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EXON 49 0 0 5 54 109 0 0 0 109 5 54 0 109 1_505660 +1 505926 505926 6.5 6.5%_46r 1 505926 UMAG_00195 EXON 43 0 0 3 46 81 0 0 0 81 3 46 0 81 1_505926 +1 2300112 2300112 11.6 11.6%_43r 1 2300112 UMAG_00768 EXON 38 0 0 5 43 66 0 0 0 66 5 43 0 66 1_2300112 +1 2356577 2356577 7.8 7.8%_64r 1 2356577 UMAG_00789 EXON 0 59 5 0 64 0 139 0 0 139 5 64 0 139 1_2356577 +1 1374368 1374368 5.0 5.0%_20r 1 1374368 UMAG_10121 EXON 0 19 1 0 20 0 45 0 0 45 1 20 0 45 1_1374368 +1 2022497 2022497 6.1 6.1%_33r 1 2022497 UMAG_00689 EXON 0 31 2 0 33 0 88 0 0 88 2 33 0 88 1_2022497 +1 1588739 1588739 5.4 5.4%_74r 1 1588739 UMAG_00545 EXON 70 0 0 4 74 492 0 0 0 492 4 74 0 492 1_1588739 +1 1567066 1567066 5.3 5.3%_38r 1 1567066 UMAG_00539 EXON 0 36 2 0 38 0 82 0 0 82 2 38 0 82 1_1567066 +1 1567522 1567522 5.6 5.6%_36r 1 1567522 UMAG_00539 EXON 0 34 2 0 36 0 97 0 0 97 2 36 0 97 1_1567522 +1 289113 289113 8.3 8.3%_84r 1 289113 UMAG_00115 EXON 77 0 0 7 84 443 0 0 0 443 7 84 0 443 1_289113 +1 284560 284560 6.9 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EXON 47 0 0 3 50 85 0 0 0 85 3 50 0 85 3_258195 +3 260164 260164 7.8 7.8%_51r 3 260164 UMAG_01518 EXON 47 0 0 4 51 136 0 0 0 136 4 51 0 136 3_260164 +3 200649 200649 5.0 5.0%_20r 3 200649 UMAG_01503 EXON 0 19 1 0 20 0 58 0 0 58 1 20 0 58 3_200649 +3 202310 202310 5.1 5.1%_39r 3 202310 UMAG_01503 EXON 0 37 2 0 39 0 53 0 0 53 2 39 0 53 3_202310 +3 144986 144986 5.3 5.3%_38r 3 144986 UMAG_11670 EXON 36 0 0 2 38 105 0 0 0 105 2 38 0 105 3_144986 +3 1328755 1328755 5.0 5.0%_60r 3 1328755 UMAG_12177 EXON 57 0 0 3 60 315 0 0 0 315 3 60 0 315 3_1328755 +3 1329293 1329293 13.3 13.3%_75r 3 1329293 UMAG_12177 EXON 65 0 0 10 75 467 0 0 0 467 10 75 0 467 3_1329293 +3 1251105 1251105 38.9 38.9%_36r 3 1251105 UMAG_01888 EXON 0 22 14 0 36 0 83 0 0 83 14 36 0 83 3_1251105 +3 1251126 1251126 6.8 6.8%_44r 3 1251126 UMAG_01888 EXON 0 41 3 0 44 0 113 0 0 113 3 44 0 113 3_1251126 +3 1426337 1426337 11.1 11.1%_36r 3 1426337 UMAG_11373 EXON 32 0 0 4 36 66 0 0 0 66 4 36 0 66 3_1426337 +3 534590 534590 9.5 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3 24 0 36 3_882553 +3 1230031 1230031 5.4 5.4%_37r 3 1230031 UMAG_01877 EXON 0 35 2 0 37 0 55 0 0 55 2 37 0 55 3_1230031 +3 1230647 1230647 7.1 7.1%_28r 3 1230647 UMAG_01877 EXON 0 25 2 1 28 0 74 0 0 74 2 28 0 74 3_1230647 +3 1231291 1231291 8.3 8.3%_24r 3 1231291 UMAG_01877 EXON 0 22 2 0 24 0 37 0 0 37 2 24 0 37 3_1231291 +3 502103 502103 6.5 6.5%_46r 3 502103 UMAG_11693 EXON 0 43 3 0 46 0 65 0 0 65 3 46 0 65 3_502103 +3 65148 65148 24.7 24.7%_85r 3 65148 UMAG_01463 EXON 63 0 1 21 85 