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My classwork from bimm143
Class13
Samuel Fisher (A18131929)
Background
Today we will perform an RNASeq analysis on the effects of dexamethasone (hereafter “dex”), a common steroid, on airway smooth muscle (ASM) cell lines.
Data Import
We need two things for this analysis:
1) countData: a table with genes as rows and samples/experiments as columns
2) colData: metadata about the columns (i.e. samples) in the main countData object
counts <- read.csv("airway_scaledcounts.csv", row.names=1)
metadata <- read.csv("airway_metadata.csv")
Let’s have a peak at the objects:
metadata
id dex celltype geo_id
1 SRR1039508 control N61311 GSM1275862
2 SRR1039509 treated N61311 GSM1275863
3 SRR1039512 control N052611 GSM1275866
4 SRR1039513 treated N052611 GSM1275867
5 SRR1039516 control N080611 GSM1275870
6 SRR1039517 treated N080611 GSM1275871
7 SRR1039520 control N061011 GSM1275874
8 SRR1039521 treated N061011 GSM1275875
counts
SRR1039508 SRR1039509 SRR1039512 SRR1039513 SRR1039516
ENSG00000000003 723 486 904 445 1170
ENSG00000000005 0 0 0 0 0
ENSG00000000419 467 523 616 371 582
ENSG00000000457 347 258 364 237 318
ENSG00000000460 96 81 73 66 118
ENSG00000000938 0 0 1 0 2
ENSG00000000971 3413 3916 6000 4308 6424
ENSG00000001036 2328 1714 2640 1381 2165
ENSG00000001084 670 372 692 448 917
ENSG00000001167 426 295 531 178 740
ENSG00000001460 191 182 287 137 256
ENSG00000001461 2232 1969 5252 2698 2699
ENSG00000001497 556 513 619 355 666
ENSG00000001561 74 53 212 160 22
ENSG00000001617 560 372 934 436 706
ENSG00000001626 16 9 14 8 8
ENSG00000001629 1491 1204 2438 1041 2838
ENSG00000001630 1014 807 1135 689 1374
ENSG00000001631 694 725 1052 475 1137
ENSG00000002016 256 202 263 109 308
ENSG00000002079 1 0 7 7 1
ENSG00000002330 760 653 796 431 769
ENSG00000002549 1460 1274 1280 980 1428
ENSG00000002586 7314 7067 9639 6221 11373
ENSG00000002587 2 3 1 1 1
ENSG00000002726 1 0 4 2 0
ENSG00000002745 5 5 28 9 2
ENSG00000002746 172 160 104 86 130
ENSG00000002822 391 306 415 185 424
ENSG00000002834 7577 7641 9125 7305 7858
ENSG00000002919 230 225 273 207 247
ENSG00000002933 1296 1542 594 433 36
ENSG00000003056 1954 1665 1960 1309 2096
ENSG00000003096 644 330 590 178 555
ENSG00000003137 140 55 235 75 31
ENSG00000003147 9 4 9 8 8
ENSG00000003249 85 73 221 84 107
ENSG00000003393 900 728 875 514 979
ENSG00000003400 101 71 121 104 97
ENSG00000003402 2065 3648 2340 2991 2828
ENSG00000003436 4665 4109 6899 3253 9138
ENSG00000003509 306 249 379 203 410
ENSG00000003756 3095 2413 3940 1763 3701
ENSG00000003987 25 28 34 35 23
ENSG00000003989 61 51 13 3 44
ENSG00000004059 1589 1893 1519 1136 1747
ENSG00000004139 311 354 326 168 370
ENSG00000004142 1501 1570 1618 981 1746
ENSG00000004399 4107 4116 3858 2064 5515
ENSG00000004455 1349 1098 1549 733 1568
ENSG00000004468 8 8 2 3 8
ENSG00000004478 552 638 727 396 870
ENSG00000004487 1345 1018 1705 788 1796
ENSG00000004534 1246 1002 2084 930 1715
ENSG00000004660 98 58 59 38 87
ENSG00000004700 1376 1618 1714 1127 1293
ENSG00000004766 567 383 533 365 638
ENSG00000004776 14175 12857 13950 8278 11255
ENSG00000004777 206 106 102 62 78
ENSG00000004779 330 340 486 265 411
ENSG00000004799 82 835 127 1130 226
ENSG00000004809 2 2 3 2 1
ENSG00000004838 19 8 28 8 25
ENSG00000004846 33 9 75 12 9
ENSG00000004848 0 1 0 0 0
ENSG00000004864 141 141 104 110 226
ENSG00000004866 522 387 1077 288 600
ENSG00000004897 1662 1367 2114 1041 1980
ENSG00000004939 9 7 12 3 17
ENSG00000004948 0 0 0 0 0
ENSG00000004961 444 345 541 289 651
ENSG00000004975 1330 954 1146 517 1330
ENSG00000005001 0 0 0 0 0
ENSG00000005007 2288 2284 2515 1544 2227
ENSG00000005020 881 710 781 499 752
ENSG00000005022 1581 1253 1555 839 1472
ENSG00000005059 200 206 242 206 194
ENSG00000005073 1 0 1 0 1
ENSG00000005075 631 693 719 378 730
ENSG00000005100 440 437 554 357 530
ENSG00000005102 1 0 0 0 3
ENSG00000005108 10 12 6 5 8
ENSG00000005156 370 170 370 178 402
ENSG00000005175 334 346 318 119 596
ENSG00000005187 24 26 35 24 33
ENSG00000005189 78 61 89 56 114
ENSG00000005194 438 373 620 314 522
ENSG00000005206 748 656 767 377 747
ENSG00000005238 801 634 853 408 991
ENSG00000005243 1171 949 1236 746 931
ENSG00000005249 227 247 279 328 284
ENSG00000005302 441 547 488 338 579
ENSG00000005339 2660 1793 3240 1690 3210
ENSG00000005379 213 128 120 134 150
ENSG00000005381 3 30 7 1 0
ENSG00000005421 0 0 0 0 1
ENSG00000005436 313 303 292 239 343
ENSG00000005448 241 214 267 158 233
ENSG00000005469 503 396 344 191 629
ENSG00000005471 67 15 61 16 18
ENSG00000005483 1863 1563 1921 1107 2491
ENSG00000005486 741 620 1121 562 1113
ENSG00000005513 10 2 5 0 4
ENSG00000005700 543 431 790 393 657
ENSG00000005801 210 212 298 161 321
ENSG00000005810 3126 2627 3159 2108 3415
ENSG00000005812 1493 1459 1472 1359 1192
