotype426 
            3             3             3             3             3             3             3 
 clonotype427  clonotype428  clonotype429   clonotype43  clonotype430  clonotype431  clonotype432 
            2             3             2             7             3             3             3 
 clonotype433  clonotype434  clonotype435  clonotype436  clonotype437  clonotype438  clonotype439 
            2             3             3             2             2             3             3 
  clonotype44  clonotype440  clonotype441  clonotype442  clonotype443  clonotype444  clonotype445 
            7             3             3             3             3             3             3 
 clonotype446  clonotype447  clonotype448  clonotype449   clonotype45  clonotype450  clonotype451 
            3             3             3             3             6             3             3 
 clonotype452  clonotype453  clonotype454  clonotype455  clonotype456  clonotype457  clonotype458 
            3             3             2             3             1             3             2 
 clonotype459   clonotype46  clonotype460  clonotype461  clonotype462  clonotype463  clonotype464 
            3             7             3             3             3             3             3 
 clonotype465  clonotype466  clonotype467  clonotype468  clonotype469   clonotype47  clonotype470 
            3             3             3             3             3             7             3 
 clonotype471  clonotype472  clonotype473  clonotype474  clonotype475  clonotype476  clonotype477 
            3             3             3             3             3             1             2 
 clonotype478  clonotype479   clonotype48  clonotype480  clonotype481  clonotype482  clonotype483 
            2             2             6             3             3             3             3 
 clonotype484  clonotype485  clonotype486  clonotype487  clonotype488  clonotype489   clonotype49 
            3             3             3             3             3             3             7 
 clonotype490  clonotype491  clonotype492  clonotype493  clonotype494  clonotype495  clonotype496 
            2             3             3             3             3             3             3 
 clonotype497  clonotype498  clonotype499    clonotype5   clonotype50  clonotype500 
            3             2             2            77             6             3 
 [ reached 'max' / getOption("max.print") -- omitted 554 entries ]

3.2 Identify expanded malignant T cells

# Since your Seurat metadata Sample_ID only contains "SS" and "CTRL",
# subset malignant cells only within the "SS" samples (Sézary syndrome samples)
ss_samples <- c("SS")  

malignant_cells_list <- list()

for (s in ss_samples) {
  # Subset Seurat object to the sample of interest
  sample_cells <- subset(merged_seurat, subset = Sample_ID == s)
  
  # Check that clonotype metadata exists and is not empty
  if (!"clonotype" %in% colnames(sample_cells@meta.data)) next
  if (length(sample_cells$clonotype) == 0) next
  
  # Count clonotype frequencies
  clonotype_counts <- table(sample_cells$clonotype)
  
  # Identify expanded clones with clone size >= 5 cells
  expanded_clones <- names(clonotype_counts[clonotype_counts >= 5])
  if (length(expanded_clones) == 0) next
  
  # Subset cells belonging to expanded clonotypes (putative malignant cells)
  malignant <- subset(sample_cells, subset = clonotype %in% expanded_clones)
  
  # Add to list if nonempty
  if (ncol(malignant) > 0) malignant_cells_list[[s]] <- malignant
}

# Remove null elements and unname list
malignant_cells_list <- malignant_cells_list[!sapply(malignant_cells_list, is.null)]
malignant_cells_list <- unname(malignant_cells_list)

# Merge all malignant Sezary cells across samples into one Seurat object
sezary_cells <- Reduce(function(x, y) merge(x, y), malignant_cells_list)

3.3 highlight top clones

Idents(sezary_cells) <- "sample"

library(scRepertoire)
library(Seurat)

# ================================
# Clonal homeostasis (size distribution)
# ================================
clonalHomeostasis(sezary_cells, cloneCall = "clonotype")

# ================================
#  Clonal proportion / relative abundance
# ================================
clonalProportion(sezary_cells, cloneCall = "clonotype")

NA
NA

3.4 Merge all Sézary malignant cells with healthy controls

# ================================
# Add tissue grouping column
# ================================
ss_vs_hc$tissue_group <- case_when(
  ss_vs_hc$orig.ident %in% c("Control") ~ "HC",   # assuming Control is blood
  ss_vs_hc$orig.ident %in% c("SS_P2", "SS_P3", "SS_P4", "SS_P5", "SS_P6") ~ "SS",  # or Skin if known
  TRUE ~ "Other"
)