802 0 0 1 803 21 85 1 803 3_65148 +3 1560197 1560197 5.4 5.4%_56r 3 1560197 UMAG_02010 EXON 53 0 0 3 56 141 0 0 0 141 3 56 0 141 3_1560197 +3 1561274 1561274 16.4 16.4%_55r 3 1561274 UMAG_02010 EXON 46 0 0 9 55 125 0 0 0 125 9 55 0 125 3_1561274 +3 1049471 1049471 6.8 6.8%_44r 3 1049471 UMAG_01803 EXON 41 0 0 3 44 98 0 0 0 98 3 44 0 98 3_1049471 +3 431772 431772 18.2 18.2%_22r 3 431772 UMAG_01574 EXON 18 0 0 4 22 20 0 0 0 20 4 22 0 20 3_431772 +3 838438 838438 7.3 7.3%_55r 3 838438 UMAG_11363 EXON 0 51 4 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0 53 0 135 0 0 135 3 53 0 135 1_2389244 +1 174243 174243 6.1 6.1%_33r 1 174243 UMAG_00068 EXON 0 31 2 0 33 0 56 0 0 56 2 33 0 56 1_174243 +1 176347 176347 11.1 11.1%_27r 1 176347 UMAG_00068 EXON 0 24 3 0 27 0 25 0 0 25 3 27 0 25 1_176347 +1 892339 892339 32.3 32.3%_31r 1 892339 UMAG_10098 EXON 21 0 0 10 31 56 0 0 0 56 10 31 0 56 1_892339 +1 893049 893049 7.3 7.3%_55r 1 893049 UMAG_10098 EXON 51 0 0 4 55 63 0 0 0 63 4 55 0 63 1_893049 +1 1610640 1610640 5.0 5.0%_20r 1 1610640 UMAG_10461 EXON 19 0 0 1 20 42 0 0 0 42 1 20 0 42 1_1610640 +1 1611350 1611350 19.0 19.0%_21r 1 1611350 UMAG_10461 EXON 17 0 0 4 21 35 0 0 0 35 4 21 0 35 1_1611350 +1 1611710 1611710 6.9 6.9%_29r 1 1611710 UMAG_10461 EXON 27 0 0 2 29 30 0 0 0 30 2 29 0 30 1_1611710 +1 1613946 1613946 6.6 6.6%_61r 1 1613946 UMAG_10461 EXON 57 0 0 4 61 121 0 0 0 121 4 61 0 121 1_1613946 +1 1005943 1005943 13.1 13.1%_61r 1 1005943 UMAG_00362 EXON 0 53 8 0 61 0 97 0 0 97 8 61 0 97 1_1005943 +1 1298334 1298334 60.7 60.7%_28r 1 1298334 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5.9%_34r 1 2300112 UMAG_00768 EXON 32 0 0 2 34 52 0 0 0 52 2 34 0 52 1_2300112 +1 1134921 1134921 7.9 7.9%_101r 1 1134921 UMAG_11821 EXON 93 0 0 8 101 1267 0 2 0 1269 8 101 0 1269 1_1134921 +1 1607761 1607761 6.1 6.1%_33r 1 1607761 UMAG_00551 EXON 31 0 0 2 33 59 0 0 0 59 2 33 0 59 1_1607761 +1 1422233 1422233 16.1 16.1%_31r 1 1422233 UMAG_00492 EXON 26 0 0 5 31 51 0 0 0 51 5 31 0 51 1_1422233 +1 622079 622079 8.6 8.6%_35r 1 622079 UMAG_11428 EXON 32 0 0 3 35 62 0 0 0 62 3 35 0 62 1_622079 +1 1180900 1180900 6.2 6.2%_32r 1 1180900 UMAG_10105 EXON 0 30 2 0 32 0 47 0 0 47 2 32 0 47 1_1180900 +1 24322 24322 15.6 15.6%_45r 1 24322 UMAG_00006 EXON 0 38 7 0 45 0 70 0 0 70 7 45 0 70 1_24322 +1 652466 652466 17.6 17.6%_34r 1 652466 UMAG_10956 EXON 28 0 0 6 34 48 0 0 0 48 6 34 0 48 1_652466 +1 1067134 1067134 8.8 8.8%_102r 1 1067134 UMAG_00381 EXON 93 0 0 9 102 706 0 0 1 707 9 102 1 707 1_1067134 +1 1358087 1358087 19.5 19.5%_41r 1 1358087 UMAG_10120 EXON 0 33 8 0 41 0 121 0 0 121 8 41 0 121 1_1358087 +1 2003157 