ENSG00000005844 5 0 1 3 9
ENSG00000005882 954 877 1254 681 1413
ENSG00000005884 701 795 1582 1106 1392
ENSG00000005889 792 584 766 404 852
ENSG00000005893 5053 4502 6271 3792 5773
ENSG00000005961 9 13 24 5 11
ENSG00000005981 4 6 4 2 8
ENSG00000006007 2249 2181 3008 2143 3340
ENSG00000006015 544 481 416 195 261
ENSG00000006016 154 112 91 82 92
ENSG00000006025 226 178 229 95 202
ENSG00000006042 3108 2545 3486 2057 3876
ENSG00000006047 0 1 1 1 1
ENSG00000006059 0 0 0 0 0
ENSG00000006062 335 294 333 115 415
ENSG00000006071 0 0 0 0 0
ENSG00000006116 0 0 1 0 2
ENSG00000006118 528 369 604 282 832
ENSG00000006125 7030 5372 7725 5197 7765
ENSG00000006128 0 4 1 0 0
ENSG00000006194 527 508 612 377 665
ENSG00000006210 9 19 9 6 9
ENSG00000006282 1765 1372 1340 752 1610
ENSG00000006283 100 46 48 11 124
ENSG00000006327 523 347 708 250 767
ENSG00000006377 0 0 0 1 0
ENSG00000006432 8 5 12 8 11
ENSG00000006451 1833 949 1094 568 1629
ENSG00000006453 214 151 260 150 272
ENSG00000006459 381 299 461 266 459
ENSG00000006468 190 109 167 133 136
ENSG00000006530 251 254 292 173 444
ENSG00000006534 885 1132 1380 847 1439
ENSG00000006555 9 4 6 2 2
ENSG00000006576 946 718 1612 711 1267
ENSG00000006606 3 3 8 1 8
ENSG00000006607 709 903 971 860 812
ENSG00000006611 0 0 0 0 0
ENSG00000006625 195 186 196 84 254
ENSG00000006634 132 72 104 74 90
ENSG00000006638 76 54 105 46 65
ENSG00000006652 922 788 1022 597 1662
ENSG00000006659 1 0 0 1 3
ENSG00000006695 184 140 200 141 220
ENSG00000006704 337 281 380 164 274
ENSG00000006712 985 879 1550 719 1562
ENSG00000006715 2045 1560 2949 1410 2865
ENSG00000006740 42 29 33 20 52
ENSG00000006744 836 931 1008 636 1253
ENSG00000006747 18 29 35 12 18
ENSG00000006756 538 423 591 371 664
ENSG00000006757 161 155 176 105 223
ENSG00000006788 5 37 0 1 5
ENSG00000006831 1055 1009 1170 894 1174
ENSG00000006837 72 56 90 80 100
ENSG00000007001 2 4 5 0 3
ENSG00000007038 0 0 2 1 0
ENSG00000007047 641 654 925 519 1247
ENSG00000007062 3 2 6 1 3
ENSG00000007080 633 715 707 402 797
ENSG00000007129 0 0 0 0 0
ENSG00000007168 3486 3399 4955 2670 4943
ENSG00000007171 0 2 0 0 2
ENSG00000007174 1 3 3 4 4
ENSG00000007202 6595 5389 7849 4289 7838
ENSG00000007216 0 0 0 0 0
ENSG00000007237 378 153 304 37 282
ENSG00000007255 140 155 124 118 130
ENSG00000007264 3 3 4 1 2
ENSG00000007306 0 0 0 0 0
ENSG00000007312 1 2 0 4 5
ENSG00000007314 0 1 2 1 1
ENSG00000007341 274 172 282 171 302
ENSG00000007350 0 0 0 0 0
ENSG00000007372 29 62 32 11 29
ENSG00000007376 278 265 222 137 341
ENSG00000007384 2000 1978 1951 983 1940
ENSG00000007392 792 659 766 430 857
ENSG00000007402 21 24 24 29 12
ENSG00000007516 6 6 6 14 25
ENSG00000007520 655 565 632 317 602
ENSG00000007541 710 669 694 361 651
ENSG00000007545 615 679 771 383 663
ENSG00000007866 1522 2112 1701 1244 1350
ENSG00000007908 0 0 0 0 0
ENSG00000007923 880 885 1095 611 1375
ENSG00000007933 782 826 2205 1633 223
ENSG00000007944 368 247 535 185 520
ENSG00000007952 11 15 7 8 10
ENSG00000007968 4 6 6 3 3
ENSG00000008018 1674 1494 1789 1115 1595
ENSG00000008056 7 23 24 15 15
ENSG00000008083 274 260 283 227 300
ENSG00000008086 323 293 585 198 428
ENSG00000008118 2 4 13 2 14
ENSG00000008128 574 559 657 336 345
ENSG00000008130 1345 2061 1441 1443 1357
ENSG00000008196 1 1 6 1 0
ENSG00000008197 0 0 0 0 0
ENSG00000008226 8 3 13 5 6
ENSG00000008256 1746 3230 3066 3432 2924
ENSG00000008277 28 19 18 20 15
ENSG00000008282 1959 1841 1966 1707 1827
ENSG00000008283 594 538 645 336 565
ENSG00000008294 2688 2195 3950 1736 3465
ENSG00000008300 55 76 127 85 104
ENSG00000008311 478 972 549 670 423
ENSG00000008323 8 12 7 3 16
ENSG00000008324 112 157 85 106 105
ENSG00000008382 156 124 167 87 198
ENSG00000008394 2589 2703 3127 1895 8070
ENSG00000008405 296 308 269 208 288
ENSG00000008438 0 6 1 1 1
ENSG00000008441 14126 9551 13469 6036 12014
ENSG00000008513 2256 1341 1760 554 2489
ENSG00000008516 19 22 31 6 23
ENSG00000008517 179 82 210 63 66
ENSG00000008710 5059 5956 5307 3517 4361
ENSG00000008735 15 22 21 13 19
ENSG00000008838 1320 1003 1229 659 1355
ENSG00000008853 644 727 956 562 837
ENSG00000008869 796 639 799 505 778
ENSG00000008952 4233 4365 5671 3341 6442
ENSG00000008988 13036 11874 13193 7244 11543
ENSG00000009307 15086 12706 19963 11668 20102
ENSG00000009335 1932 2021 2275 1634 2561
ENSG00000009413 5320 10463 6250 7792 6761
ENSG00000009694 18 7 2 3 4
ENSG00000009709 0 0 0 0 0
ENSG00000009724 40 30 60 24 46
ENSG00000009765 12 30 4 1 1
ENSG00000009780 150 187 173 146 165
ENSG00000009790 3 6 6 3 10
ENSG00000009830 381 460 546 285 431
ENSG00000009844 829 675 1092 532 1201
ENSG00000009950 10 21 6 2 3
ENSG00000009954 2827 2653 3352 2041 3688
ENSG00000010017 1510 2278 1804 2001 2124
ENSG00000010030 30 15 22 11 17
ENSG00000010072 194 143 203 105 216
ENSG00000010165 959 950 1177 688 1089