# Quick check
table(ss_vs_hc$tissue_group)

   HC    SS 
 4437 33393 

# ================================
# Visualize CLIC1 expression
# ================================

# FeaturePlot with red gradient
FeaturePlot(ss_vs_hc, features = "CLIC1", reduction = "umap",
            cols = c("grey90", "red"), label = TRUE, repel = T)

# DotPlot by sample_id with red-grey gradient
DotPlot(ss_vs_hc, features = "CLIC1", group.by = "Sample_ID") +
  RotatedAxis() +
  scale_color_gradient(low = "grey90", high = "red") +
  ggtitle("CLIC1 Expression by Sample") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# DotPlot by tissue_group with red-grey gradient
DotPlot(ss_vs_hc, features = "CLIC1", group.by = "orig.ident") +
  RotatedAxis() +
  scale_color_gradient(low = "grey90", high = "red") +
  ggtitle("CLIC1 Expression by Tissue") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# ================================
# Visualize CLIC1
# ================================
# FeaturePlot
FeaturePlot(ss_vs_hc, features = "CLIC1", reduction = "umap")

# DotPlot grouped by sample
DotPlot(ss_vs_hc, features = "CLIC1", group.by = "Sample_ID") + RotatedAxis()

# DotPlot grouped by tissue
DotPlot(ss_vs_hc, features = "CLIC1", group.by = "orig.ident") + RotatedAxis()

3.5 Visualize CLIC1

# DimPlot by sample
DimPlot(ss_vs_hc, group.by = "Sample_ID", reduction = "umap")

# FeaturePlot for CLIC1
FeaturePlot(ss_vs_hc, features = "CLIC1", reduction = "umap")

# DotPlot for CLIC1 across groups
DotPlot(ss_vs_hc, features = "CLIC1", group.by = "Sample_ID") + 
  theme(axis.text.x = element_text(angle = 45, hjust = 1))

# FeaturePlot with red gradient
FeaturePlot(ss_vs_hc, features = "CLIC1", reduction = "umap",
            cols = c("grey90", "red"), label = TRUE, repel = T)

3.6 FeaturePlots for Top50 UP

top_50_up <- read.csv("top_50_upregulated.csv")        # or read.delim("top_50_up.tsv")
top_50_down <- read.csv("top_50_downregulated.csv")



FeaturePlot(ss_vs_hc, 
             features = top_50_up$gene[1:10], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_up$gene[11:20], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_up$gene[21:30], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_up$gene[31:40], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_up$gene[41:50], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

3.7 FeaturePlots for Top50 DOWN

FeaturePlot(ss_vs_hc, 
             features = top_50_down$gene[1:10], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_down$gene[11:20], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_down$gene[21:30], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_down$gene[31:40], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_down$gene[41:50], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

NA
NA
NA

3.8 Visualization of Potential biomarkers-Upregulated

# Vector of genes to plot
up_genes <- c("CLIC1", "COX5A","GTSF1", "MAD2L1","MYBL2","MYL6B","NME1","PLK1", "PYCR1", "SLC25A5", "SRI", "TUBA1C", "UBE2T", "YWHAH")

# DotPlot with custom firebrick-red gradient
DotPlot(ss_vs_hc, features = up_genes) +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Upregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# DotPlot with custom firebrick-red gradient
DotPlot(ss_vs_hc, features = up_genes, group.by = "Sample_ID") +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Upregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# DotPlot with custom firebrick-red gradient
DotPlot(ss_vs_hc, features = up_genes, group.by = "orig.ident") +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Upregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

3.9 Visualization of Potential biomarkers-Downregulated

# Downregulated genes
down_genes <- c("TXNIP", "RASA3", "RIPOR2", 
                "ZFP36", "ZFP36L1", "ZFP36L2",
                "PRMT2", "MAX", "PIK3IP1", 
                "BTG1", "CDKN1B")

# DotPlot with firebrick color for high expression
DotPlot(ss_vs_hc, features = down_genes) +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Downregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# DotPlot with firebrick color for high expression
DotPlot(ss_vs_hc, features = down_genes, group.by = "Sample_ID") +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Downregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# DotPlot with firebrick color for high expression
DotPlot(ss_vs_hc, features = down_genes, group.by = "orig.ident") +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Downregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