2003157 10.3 10.3%_39r 1 2003157 UMAG_00679 EXON 35 0 0 4 39 54 0 0 0 54 4 39 0 54 1_2003157 +1 1747372 1747372 7.1 7.1%_28r 1 1747372 UMAG_00596 EXON 26 0 0 2 28 22 0 0 0 22 2 28 0 22 1_1747372 +1 1588739 1588739 5.0 5.0%_80r 1 1588739 UMAG_00545 EXON 76 0 0 4 80 444 0 0 2 446 4 80 2 446 1_1588739 +1 2206541 2206541 22.7 22.7%_22r 1 2206541 UMAG_00742 EXON 17 0 0 5 22 51 0 0 0 51 5 22 0 51 1_2206541 +1 1794603 1794603 9.5 9.5%_63r 1 1794603 UMAG_00612 EXON 0 57 6 0 63 0 114 0 0 114 6 63 0 114 1_1794603 +1 1794884 1794884 5.1 5.1%_39r 1 1794884 UMAG_00612 EXON 0 37 2 0 39 0 72 0 0 72 2 39 0 72 1_1794884 +1 1795474 1795474 11.1 11.1%_27r 1 1795474 UMAG_00612 EXON 0 24 3 0 27 0 45 0 0 45 3 27 0 45 1_1795474 +1 277572 277572 6.0 6.0%_50r 1 277572 UMAG_00111 EXON 0 47 3 0 50 0 205 0 0 205 3 50 0 205 1_277572 +1 1702034 1702034 21.4 21.4%_42r 1 1702034 UMAG_00582 EXON 33 0 0 9 42 131 0 0 0 131 9 42 0 131 1_1702034 +1 1704706 1704706 9.8 9.8%_61r 1 1704706 UMAG_00582 EXON 55 0 0 6 61 109 0 0 0 109 6 61 0 109 1_1704706 +1 1320668 1320668 5.5 5.5%_55r 1 1320668 UMAG_00463 EXON 52 0 0 3 55 50 0 0 0 50 3 55 0 50 1_1320668 +1 1000210 1000210 6.5 6.5%_31r 1 1000210 UMAG_00360 EXON 0 29 2 0 31 0 39 0 0 39 2 31 0 39 1_1000210 +1 287247 287247 14.6 14.6%_41r 1 287247 UMAG_00114 EXON 0 35 6 0 41 0 84 0 0 84 6 41 0 84 1_287247 +1 1448445 1448445 10.0 10.0%_20r 1 1448445 UMAG_00499 EXON 0 18 2 0 20 0 28 0 0 28 2 20 0 28 1_1448445 +1 1417259 1417259 11.9 11.9%_42r 1 1417259 UMAG_00490 EXON 37 0 0 5 42 91 0 0 0 91 5 42 0 91 1_1417259 +1 14700 14700 5.4 5.4%_37r 1 14700 UMAG_00003 EXON 35 0 0 2 37 53 0 0 0 53 2 37 0 53 1_14700 +1 534131 534131 19.0 19.0%_63r 1 534131 UMAG_00204 EXON 51 0 0 12 63 152 0 0 0 152 12 63 0 152 1_534131 +1 2032740 2032740 9.1 9.1%_22r 1 2032740 UMAG_15016 EXON 20 0 0 2 22 25 0 0 0 25 2 22 0 25 1_2032740 +1 412116 412116 11.6 11.6%_43r 1 412116 UMAG_00161 EXON 0 38 5 0 43 0 63 0 0 63 5 43 0 63 1_412116 +1 413557 413557 13.3 13.3%_30r 1 413557 UMAG_00161 EXON 0 26 4 0 30 0 41 0 0 41 4 30 0 41 1_413557 +1 2294098 2294098 8.1 8.1%_37r 1 2294098 UMAG_00766 EXON 34 0 0 3 37 59 0 0 0 59 3 37 0 59 1_2294098 +1 2294231 2294231 5.0 5.0%_20r 1 2294231 UMAG_00766 EXON 19 0 0 1 20 59 0 1 0 60 1 20 0 60 1_2294231 +1 353598 353598 8.7 8.7%_23r 1 353598 UMAG_00140 EXON 0 21 2 0 23 0 47 0 0 47 2 23 0 47 1_353598 +1 1858427 1858427 14.0 14.0%_50r 1 1858427 UMAG_00635 EXON 0 43 7 0 50 0 126 0 0 126 7 50 0 126 1_1858427 +1 1638775 1638775 7.8 7.8%_51r 1 1638775 UMAG_00562 EXON 0 47 4 0 51 0 116 0 0 116 4 51 0 116 1_1638775 +1 549521 549521 15.0 15.0%_20r 1 549521 UMAG_10054 EXON 17 0 0 3 20 25 0 0 0 25 3 20 