ENSG00000010219 262 189 326 142 314
ENSG00000010244 2884 2599 2943 1669 3710
ENSG00000010256 1649 1795 1587 1379 1940
ENSG00000010270 888 800 884 504 964
ENSG00000010278 3133 3438 3747 3527 2633
ENSG00000010282 0 0 0 0 0
ENSG00000010292 1407 1139 1356 827 1409
ENSG00000010295 1471 1280 1679 865 897
ENSG00000010310 27 13 30 12 22
ENSG00000010318 61 71 81 71 71
ENSG00000010319 1 0 1 0 0
ENSG00000010322 2581 2022 3157 1472 3739
ENSG00000010327 0 0 1 1 0
ENSG00000010361 273 226 312 170 208
ENSG00000010379 0 1 7 1 0
ENSG00000010404 2447 2016 3319 1999 3261
ENSG00000010438 7 11 12 6 19
ENSG00000010539 140 128 166 108 183
ENSG00000010610 37 18 97 53 122
ENSG00000010626 151 103 210 111 179
ENSG00000010671 0 0 0 0 3
ENSG00000010704 508 302 674 303 503
ENSG00000010803 770 660 858 531 995
ENSG00000010810 1783 1707 1607 882 2566
ENSG00000010818 1884 1023 1633 835 1992
ENSG00000010932 16 33 70 46 0
ENSG00000011007 1470 1365 1388 954 1533
ENSG00000011009 451 500 518 265 579
ENSG00000011021 879 666 655 397 719
ENSG00000011028 17930 13855 25972 10769 17814
ENSG00000011052 307 195 298 80 274
ENSG00000011083 0 0 1 1 1
ENSG00000011105 3095 2676 3524 2493 3490
ENSG00000011114 721 462 606 398 686
ENSG00000011132 198 220 301 143 258
ENSG00000011143 207 145 226 100 159
ENSG00000011198 586 1488 820 1040 1219
ENSG00000011201 2 0 1 0 0
ENSG00000011243 823 694 963 430 1072
ENSG00000011258 473 282 405 224 464
ENSG00000011260 489 465 369 295 444
ENSG00000011275 1680 1455 1932 1070 2206
ENSG00000011295 428 369 466 329 459
ENSG00000011304 3395 3914 3193 2347 4152
ENSG00000011332 25 12 18 18 10
ENSG00000011347 174 87 157 69 217
ENSG00000011376 547 454 448 278 436
ENSG00000011405 1181 1300 1462 843 1906
ENSG00000011422 399 368 489 250 347
ENSG00000011426 265 251 239 138 346
ENSG00000011451 1536 1653 1596 899 1630
ENSG00000011454 1864 1776 2363 2292 2468
ENSG00000011465 218156 190421 427652 214758 302872
ENSG00000011478 95 138 138 133 143
ENSG00000011485 818 1191 963 672 1131
ENSG00000011523 752 710 999 580 1002
ENSG00000011566 851 1132 1434 1153 1367
ENSG00000011590 6 7 7 0 3
ENSG00000011600 0 1 4 3 0
ENSG00000011638 975 808 771 661 837
ENSG00000011677 0 2 1 2 1
ENSG00000012048 332 109 485 89 411
ENSG00000012061 1160 913 1519 599 1388
ENSG00000012124 4 4 4 0 7
ENSG00000012171 1860 956 2108 723 1281
ENSG00000012174 722 620 848 523 856
ENSG00000012211 109 139 138 65 134
ENSG00000012223 0 0 0 1 3
ENSG00000012232 1271 1030 1370 885 1652
ENSG00000012504 0 0 0 0 0
ENSG00000012660 2069 1877 2572 2274 2236
ENSG00000012779 0 1 0 0 0
ENSG00000012817 887 816 993 483 1227
ENSG00000012822 4687 5376 6913 5128 6323
ENSG00000012963 333 305 451 250 320
ENSG00000012983 1997 1575 2569 1205 2816
ENSG00000013016 2070 3397 1763 885 1808
ENSG00000013275 503 522 518 317 503
ENSG00000013288 3106 2541 4085 2321 3913
ENSG00000013293 222 63 389 114 424
ENSG00000013297 6526 1575 5336 1498 5069
ENSG00000013306 1015 1023 1095 602 1303
ENSG00000013364 9639 8804 9096 5778 8309
ENSG00000013374 1208 1116 1788 1043 2071
ENSG00000013375 1452 984 1762 707 1874
ENSG00000013392 226 142 275 153 299
ENSG00000013441 2515 2754 2337 2351 3115
ENSG00000013503 336 295 369 143 399
ENSG00000013523 428 303 483 192 530
ENSG00000013561 770 854 1054 783 1104
ENSG00000013563 406 361 770 440 799
ENSG00000013573 331 229 264 126 240
ENSG00000013583 1258 1289 1497 1299 1670
ENSG00000013588 594 450 975 541 802
ENSG00000013619 757 1112 1256 823 1296
ENSG00000013725 11 10 6 7 13
ENSG00000013810 95 134 124 65 69
ENSG00000014123 934 716 1014 621 1088
ENSG00000014138 190 108 276 84 152
ENSG00000014164 578 498 546 286 523
ENSG00000014216 3114 3533 3956 2480 3546
ENSG00000014257 26 16 21 5 23
ENSG00000014641 1613 1301 1892 1105 1768
ENSG00000014824 1102 1101 1449 1056 1672
ENSG00000014914 597 348 775 439 642
ENSG00000014919 714 510 728 441 830
ENSG00000015133 5 10 1 3 4
ENSG00000015153 183 279 273 201 320
ENSG00000015171 2042 1927 2663 1381 2867
ENSG00000015285 7 14 12 2 7
ENSG00000015413 0 3 0 1 2
ENSG00000015475 270 322 406 163 525
ENSG00000015479 5543 3333 6399 2983 7971
ENSG00000015520 22 4 23 1 65
ENSG00000015532 724 598 782 431 768
ENSG00000015568 210 112 173 85 295
ENSG00000015592 0 3 0 2 1
ENSG00000015676 1829 1527 1866 1061 1773
ENSG00000016082 0 0 1 0 0
ENSG00000016391 155 25 157 30 89
ENSG00000016402 22 6 8 3 13
ENSG00000016490 0 1 0 0 0
ENSG00000016602 1 0 0 0 0
ENSG00000016864 1537 1445 1959 1185 1508
ENSG00000017260 2792 2045 3487 2004 3693
ENSG00000017427 1100 832 1138 766 513
ENSG00000017483 703 396 566 123 528
ENSG00000017797 2930 3683 2819 2432 3442
ENSG00000018189 1110 845 1108 655 1404
ENSG00000018236 101 47 27 31 58
ENSG00000018280 23 15 19 13 17
ENSG00000018408 3114 2596 2997 1784 3449
ENSG00000018510 850 656 846 462 788
ENSG00000018607 0 0 0 0 0