NA
NA
NA

---

4 Differential Expression

4.1 Compare condition using RNA assay-ss_vs_hc

4.2 Compare condition using RNA assay-Merged seurat

# Load required libraries
library(Seurat)
library(dplyr)
library(tibble)

table(merged_seurat$Sample_ID)

 CTRL    SS 
 4437 46658 

combined_seu <- merged_seurat

# Join the layers of the RNA assay
combined_seu <- JoinLayers(combined_seu, assay = "RNA")

# Ensure your identity class is set to disease status
Idents(combined_seu) <- "Sample_ID"  # e.g., levels: "SS", "Control"

# Run differential expression between SS vs Control
markers_disease <- FindMarkers(
  object = combined_seu,
  ident.1 = "SS",
  ident.2 = "CTRL",
  assay = "RNA",
  logfc.threshold = 0,
  min.pct = 0,
  test.use = "wilcox"  
)

# Save results to CSV
write.csv(markers_disease, file = "MergedSeurat_DE_SS_vs_Healthy_Borcherding2023.csv", row.names = TRUE)

# Get log-normalized expression matrix (RNA assay)
expression_data_RNA <- GetAssayData(combined_seu, assay = "RNA", slot = "data")

# Get cell names for each group
ss_cells <- WhichCells(combined_seu, idents = "SS")
healthy_cells <- WhichCells(combined_seu, idents = "CTRL")

# Function to add mean expression per group
calculate_mean_expression <- function(markers, group1_cells, group2_cells, expression_data) {
  group1_mean <- rowMeans(expression_data[, group1_cells, drop = FALSE], na.rm = TRUE)
  group2_mean <- rowMeans(expression_data[, group2_cells, drop = FALSE], na.rm = TRUE)
  markers <- markers %>%
    rownames_to_column("gene") %>%
    mutate(
      mean_expr_SS = group1_mean[gene],
      mean_expr_Healthy = group2_mean[gene],
      log2FC_manual = log2(mean_expr_SS + 1) - log2(mean_expr_Healthy + 1)
    )
  return(markers)
}

# Apply the function and save final result
markers_disease_with_mean <- calculate_mean_expression(markers_disease, ss_cells, healthy_cells, expression_data_RNA)
write.csv(markers_disease_with_mean, "MergedSeurat_DE_SS_vs_Healthy_with_MeanExpr_Borcherding2023.csv", row.names = FALSE)

---

5 Save Final Seurat object

sessionInfo()

R version 4.5.2 (2025-10-31)
Platform: x86_64-pc-linux-gnu
Running under: Ubuntu 24.04.3 LTS

Matrix products: default
BLAS:   /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.12.0 
LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.12.0  LAPACK version 3.12.0

locale:
 [1] LC_CTYPE=en_GB.UTF-8       LC_NUMERIC=C               LC_TIME=fr_FR.UTF-8        LC_COLLATE=en_GB.UTF-8    
 [5] LC_MONETARY=fr_FR.UTF-8    LC_MESSAGES=en_GB.UTF-8    LC_PAPER=fr_FR.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C             LC_MEASUREMENT=fr_FR.UTF-8 LC_IDENTIFICATION=C       

time zone: Europe/Paris
tzcode source: system (glibc)

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] stringr_1.6.0      readr_2.1.5        tidyr_1.3.1        patchwork_1.3.2    rstatix_0.7.3      ggpubr_0.6.2      
 [7] scRepertoire_2.6.1 ggplot2_4.0.0      tibble_3.3.0       dplyr_1.1.4        Seurat_5.3.1       SeuratObject_5.2.0
[13] sp_2.2-0          