0 25 1_549521 +1 549587 549587 17.2 17.2%_29r 1 549587 UMAG_10054 EXON 24 0 0 5 29 59 0 0 0 59 5 29 0 59 1_549587 +1 549590 549590 20.0 20.0%_30r 1 549590 UMAG_10054 EXON 24 0 0 6 30 59 0 0 0 59 6 30 0 59 1_549590 +1 1931636 1931636 17.2 17.2%_29r 1 1931636 UMAG_15012 EXON 24 0 0 5 29 46 0 0 0 46 5 29 0 46 1_1931636 +1 773712 773712 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5 46 0 79 1_1828993 +1 365836 365836 10.8 10.8%_37r 1 365836 UMAG_00145 EXON 0 33 4 0 37 0 53 0 0 53 4 37 0 53 1_365836 +1 366769 366769 6.5 6.5%_31r 1 366769 UMAG_00145 EXON 0 29 2 0 31 0 33 0 0 33 2 31 0 33 1_366769 +1 2007558 2007558 5.9 5.9%_34r 1 2007558 UMAG_11618 EXON 0 32 2 0 34 0 43 0 0 43 2 34 0 43 1_2007558 +1 2008440 2008440 8.1 8.1%_37r 1 2008440 UMAG_11618 EXON 0 34 3 0 37 0 39 0 0 39 3 37 0 39 1_2008440 +1 2368222 2368222 6.3 6.3%_63r 1 2368222 UMAG_10788 EXON 59 0 0 4 63 317 0 0 0 317 4 63 0 317 1_2368222 +1 2370235 2370235 10.5 10.5%_86r 1 2370235 UMAG_10788 EXON 77 0 0 9 86 314 0 0 0 314 9 86 0 314 1_2370235 +1 2370305 2370305 11.4 11.4%_105r 1 2370305 UMAG_10788 EXON 93 0 0 12 105 543 0 0 0 543 12 105 0 543 1_2370305 +1 2370872 2370872 5.7 5.7%_105r 1 2370872 UMAG_10788 EXON 99 0 0 6 105 780 0 0 0 780 6 105 0 780 1_2370872 +1 589687 589687 6.1 6.1%_33r 1 589687 UMAG_00225 EXON 0 31 2 0 33 0 43 0 0 43 2 33 0 43 1_589687 +1 589723 589723 6.2 6.2%_32r 1 589723 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13.6%_59r 2 1136831 UMAG_01178 EXON 51 0 0 8 59 100 0 0 0 100 8 59 0 100 2_1136831 +2 663571 663571 8.8 8.8%_34r 2 663571 UMAG_15057 EXON 0 31 3 0 34 0 53 0 0 53 3 34 0 53 2_663571 +2 664140 664140 5.3 5.3%_38r 2 664140 UMAG_15057 EXON 0 36 2 0 38 0 32 0 0 32 2 38 0 32 2_664140 +2 252465 252465 9.2 9.2%_65r 2 252465 UMAG_10613 EXON 0 59 6 0 65 0 279 0 0 279 6 65 0 279 2_252465 +2 1307803 1307803 5.3 5.3%_75r 2 1307803 UMAG_01234 EXON 71 0 0 4 75 353 0 0 0 353 4 75 0 353 2_1307803 +2 1307825 1307825 24.7 24.7%_77r 2 1307825 UMAG_01234 EXON 58 0 0 19 77 356 0 0 0 356 19 77 0 356 2_1307825 +2 224620 224620 16.7 16.7%_24r 2 224620 UMAG_00894 EXON 0 20 4 0 24 0 19 0 0 19 4 24 0 19 2_224620 +2 1701976 1701976 7.0 7.0%_57r 2 1701976 UMAG_01382 EXON 53 0 0 4 57 103 0 0 0 103 4 57 0 103 2_1701976 +2 1064738 1064738 28.3 28.3%_60r 2 1064738 UMAG_11651 EXON 43 0 0 17 60 173 0 0 0 173 17 60 0 173 2_1064738 +2 671961 671961 5.0 5.0%_20r 2 671961 UMAG_01032 EXON 19 0 0 1 20 16 0 0 0 16 1 20 0 16 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0 27 2_778815 +2 1104816 1104816 10.7 10.7%_56r 2 1104816 UMAG_01168 EXON 0 50 6 0 56 0 97 0 2 99 6 56 0 99 2_1104816 +2 1105783 1105783 8.2 8.2%_49r 2 