ENSG00000018610 315 261 355 164 350
ENSG00000018625 285 146 3356 855 262
ENSG00000018699 255 208 268 246 324
ENSG00000018869 63 65 71 58 74
ENSG00000019102 1 0 3 7 16
ENSG00000019144 3326 4395 3783 2512 3457
ENSG00000019169 0 2 0 0 0
ENSG00000019186 65 5 8 0 9
ENSG00000019485 314 332 320 171 264
ENSG00000019505 1 0 2 2 2
ENSG00000019549 3353 5423 2830 1636 3797
ENSG00000019582 22 25 101 40 30
ENSG00000019991 1517 926 1399 338 2590
ENSG00000019995 1356 2658 1592 1916 2005
ENSG00000020129 1459 974 1272 481 1423
ENSG00000020181 11137 6620 11659 5319 11307
ENSG00000020219 0 0 0 0 1
ENSG00000020256 373 291 413 170 403
ENSG00000020426 196 181 311 181 329
ENSG00000020577 2269 4046 1925 2507 1984
ENSG00000020633 143 176 90 60 7
ENSG00000020922 562 464 553 376 543
ENSG00000021300 19 6 22 8 15
ENSG00000021355 588 567 662 457 610
ENSG00000021461 3 2 6 2 2
ENSG00000021488 5 4 4 1 7
ENSG00000021574 527 443 563 357 587
ENSG00000021645 9 22 47 71 13
ENSG00000021762 1443 1806 1032 759 1363
ENSG00000021776 1228 1276 1378 1016 1793
ENSG00000021826 404 261 461 226 302
ENSG00000021852 0 0 0 0 0
ENSG00000022267 7166 8589 11992 8443 8451
ENSG00000022277 1947 1718 2425 1378 2380
ENSG00000022355 0 0 0 0 0
ENSG00000022556 5 1 4 1 5
ENSG00000022567 146 133 156 96 186
ENSG00000022840 2564 2576 3094 2034 2940
ENSG00000022976 205 124 206 104 188
ENSG00000023041 674 637 697 481 805
ENSG00000023171 62 16 120 29 247
ENSG00000023191 2903 2739 2997 1614 2028
ENSG00000023228 1597 1657 2041 1173 2123
ENSG00000023287 2603 2406 2881 2069 3485
ENSG00000023318 1340 1261 1476 1068 1576
ENSG00000023330 1025 840 1159 549 1519
ENSG00000023445 78 83 59 29 66
ENSG00000023516 1937 1549 2110 1219 2267
ENSG00000023572 96 108 97 85 91
ENSG00000023608 230 338 430 368 426
ENSG00000023697 460 502 510 357 569
ENSG00000023734 1655 1993 2171 1574 2429
ENSG00000023839 23 33 23 6 9
ENSG00000023892 7 2 5 8 11
ENSG00000023902 654 766 618 474 863
ENSG00000023909 1079 1615 1324 1867 1347
ENSG00000024048 1322 1162 1558 997 1412
ENSG00000024422 10748 11310 12572 7587 13761
ENSG00000024526 22 33 22 17 28
ENSG00000024862 210 231 287 192 251
ENSG00000025039 3 11 8 6 19
ENSG00000025156 302 237 300 146 339
ENSG00000025293 1417 1420 1516 908 2042
ENSG00000025423 235 61 605 58 104
ENSG00000025434 403 492 513 311 399
ENSG00000025708 136 405 110 122 136
ENSG00000025770 426 364 444 191 357
ENSG00000025772 840 574 933 396 1528
ENSG00000025796 3318 2361 4840 2777 4059
ENSG00000025800 1962 1835 2372 1649 2626
ENSG00000026025 98471 64439 119665 60034 117016
ENSG00000026036 322 170 381 59 99
ENSG00000026103 991 874 1425 660 1395
ENSG00000026297 266 148 288 133 295
ENSG00000026508 19783 12248 29627 14247 30110
ENSG00000026559 34 20 16 4 47
ENSG00000026652 556 312 359 125 600
ENSG00000026751 0 1 2 4 1
ENSG00000026950 1003 485 1098 400 1019
ENSG00000027001 548 370 794 309 651
ENSG00000027075 78 43 75 52 83
ENSG00000027644 1 0 1 0 2
ENSG00000027697 1353 1607 1341 1244 1561
ENSG00000027847 453 411 568 271 484
ENSG00000027869 1 0 1 0 0
ENSG00000028116 225 247 275 180 332
ENSG00000028137 403 613 596 489 282
ENSG00000028203 997 858 1720 1051 1312
ENSG00000028277 68 29 38 3 227
ENSG00000028310 524 498 718 373 721
ENSG00000028528 3551 2885 4343 2343 4526
ENSG00000028839 490 440 496 284 682
ENSG00000029153 311 218 422 245 450
ENSG00000029363 3179 2186 4444 2097 4022
ENSG00000029364 1468 1022 1628 933 1712
ENSG00000029534 69 30 156 114 127
ENSG00000029559 0 0 5 0 0
ENSG00000029639 129 100 128 83 105
ENSG00000029725 1589 1435 2424 1297 2494
ENSG00000029993 495 318 928 398 898
ENSG00000030066 978 850 1050 610 1006
ENSG00000030110 183 186 193 122 204
ENSG00000030304 35 54 41 14 19
ENSG00000030419 227 139 239 158 201
ENSG00000030582 10961 7853 13343 6964 13342
ENSG00000031003 1339 1017 1665 903 1486
ENSG00000031081 464 216 463 125 496
ENSG00000031691 58 56 83 53 114
ENSG00000031698 3373 2391 4016 1609 3679
ENSG00000031823 1565 1070 2421 781 2243
ENSG00000032219 701 523 782 409 800
ENSG00000032389 238 207 314 113 214
ENSG00000032444 1317 1167 1821 1105 1788
ENSG00000032742 287 148 344 122 334
ENSG00000033011 521 473 622 335 625
ENSG00000033030 395 425 464 272 454
ENSG00000033050 1036 1349 1257 922 1615
ENSG00000033100 2425 1977 2652 1648 2793
ENSG00000033122 9 3 9 1 10
ENSG00000033170 1457 1386 2170 991 2306
ENSG00000033178 979 515 1532 435 1241
ENSG00000033327 1158 1135 1271 1112 1349
ENSG00000033627 758 1319 961 857 915
ENSG00000033800 1118 900 1206 842 1074
ENSG00000033867 2526 1161 3813 1080 3589
ENSG00000034053 46 53 21 4 48
ENSG00000034152 886 1178 924 896 671
ENSG00000034239 14 9 17 4 10
ENSG00000034510 11323 10854 17313 8263 18277
ENSG00000034533 142 98 152 64 186
ENSG00000034677 837 833 1106 657 792
ENSG00000034693 345 210 420 192 614
ENSG00000034713 1332 1382 1392 1186 1389