loaded via a namespace (and not attached):
  [1] RcppAnnoy_0.0.22            splines_4.5.2               later_1.4.4                 polyclip_1.10-7            
  [5] fastDummies_1.7.5           lifecycle_1.0.4             vroom_1.6.6                 globals_0.18.0             
  [9] lattice_0.22-7              MASS_7.3-65                 backports_1.5.0             magrittr_2.0.4             
 [13] limma_3.66.0                sass_0.4.10                 plotly_4.11.0               rmarkdown_2.30             
 [17] jquerylib_0.1.4             yaml_2.3.10                 httpuv_1.6.16               otel_0.2.0                 
 [21] sctransform_0.4.2           spam_2.11-1                 spatstat.sparse_3.1-0       reticulate_1.44.0          
 [25] cowplot_1.2.0               pbapply_1.7-4               RColorBrewer_1.1-3          abind_1.4-8                
 [29] rvest_1.0.5                 Rtsne_0.17                  GenomicRanges_1.62.0        presto_1.0.0               
 [33] purrr_1.2.0                 ggraph_2.2.2                BiocGenerics_0.56.0         hash_2.2.6.3               
 [37] tweenr_2.0.3                evmix_2.12                  IRanges_2.44.0              S4Vectors_0.48.0           
 [41] ggrepel_0.9.6               irlba_2.3.5.1               listenv_0.10.0              spatstat.utils_3.2-0       
 [45] iNEXT_3.0.2                 MatrixModels_0.5-4          goftest_1.2-3               RSpectra_0.16-2            
 [49] spatstat.random_3.4-2       fitdistrplus_1.2-4          parallelly_1.45.1           codetools_0.2-20           
 [53] DelayedArray_0.36.0         xml2_1.4.1                  ggforce_0.5.0               tidyselect_1.2.1           
 [57] farver_2.1.2                viridis_0.6.5               matrixStats_1.5.0           stats4_4.5.2               
 [61] spatstat.explore_3.5-3      Seqinfo_1.0.0               jsonlite_2.0.0              Formula_1.2-5              
 [65] tidygraph_1.3.1             progressr_0.18.0            ggridges_0.5.7              ggalluvial_0.12.5          
 [69] survival_3.8-3              tools_4.5.2                 ica_1.0-3                   Rcpp_1.1.0                 
 [73] glue_1.8.0                  gridExtra_2.3               SparseArray_1.10.1          xfun_0.54                  
 [77] MatrixGenerics_1.22.0       withr_3.0.2                 fastmap_1.2.0               SparseM_1.84-2             
 [81] digest_0.6.38               R6_2.6.1                    mime_0.13                   scattermore_1.2            
 [85] tensor_1.5.1                dichromat_2.0-0.1           spatstat.data_3.1-9         utf8_1.2.6                 
 [89] generics_0.1.4              data.table_1.17.8           graphlayouts_1.2.2          httr_1.4.7                 
 [93] htmlwidgets_1.6.4           S4Arrays_1.10.0             uwot_0.2.3                  pkgconfig_2.0.3            
 [97] gtable_0.3.6                rsconnect_1.6.0             lmtest_0.9-40               S7_0.2.0                   
[101] SingleCellExperiment_1.32.0 XVector_0.50.0              htmltools_0.5.8.1           carData_3.0-5              
[105] dotCall64_1.2               scales_1.4.0                Biobase_2.70.0              png_0.1-8                  
[109] spatstat.univar_3.1-4       ggdendro_0.2.0              knitr_1.50                  rstudioapi_0.17.1          
[113] tzdb_0.5.0                  reshape2_1.4.5              rjson_0.2.23                nlme_3.1-168               
[117] zoo_1.8-14                  cachem_1.1.0                KernSmooth_2.23-26          parallel_4.5.2             
[121] miniUI_0.1.2                pillar_1.11.1               grid_4.5.2                  vctrs_0.6.5                
[125] RANN_2.6.2                  promises_1.4.0              car_3.1-3                   xtable_1.8-4               
[129] cluster_2.1.8.1             evaluate_1.0.5              cli_3.6.5                   compiler_4.5.2             
[133] crayon_1.5.3                rlang_1.1.6                 future.apply_1.20.0         ggsignif_0.6.4             
[137] labeling_0.4.3              immApex_1.4.0               plyr_1.8.9                  stringi_1.8.7              
[141] viridisLite_0.4.2           deldir_2.0-4                gsl_2.1-9                   lazyeval_0.2.2             
[145] spatstat.geom_3.6-0         quantreg_6.1                Matrix_1.7-4                RcppHNSW_0.6.0             
[149] hms_1.1.4                   bit64_4.6.0-1               future_1.67.0               statmod_1.5.1              
[153] shiny_1.11.1                SummarizedExperiment_1.40.0 ROCR_1.0-11                 igraph_2.2.1               
[157] broom_1.0.10                memoise_2.0.1               bslib_0.9.0                 bit_4.6.0                  

---