1105783 UMAG_01168 EXON 0 45 4 0 49 0 105 0 0 105 4 49 0 105 2_1105783 +2 1106236 1106236 11.4 11.4%_44r 2 1106236 UMAG_01168 EXON 0 39 5 0 44 0 74 0 0 74 5 44 0 74 2_1106236 +2 370849 370849 5.7 5.7%_105r 2 370849 UMAG_10625 EXON 0 99 6 0 105 0 1830 2 4 1836 6 105 2 1836 2_370849 +2 688169 688169 7.0 7.0%_71r 2 688169 UMAG_11267 EXON 0 66 5 0 71 0 262 0 2 264 5 71 0 264 2_688169 +2 1491534 1491534 6.2 6.2%_32r 2 1491534 UMAG_01295 EXON 30 0 0 2 32 35 0 0 0 35 2 32 0 35 2_1491534 +2 761531 761531 16.1 16.1%_56r 2 761531 UMAG_01064 EXON 0 47 9 0 56 2 127 0 0 129 9 56 0 129 2_761531 +2 178405 178405 6.9 6.9%_29r 2 178405 UMAG_00884 EXON 0 27 2 0 29 0 52 0 0 52 2 29 0 52 2_178405 +2 1275173 1275173 6.1 6.1%_49r 2 1275173 UMAG_01224 EXON 46 0 0 3 49 76 0 0 0 76 3 49 0 76 2_1275173 +2 84975 84975 10.4 10.4%_48r 2 84975 UMAG_00855 EXON 0 43 5 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UMAG_01123 EXON 0 23 6 0 29 0 45 0 0 45 6 29 0 45 2_949255 +2 517827 517827 17.9 17.9%_28r 2 517827 UMAG_00987 EXON 23 0 0 5 28 32 0 0 0 32 5 28 0 32 2_517827 +2 517936 517936 6.3 6.3%_63r 2 517936 UMAG_00987 EXON 59 0 0 4 63 98 0 0 0 98 4 63 0 98 2_517936 +2 519882 519882 17.8 17.8%_45r 2 519882 UMAG_00987 EXON 37 0 0 8 45 56 0 0 0 56 8 45 0 56 2_519882 +2 1538370 1538370 6.9 6.9%_29r 2 1538370 UMAG_10236 EXON 0 27 2 0 29 0 32 0 0 32 2 29 0 32 2_1538370 +2 1271320 1271320 22.2 22.2%_27r 2 1271320 UMAG_10594 EXON 0 21 6 0 27 0 30 0 0 30 6 27 0 30 2_1271320 +2 677918 677918 5.0 5.0%_20r 2 677918 UMAG_01033 EXON 19 0 0 1 20 34 0 0 0 34 1 20 0 34 2_677918 +2 681015 681015 7.5 7.5%_53r 2 681015 UMAG_01033 EXON 49 0 0 4 53 105 0 0 0 105 4 53 0 105 2_681015 +2 1254198 1254198 6.7 6.7%_30r 2 1254198 UMAG_01219 EXON 28 0 0 2 30 36 0 0 0 36 2 30 0 36 2_1254198 +2 1624642 1624642 7.9 7.9%_89r 2 1624642 UMAG_11309 EXON 0 82 7 0 89 0 364 0 0 364 7 89 0 364 2_1624642 +2 1574100 1574100 11.1 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2_415547 +2 418088 418088 5.0 5.0%_40r 2 418088 UMAG_00957 EXON 0 38 2 0 40 0 83 0 0 83 2 40 0 83 2_418088 +2 268777 268777 7.9 7.9%_38r 2 268777 UMAG_00907 EXON 0 35 3 0 38 0 51 0 0 51 3 38 0 51 2_268777 +2 1146958 1146958 5.3 5.3%_38r 2 1146958 UMAG_01181 EXON 36 0 0 2 38 56 0 0 0 56 2 38 0 56 2_1146958 +2 1147862 1147862 14.6 14.6%_48r 2 1147862 UMAG_01181 EXON 41 0 0 7 48 95 0 0 0 95 7 48 0 95 2_1147862 +2 55559 55559 7.1 7.1%_28r 2 55559 UMAG_11249 EXON 26 0 0 2 28 28 0 0 0 28 2 28 0 28 2_55559 +2 509689 509689 6.7 6.7%_45r 2 509689 UMAG_00984 EXON 42 0 0 3 45 79 0 0 0 79 3 45 0 79 2_509689 +2 1557491 1557491 9.1 9.1%_44r 2 1557491 UMAG_01327 EXON 0 40 4 0 44 0 71 0 0 71 4 44 0 