ENSG00000034971 339 260 1033 645 99
ENSG00000035115 650 352 583 252 603
ENSG00000035141 364 374 382 261 378
ENSG00000035403 11492 16965 17242 16658 9835
ENSG00000035499 21 15 19 2 16
ENSG00000035664 168 517 631 966 180
ENSG00000035681 1162 868 1307 569 1407
ENSG00000035687 866 750 1039 673 1180
ENSG00000035720 0 0 0 0 0
ENSG00000035862 51281 43711 66689 48258 67911
ENSG00000035928 1042 988 1126 736 1216
ENSG00000036054 1278 1230 1180 854 1433
ENSG00000036257 1672 1488 2047 1331 2419
ENSG00000036448 7 13 6 1 6
ENSG00000036473 0 0 0 0 1
ENSG00000036530 43 24 45 23 32
ENSG00000036549 1008 800 951 543 903
ENSG00000036565 3 5 1 3 5
ENSG00000036672 20 8 11 13 23
ENSG00000036828 0 0 0 0 0
ENSG00000037042 440 347 568 309 519
ENSG00000037241 296 310 297 218 367
ENSG00000037280 9 17 15 2 15
ENSG00000037474 766 742 806 480 1098
ENSG00000037637 632 624 701 477 635
ENSG00000037749 386 373 540 370 479
ENSG00000037757 397 372 434 255 442
ENSG00000037897 71 52 116 42 125
ENSG00000037965 6 7 8 4 23
ENSG00000038002 792 462 806 343 561
ENSG00000038210 410 437 390 274 319
ENSG00000038219 2660 1743 2596 1621 2859
ENSG00000038274 1642 1880 1446 1509 1562
ENSG00000038295 13 7 35 7 5
ENSG00000038358 1004 878 1338 715 1245
ENSG00000038382 6242 5674 7550 3781 6347
ENSG00000038427 9784 7411 12674 4848 11144
ENSG00000038532 743 486 796 424 953
ENSG00000038945 112 46 112 23 328
ENSG00000039068 12 1 8 7 13
ENSG00000039123 1387 1084 1806 785 1781
ENSG00000039139 177 237 306 65 274
ENSG00000039319 1050 848 1441 651 1453
ENSG00000039523 1783 2025 1795 1130 1936
ENSG00000039537 2 22 13 27 16
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ENSG00000167476 11 4 3
ENSG00000167483 61 35 37
ENSG00000167487 142 136 128
ENSG00000167491 1644 1024 1117
ENSG00000167494 0 0 0
ENSG00000167508 473 257 294
ENSG00000167513 20 25 23
ENSG00000167515 962 551 431
ENSG00000167522 7129 3647 4039
ENSG00000167523 100 62 75
ENSG00000167524 67 37 41
ENSG00000167525 47 29 39
ENSG00000167526 22292 16402 17336
ENSG00000167528 396 278 276
ENSG00000167531 0 0 0
ENSG00000167535 1630 1194 1292
ENSG00000167536 129 74 65
ENSG00000167543 720 415 783
ENSG00000167548 3782 1644 2202
ENSG00000167549 2125 588 3925
ENSG00000167550 32 11 23
ENSG00000167552 8558 7824 3240
ENSG00000167553 3520 1815 1017
ENSG00000167554 129 98 82
ENSG00000167555 1062 616 638
ENSG00000167562 231 147 100
ENSG00000167565 378 216 236
ENSG00000167566 1041 662 608
ENSG00000167578 363 207 282
ENSG00000167580 0 0 0
ENSG00000167588 7 8 3
ENSG00000167595 209 179 218
ENSG00000167600 40 76 57
ENSG00000167601 6683 4193 5035
ENSG00000167604 53 31 40
ENSG00000167608 4 0 0
ENSG00000167612 7 2 0
ENSG00000167613 0 5 0
ENSG00000167614 1 0 3
ENSG00000167615 3111 1550 2049
ENSG00000167617 73 36 44
ENSG00000167618 0 0 0
ENSG00000167619 5 12 3
ENSG00000167625 434 184 233
ENSG00000167632 610 388 472
ENSG00000167633 0 0 0
ENSG00000167634 0 0 0
ENSG00000167635 1719 1060 852
ENSG00000167637 171 108 87
ENSG00000167641 923 30 232
ENSG00000167642 309 71 180
ENSG00000167644 2 0 0
ENSG00000167645 1508 315 722
ENSG00000167646 5 7 4
ENSG00000167653 47 4 3
ENSG00000167654 12 11 9
ENSG00000167656 0 0 0
ENSG00000167657 2013 1159 1418
ENSG00000167658 71629 54599 60714
ENSG00000167664 3 1 0
ENSG00000167670 177 130 122
ENSG00000167671 4129 2247 2589
ENSG00000167674 1734 1159 1214
ENSG00000167676 84 86 53
ENSG00000167680 30 48 6
ENSG00000167685 556 432 323
ENSG00000167693 3216 1025 987
ENSG00000167695 883 506 423
ENSG00000167699 721 606 630
ENSG00000167700 224 116 118
ENSG00000167701 52 23 22
ENSG00000167702 519 226 490
ENSG00000167703 129 55 82
ENSG00000167705 139 55 75
ENSG00000167711 5 6 15
ENSG00000167716 1172 876 942
ENSG00000167720 421 321 314
ENSG00000167721 2460 1089 1036
ENSG00000167723 64 51 40
ENSG00000167733 233 162 164
ENSG00000167740 732 553 580
ENSG00000167741 6 2 2
ENSG00000167747 241 260 192
ENSG00000167748 0 0 0
ENSG00000167749 3 2 23
ENSG00000167751 1 1 2
ENSG00000167754 0 0 0
ENSG00000167755 0 0 0
ENSG00000167757 4 7 5
ENSG00000167759 0 1 0
ENSG00000167766 1218 952 684
ENSG00000167767 47 8 3
ENSG00000167768 0 0 0
ENSG00000167769 1 0 2
ENSG00000167770 1536 847 1009
ENSG00000167771 39 67 20
ENSG00000167772 1435 1440 1639
ENSG00000167774 18 0 0
ENSG00000167775 409 274 248
ENSG00000167778 1217 639 650
ENSG00000167779 2846 4859 6703
ENSG00000167780 0 3 8
ENSG00000167785 560 358 424
ENSG00000167791 0 0 0
ENSG00000167792 1827 1167 1293
ENSG00000167797 657 165 363
ENSG00000167798 0 0 0
ENSG00000167799 21 19 25
ENSG00000167800 0 1 0
ENSG00000167807 26 14 4
ENSG00000167815 1679 1105 926
ENSG00000167822 0 0 0
ENSG00000167825 0 0 0
ENSG00000167840 105 78 87
ENSG00000167842 292 257 234
ENSG00000167850 0 0 0
ENSG00000167851 0 0 0
ENSG00000167858 0 0 0
ENSG00000167861 30 5 15
ENSG00000167862 340 142 200
ENSG00000167863 2510 1153 1355
ENSG00000167874 7 6 1
ENSG00000167880 6 0 9
ENSG00000167881 1987 1195 1042
ENSG00000167889 4 5 5
ENSG00000167895 1 1 0
ENSG00000167900 85 73 54
ENSG00000167904 249 267 172
ENSG00000167910 0 2 1
ENSG00000167914 4 3 4
ENSG00000167916 0 0 0
ENSG00000167920 167 168 208
ENSG00000167925 434 286 400
ENSG00000167930 3250 2025 2554
ENSG00000167941 2 0 0
ENSG00000167945 0 2 5
ENSG00000167962 1165 726 798
ENSG00000167964 14 15 8
ENSG00000167965 870 524 489
ENSG00000167967 577 312 381
ENSG00000167968 8 6 9
ENSG00000167969 600 370 490
ENSG00000167971 4 4 17
ENSG00000167972 77 141 226
ENSG00000167977 485 261 326
ENSG00000167978 16781 9008 10817
ENSG00000167981 119 66 60
ENSG00000167984 36 22 22
ENSG00000167985 347 179 250
ENSG00000167986 11148 7417 8312
ENSG00000167987 477 304 281
ENSG00000167992 187 393 111
ENSG00000167994 1029 747 907
ENSG00000167995 381 555 324
ENSG00000167996 211679 120693 129225
ENSG00000168000 244 237 211
ENSG00000168002 786 570 592
ENSG00000168003 1468 1570 1526
ENSG00000168004 1 13 36
ENSG00000168005 571 327 346
ENSG00000168010 342 277 140
ENSG00000168014 923 489 431
ENSG00000168016 2796 1721 1907
ENSG00000168026 81 106 125
ENSG00000168028 18339 13265 12845
ENSG00000168032 83 97 94
ENSG00000168036 7479 4748 5081
ENSG00000168038 82 47 78
ENSG00000168040 553 357 341
ENSG00000168056 7568 5464 5838
ENSG00000168060 198 176 167
ENSG00000168061 70 91 79
ENSG00000168062 43 27 14
ENSG00000168065 2 2 3
ENSG00000168066 4511 2303 2490
ENSG00000168067 314 208 269
ENSG00000168070 0 0 1
ENSG00000168071 69 31 26
ENSG00000168077 2367 2154 1730
ENSG00000168078 14 30 19
ENSG00000168079 64 302 934
ENSG00000168081 0 0 0
ENSG00000168090 1925 1144 1042
ENSG00000168092 2568 1163 1592
ENSG00000168096 480 272 295
ENSG00000168101 368 166 238
ENSG00000168116 272 128 185
ENSG00000168118 969 676 976
ENSG00000168122 0 0 0
ENSG00000168124 2 0 0
ENSG00000168126 1 0 0
ENSG00000168129 0 0 0
ENSG00000168131 0 0 0
ENSG00000168135 0 0 1
ENSG00000168137 3295 2445 1583
ENSG00000168140 1878 2282 2256
ENSG00000168143 0 0 0
ENSG00000168148 2 0 3
ENSG00000168152 86 106 95
ENSG00000168158 2 5 1
ENSG00000168159 2418 1659 1457
ENSG00000168172 2764 1508 1683
ENSG00000168175 2257 910 1251
ENSG00000168209 1808 1322 2116
ENSG00000168214 5855 7176 4385
ENSG00000168216 1390 868 910
ENSG00000168228 252 157 175
ENSG00000168229 1 0 1
ENSG00000168234 997 420 731
ENSG00000168237 57 38 41
ENSG00000168243 3 5 1
ENSG00000168246 882 715 675
ENSG00000168255 1046 798 859
ENSG00000168256 850 495 648
ENSG00000168259 1542 1074 1125
ENSG00000168263 1 0 1
ENSG00000168264 1906 1810 1382
ENSG00000168267 0 0 0
ENSG00000168268 1768 977 1146
ENSG00000168269 1 0 0
ENSG00000168273 99 93 65
ENSG00000168275 222 93 131
ENSG00000168280 5 10 3
ENSG00000168282 1275 940 886
ENSG00000168283 1814 1148 1253
ENSG00000168286 594 388 466
ENSG00000168288 2150 1203 1361
ENSG00000168291 1457 699 873
ENSG00000168297 2176 1552 1475
ENSG00000168298 1 2 0
ENSG00000168300 2017 1814 1679
ENSG00000168301 180 115 142
ENSG00000168303 574 214 416
ENSG00000168306 438 456 356
ENSG00000168309 73 11 201
ENSG00000168310 952 568 785
ENSG00000168314 14 11 5
ENSG00000168329 0 4 0
ENSG00000168333 0 0 0
ENSG00000168334 0 6 0
ENSG00000168348 0 0 0
ENSG00000168350 3 27 28
ENSG00000168356 9 3 8
ENSG00000168374 8835 4546 4936
ENSG00000168379 0 0 1
ENSG00000168384 10 7 5
ENSG00000168385 20448 12644 12290
ENSG00000168386 2544 1635 1294
ENSG00000168389 30 7 6
ENSG00000168393 290 218 201
ENSG00000168394 150 74 22
ENSG00000168395 410 336 332
ENSG00000168397 1270 678 723
ENSG00000168398 4949 7748 1445
ENSG00000168404 113 81 94
ENSG00000168405 163 109 209
ENSG00000168411 501 321 372
ENSG00000168412 0 0 0
ENSG00000168418 4 4 2
ENSG00000168421 15 10 15
ENSG00000168427 55 10 14
ENSG00000168434 445 333 311
ENSG00000168438 837 542 517
ENSG00000168439 2765 1402 1349
ENSG00000168447 0 0 0
ENSG00000168452 144 34 31
ENSG00000168453 1180 696 513
ENSG00000168454 5 0 0
ENSG00000168461 7061 2234 5296
ENSG00000168468 51 43 49
ENSG00000168476 290 178 230
ENSG00000168477 2181 3544 3148
ENSG00000168481 22 1 57
ENSG00000168484 0 0 1
ENSG00000168487 3337 2330 2428
ENSG00000168488 2234 1632 1622
ENSG00000168490 50 65 29
ENSG00000168491 6 11 5
ENSG00000168495 946 470 531
ENSG00000168496 180 95 105
ENSG00000168497 1185 487 842
ENSG00000168502 3420 2374 1627
ENSG00000168505 1 1 0
ENSG00000168509 1 0 0
ENSG00000168515 0 0 0
ENSG00000168517 115 103 96
ENSG00000168522 1708 1178 1317
ENSG00000168528 3089 1135 1329
ENSG00000168530 0 0 0
ENSG00000168538 1939 987 1616
ENSG00000168539 0 0 0
ENSG00000168542 64519 108876 57372
ENSG00000168546 0 3 0
ENSG00000168556 514 239 608
ENSG00000168564 510 333 446
ENSG00000168566 380 264 357
ENSG00000168569 258 153 150
ENSG00000168575 2341 1020 1121
ENSG00000168582 0 0 0