71 2_1557491 +2 1557554 1557554 5.0 5.0%_20r 2 1557554 UMAG_01327 EXON 0 19 1 0 20 0 38 0 0 38 1 20 0 38 2_1557554 +2 1754001 1754001 9.1 9.1%_22r 2 1754001 UMAG_11326 EXON 0 20 2 0 22 0 20 0 0 20 2 22 0 20 2_1754001 +2 1631511 1631511 9.4 9.4%_53r 2 1631511 UMAG_01357 EXON 0 48 5 0 53 0 72 0 0 72 5 53 0 72 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0000000000000000000000000000000000000000..9d8e666ad600a9cb4cbda5be4ba79088316235ed --- /dev/null +++ b/assays/S5_A1_HyperTRIBE_Khd4_workflow/dataset/Fig_S6_characterization_of_Khd4_Ada_GFP_editing_events.html @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:eb5bda977f323add7528ca4a39f1e13d35fcf563726af615e34938c8a00fcc2a +size 1500401 diff --git a/assays/S5_A1_HyperTRIBE_Khd4_workflow/dataset/Fig_S6_characterization_of_Khd4_Ada_GFP_editing_events.qmd b/assays/S5_A1_HyperTRIBE_Khd4_workflow/dataset/Fig_S6_characterization_of_Khd4_Ada_GFP_editing_events.qmd new file mode 100644 index 0000000000000000000000000000000000000000..70c01a4f3276d0ae55571a12058389f7666df878 --- /dev/null +++ b/assays/S5_A1_HyperTRIBE_Khd4_workflow/dataset/Fig_S6_characterization_of_Khd4_Ada_GFP_editing_events.qmd @@ -0,0 +1,355 @@ +--- +title: "01_Characterization of editing sites" +date: last-modified +author: + - name: "Srimeenakshi Sankaranarayanan" + +title-block-banner: true +format: + html: + theme: flatly + self-contained: true + code-fold: true + code-tools: true + code-summary: "Show the code" + toc: true + number-sections: true + anchor-sections: true +editor: visual +execute: + echo: true + error: false + warning: false + message: false +--- + +```{r} +# library +library(BSgenome.Umaydis.ENSMBL.UM1) # forged ustilago genome +library(ggplot2) +library(BindingSiteFinder) +library(rtracklayer) +library(ComplexHeatmap) +library(GenomicFeatures) +library(forcats) +library(tidyr) +library(dplyr) +library(tidyverse) +library(GenomicRanges) +library(magick) +library(magrittr) +library(gridExtra) +library(IRanges) +library(Biostrings) +library(ggpp) +library(gginnards) +library(ggrepel) +library(ggpubr) +library(ggforce) +library(ggrastr) +library(viridis) +library(reshape2) +library(gprofiler2) +library(ggsci) +library(ggh4x) +library(ggplotify) +library(gridExtra) +library(circlize) +library(EnrichedHeatmap) +library(UpSetR) +library(kableExtra) +library(cowplot) +library(rstatix) +library(beeswarm) +library(clusterProfiler) +library(ggseqlogo) +library(tidyHeatmap) +library(paletteer) +library(ggvenn) +library(colorspace) +library(ggpointdensity) +library(lookup) +library(rstatix) +library(ggrepel) + +``` + +```{r} +load("C:/Users/Sri/Documents/Khd4/DGE_analysis_SM_PhD/Kathis_lab/hyperTRIBE/hyperTRIBE.rds") +``` + +# Characterise