ENSG00000168589 4 12 8
ENSG00000168591 1192 749 971
ENSG00000168594 5 6 1
ENSG00000168610 13347 5601 9791
ENSG00000168612 294 216 220
ENSG00000168615 22075 15333 16521
ENSG00000168619 0 0 0
ENSG00000168621 454 91 564
ENSG00000168630 0 0 0
ENSG00000168631 0 0 0
ENSG00000168634 0 0 0
ENSG00000168646 654 321 780
ENSG00000168653 2039 1008 956
ENSG00000168658 2 2 1
ENSG00000168661 54 66 17
ENSG00000168671 0 0 0
ENSG00000168672 11 15 9
ENSG00000168675 5 6 2
ENSG00000168676 6 10 3
ENSG00000168679 292 449 266
ENSG00000168685 193 47 36
ENSG00000168701 476 221 279
ENSG00000168702 7 1 10
ENSG00000168703 1 2 1
ENSG00000168710 4553 2130 2906
ENSG00000168724 2271 1173 1592
ENSG00000168734 2232 1915 1959
ENSG00000168743 9 30 14
ENSG00000168746 0 0 0
ENSG00000168748 0 2 0
ENSG00000168754 3 0 4
ENSG00000168757 0 0 0
ENSG00000168758 879 558 1037
ENSG00000168763 525 460 455
ENSG00000168765 391 316 317
ENSG00000168769 523 357 304
ENSG00000168772 0 0 0
ENSG00000168778 395 336 346
ENSG00000168779 480 133 132
ENSG00000168781 334 512 196
ENSG00000168785 4831 3085 3132
ENSG00000168787 0 0 0
ENSG00000168792 582 340 562
ENSG00000168795 296 202 212
ENSG00000168802 2604 1647 1397
ENSG00000168803 252 201 134
ENSG00000168806 244 135 83
ENSG00000168807 3229 2161 2307
ENSG00000168811 37 30 4
ENSG00000168813 701 465 410
ENSG00000168818 702 402 443
ENSG00000168824 32 31 28
ENSG00000168826 72 61 55
ENSG00000168827 923 665 653
ENSG00000168828 1 0 2
ENSG00000168830 0 0 0
ENSG00000168843 1 186 48
ENSG00000168852 7 3 2
ENSG00000168872 1510 846 749
ENSG00000168874 629 510 929
ENSG00000168875 0 0 0
ENSG00000168876 283 212 233
ENSG00000168878 6 15 20
ENSG00000168883 1314 823 1194
ENSG00000168884 1133 445 729
ENSG00000168887 512 372 353
ENSG00000168890 810 313 701
ENSG00000168894 1064 688 659
ENSG00000168899 1092 898 1094
ENSG00000168903 0 0 0
ENSG00000168904 313 263 264
ENSG00000168906 5401 4077 4701
ENSG00000168907 3 2 4
ENSG00000168913 1 1 4
ENSG00000168916 817 663 717
ENSG00000168917 403 161 325
ENSG00000168918 16 9 7
ENSG00000168924 1114 528 630
ENSG00000168925 0 0 0
ENSG00000168928 3 3 2
ENSG00000168930 0 0 0
ENSG00000168936 1876 1192 1339
ENSG00000168938 1155 1208 972
ENSG00000168939 83 61 60
ENSG00000168944 723 522 467
ENSG00000168952 78 175 322
ENSG00000168955 263 5 13
ENSG00000168958 3554 1569 1848
ENSG00000168959 2 0 3
ENSG00000168961 6 31 1
ENSG00000168967 2 1 1
ENSG00000168970 226 192 123
ENSG00000168992 4 5 1
ENSG00000168993 8 2 0
ENSG00000168994 2392 682 2693
ENSG00000168995 0 0 0
ENSG00000169006 0 0 0
ENSG00000169016 376 182 214
ENSG00000169018 2162 1372 1206
ENSG00000169019 597 285 346
ENSG00000169020 1163 520 577
ENSG00000169021 1105 649 716
ENSG00000169026 104 57 86
ENSG00000169031 56 22 114
ENSG00000169032 2520 1131 1460
ENSG00000169035 2 1 1
ENSG00000169040 0 0 0
ENSG00000169045 5421 3082 3619
ENSG00000169047 7929 1964 4057
ENSG00000169057 2460 1667 2046
ENSG00000169059 0 0 0
ENSG00000169062 1482 1103 941
ENSG00000169064 0 0 0
ENSG00000169067 1 0 0
ENSG00000169071 2182 1151 1264
ENSG00000169075 2 1 0
ENSG00000169083 2691 1184 1376
ENSG00000169084 666 452 474
ENSG00000169085 68 21 89
ENSG00000169087 165 115 130
ENSG00000169093 1200 596 910
ENSG00000169100 10070 5675 5323
ENSG00000169105 1016 745 1009
ENSG00000169116 40 19 15
ENSG00000169118 807 419 663
ENSG00000169122 549 508 436
ENSG00000169126 8 6 4
ENSG00000169129 7 15 4
ENSG00000169131 267 223 196
ENSG00000169136 606 3089 1017
ENSG00000169139 1269 630 681
ENSG00000169154 0 0 0
ENSG00000169155 526 285 280
ENSG00000169164 0 0 0
ENSG00000169169 734 591 565
ENSG00000169174 1 2 4
[ reached 'max' / getOption("max.print") -- omitted 26195 rows ]
head(counts)
SRR1039508 SRR1039509 SRR1039512 SRR1039513 SRR1039516
ENSG00000000003 723 486 904 445 1170
ENSG00000000005 0 0 0 0 0
ENSG00000000419 467 523 616 371 582
ENSG00000000457 347 258 364 237 318
ENSG00000000460 96 81 73 66 118
ENSG00000000938 0 0 1 0 2
SRR1039517 SRR1039520 SRR1039521
ENSG00000000003 1097 806 604
ENSG00000000005 0 0 0
ENSG00000000419 781 417 509
ENSG00000000457 447 330 324
ENSG00000000460 94 102 74
ENSG00000000938 0 0 0
##Check on metadata counts correspondance
We need to check that the metadata matches the samples in our count data.
ncol(counts) == nrow(metadata)
[1] TRUE
colnames(counts) == metadata$id
[1] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Q1. How many genes are in this dataset?
nrow(counts)
[1] 38694
38694 genes in this dataset
Q2. How many “control” samples are in this dataset?
sum(metadata$dex == "control")
[1] 4
4 control samples in this dataset
Analysis Plan
We have 4 replicates per condition (“control” and “treated”) (drug and no drug respectively). We want to compare the control vs the treated to see which gene’s expression levels change when we have the drug present.
We are going to row by row (gene by gene) and see if the average value in control columns is different than the treated columns
Step 1: Find which columns in counts correspond to the “control”
Step 2: Filter / Select / Extract these columns
Step 3: Calculate an average value for each gene.
# The indices (i.e. positions) that are "control"
control.inds <- metadata$dex == "control"
# Extract/select these "control" columns from counts
control.counts <- counts[, control.inds]
# Calculate the mean for each gene (i.e. row)
control.mean <- rowMeans(control.counts)
Q. Do the same for “treated” samples - find the mean count value per gene.
treated.counts <- rowMeans(counts[, metadata$dex == "treated"])
Let’s put these two mean values into a new data.frame meancounts for
easy book-keeping and plotting.
treated.mean <- rowMeans(counts[, metadata$dex == "treated"])
meancounts <- data.frame(control.mean,
treated.mean)
head(meancounts)
control.mean treated.mean
ENSG00000000003 900.75 658.00
ENSG00000000005 0.00 0.00
ENSG00000000419 520.50 546.00
ENSG00000000457 339.75 316.50
ENSG00000000460 97.25 78.75
ENSG00000000938 0.75 0.00
Q. Make a ggplot of average counts of control vs treated
library(ggplot2)
ggplot(meancounts) +
aes(control.mean,
treated.mean) +
geom_point(alpha=0.4)
Adding logarithmic axises
library(ggplot2)
ggplot(meancounts) +
aes(control.mean,
treated.mean) +
geom_point(alpha=0.4) +
scale_x_log10() + scale_y_log10()
Warning in scale_x_log10(): log-10 transformation introduced infinite values.
Warning in scale_y_log10(): log-10 transformation introduced infinite values.
Log2 units and fold change
If we consider “treated”/“control” counts we will get a number that tells us the change.
# No change
log2(20/20)
[1] 0
# A doubling in the treated vs control
log2(40/20)
[1] 1
# A halving in the treated vs control
log2(10/20)
[1] -1
Q. Add a new column called
log2fcfor log2 fold change of treated/ control to ourmeancountsobject.
meancounts$log2fc <-
log2(meancounts$treated.mean/
meancounts$control.mean)
head(meancounts)
control.mean treated.mean log2fc
ENSG00000000003 900.75 658.00 -0.45303916
ENSG00000000005 0.00 0.00 NaN
ENSG00000000419 520.50 546.00 0.06900279
ENSG00000000457 339.75 316.50 -0.10226805
ENSG00000000460 97.25 78.75 -0.30441833
ENSG00000000938 0.75 0.00 -Inf
Remove zero count genes
Typically we would not consider zero count genes - as we have no data about them and they should be excluded from further consideration. These lead to “funky” log2 fold change values (e.g. divide by zero errors etc.)
DESeq analysis
We are missing any measure of significance from the work we have so far. Let’s do this properly with the DESeq2 package.
library(DESeq2)
The DESeq2 package, like many bioconductor packages, wants it’s input in a very specific way - a data structure setup with all the info it needs for the calculations.
dds <- DESeqDataSetFromMatrix(countData = counts,
colData = metadata,
design = ~dex)
converting counts to integer mode
Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
design formula are characters, converting to factors
The main function in this package is called DESeq() it will run the
full analysis for us on our dds input object:
dds <- DESeq(dds)
estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting model and testing
Extract our results:
res <- results(dds)
head(res)
log2 fold change (MLE): dex treated vs control
Wald test p-value: dex treated vs control
DataFrame with 6 rows and 6 columns
baseMean log2FoldChange lfcSE stat pvalue
<numeric> <numeric> <numeric> <numeric> <numeric>
ENSG00000000003 747.194195 -0.350703 0.168242 -2.084514 0.0371134
ENSG00000000005 0.000000 NA NA NA NA
ENSG00000000419 520.134160 0.206107 0.101042 2.039828 0.0413675
ENSG00000000457 322.664844 0.024527 0.145134 0.168996 0.8658000
ENSG00000000460 87.682625 -0.147143 0.256995 -0.572550 0.5669497
ENSG00000000938 0.319167 -1.732289 3.493601 -0.495846 0.6200029
padj
<numeric>
ENSG00000000003 0.163017
ENSG00000000005 NA
ENSG00000000419 0.175937
ENSG00000000457 0.961682
ENSG00000000460 0.815805
ENSG00000000938 NA
Volcano Plot
A useful summary figure of our results is often called a volcano plot. It’s basically a plot of log2 fold change values vs adjusted p-values.
Q. Use ggplot to make a first version “volcano plot” of
log2FoldChangevspadj
ggplot(res, aes(log2FoldChange, padj)) + geom_point()
Warning: Removed 23549 rows containing missing values or values outside the scale range
(`geom_point()`).
This is niot very useful because the y-axis (P-value) is not really helpful - we want to focus on low P-values.
ggplot(res) +
aes(x = log2FoldChange,
y = log10(padj)) +
geom_point()
Warning: Removed 23549 rows containing missing values or values outside the scale range
(`geom_point()`).
ggplot(res) +
aes(x = log2FoldChange,
y = -log10(padj)) +
geom_point()
Warning: Removed 23549 rows containing missing values or values outside the scale range
(`geom_point()`).
ggplot(res) +
aes(x = log2FoldChange,
y = -log10(padj)) +
geom_point() +
geom_vline(xintercept = c(-2, 2), col="red") +
geom_hline(yintercept = -log(0.05), col = "red")
Warning: Removed 23549 rows containing missing values or values outside the scale range
(`geom_point()`).
Add some plot annotation
Q. Add color to the points (genes) we care about, nice axis labels, a useful title and a nice theme.
ggplot(res) +
aes(x = log2FoldChange,
y = -log10(padj)) +
geom_point() +
geom_vline(xintercept = c(-2, 2), col="red") +
geom_hline(yintercept = -log(0.05), col = "red")
Warning: Removed 23549 rows containing missing values or values outside the scale range
(`geom_point()`).
mycols <- rep("gray", nrow(res))
mycols[res$log2FoldChange > 2] <- "blue"
mycols[res$log2FoldChange < -2 ] <- "darkgreen"
mycols[res$padj >= 0.05] <- "gray"
ggplot(res, aes(x = log2FoldChange,
y = -log10(padj))) +
geom_point(color = mycols, alpha = 0.5) +
geom_vline(xintercept = c(-2, 2), col = "red") +
geom_hline(yintercept = -log10(0.05), col = "red") +
labs(title = "Volcano Plot",
x = "Log2 Fold Change",
y = "-Log10 Adjusted P-value") +
theme_minimal()
Warning: Removed 23549 rows containing missing values or values outside the scale range
(`geom_point()`).
Save our results to a CSV file
write.csv(res, file="results.csv")