editing sites + +The editing events across transcriptome was identified as described here https://hypertribe.readthedocs.io/en/latest/ + +It is important to note that a few editing sites needed to be removed for the following reasons: - 6 sites were not on A - due to the gene extension by 300-nt, a handful of editing sites overlapped with 2 genes + +```{r} +# Import hyperTRIBE data +hyper.dir = "C:/Users/Sri/Documents/Khd4/DGE_analysis_SM_PhD/TRIBE_data/Editing_events_all/Khd4/bedgraphs/" + +# get all bedgraph files +hyper.files = list.files(hyper.dir, pattern="bedgraph", full.names=TRUE) + +# assign sample names (ATTENTION!) +names(hyper.files) = c("khd4_rep1", "khd4_rep2", "ctrl_rep1", "ctrl_rep2") +``` + +```{r} +# combine the hyperTRIBE results into a list +hyper.list = lapply(hyper.files, function(i){ + # import data + hyper = read.table(i, sep="\t") + + # keep only first 8 columns + hyper = hyper[,c(1:2,4:5,8)] + + # assign column names + colnames(hyper) = c("chr", "start", "perc.editing", "no.reads", "gene.id") + + # extract number of reads + hyper$no.reads = strsplit(hyper$no.reads, "_") %>% sapply(., "[[", 2) %>% sub("r", "", .) %>% as.integer(.) + + # add strand (from annotation) + pos = match(hyper$gene.id, genes.GR$gene_id) + hyper$strand = as.vector(strand(genes.GR))[pos] + + # add id + hyper$id = paste(hyper$chr, hyper$start, sep="_") + + # add number of motifs + sel = match(hyper$gene.id, genes.GR$gene_id) + hyper$AUACCC = genes.GR$AUACCC[sel] + hyper$AGAUCU = genes.GR$AGAUCU[sel] + hyper$GGGTAT = genes.GR$GGGTAT[sel] + hyper$ACACUC = genes.GR$ACACUC[sel] + + return(hyper) +}) + +#head(hyper.list[[1]]) + +# CHECK FOR DUPLICATED SITES -> overlapping genes due to extension +# these genes are removed at the moment (could also be resolved differently if needed) +sapply(hyper.list, function(hyper) table(duplicated(hyper$id))) +for (i in seq_along(hyper.list)){ + hyper = hyper.list[[i]] + dupls = hyper$id[duplicated(hyper$id)] + print( hyper[hyper$id %in% dupls,] ) +} + +# REMOVE DUPLICATED SITES +hyper.list = lapply(hyper.list, function(hyper){ + dupls = hyper$id[duplicated(hyper$id)] + hyper = subset(hyper, !(id %in% dupls)) + return(hyper) +}) + +# test +# sapply(hyper.list, function(hyper) table(duplicated(hyper$id))) +``` + +```{r} +# convert editing sites into GRanges object +hyper.GR = lapply(hyper.list, function(hyper){ + gr = GRanges( + seqnames=hyper$chr, + ranges=IRanges(start=hyper$start, width=1), + strand=hyper$strand, + id=hyper$id, + gene.id=hyper$gene.id, + no.reads = hyper$no.reads, + perc.editing = hyper$perc.editing + ) + return(gr) +}) %>% as(., "GRangesList") +``` + +## Get sequence of editing sites + +```{r} +# get sequence of all editing sites +seqs = lapply(hyper.GR, function(gr){ + seq = getSeq(Umaydis, gr) + return(seq) +}) + +# check distribution +sapply(seqs, table) + +#### REMOVE INDIVIDUAL SITES THAT ARE NOT ON A +hyper.GR = lapply(hyper.GR, function(gr){ + seqs = getSeq(Umaydis, gr) + gr = subset(gr, seqs == "A") + return(gr) +}) + +# check that only A's remain +seqs = lapply(hyper.GR, function(gr){ + seq = getSeq(Umaydis, gr) + return(seq) +}) + +sapply(seqs, table) +``` + +## Compare between replicates + +Reproducible editing sites overlapped between the two independent biological replicates of the Khd4-Ada-Gfp and control-Ada data were compared with each other (#1,2- replicates 1 and 2). Only editing events with 5% editing were used for the analysis. ( + +```{r} +# Venn diagram of Khd4-ada-gfp replicates +venn.list = lapply(hyper.GR[1:2], function(hyper){ + return(hyper$id) +}) + +pl1 = ggvenn(venn.list, names(hyper.GR)[1:2], fill_color = c(lighten(khd4.col, 0.2), darken(khd4.col, 0.2)), set_name_size = 2) +theme_paper()+labs(x = NULL, y = NULL) + + guides(x = "none", y = "none") + +# Venn diagram of control-Ada replicates +venn.list = lapply(hyper.GR[3:4], function(hyper){ + return(hyper$id) +}) + +pl2 = ggvenn(venn.list, names(hyper.GR)[3:4], fill_color = c(lighten(ada.col, 0.2), darken(ada.col, 0.2)), set_name_size = 2) +theme_paper()+labs(x = NULL, y = NULL) + + guides(x = "none", y = "none") + +#FigS6A +vennrep<-ggarrange(pl1, pl2, + labels = c("A", "B"), + ncol = 2, nrow = 1) +vennrep +``` + +```{r} +# KEEP ONLY REPRODUCIBLE SITES FOR FURTHER ANALYSES +# merge Khd4-Ada-Gfp replicates +khd4.tab = merge(hyper.GR$khd4_rep1 %>% mcols(), hyper.GR$khd4_rep2 %>% mcols(), by=c("id", "gene.id"), all=FALSE, suffixes=c(".rep1", ".rep2")) %>% as.data.frame(.) + +# select only sites from overlap and add combined info of both replicates +sel = match(khd4.tab$id, hyper.GR$khd4_rep1$id) +khd4.rep = hyper.GR$khd4_rep1[sel] +mcols(khd4.rep) = khd4.tab + +# merge control-Ada replicates +ctrl.tab = merge(hyper.GR$ctrl_rep1 %>% mcols(), hyper.GR$ctrl_rep2 %>% mcols(), by=c("id", "gene.id"), all=FALSE, suffixes=c(".rep1", ".rep2")) %>% as.data.frame(.) + +# select only sites from overlap and add combined info of both replicates +sel = match(ctrl.tab$id, hyper.GR$ctrl_rep1$id) +ctrl.rep = hyper.GR$ctrl_rep1[sel] +mcols(ctrl.rep) = ctrl.tab + +## NEW GRANGES OBJECT WITH ONLY REPRODUCIBLE SITES +hyper.rep = GRangesList(khd4 = khd4.rep, ctrl = ctrl.rep) + +# number of reproducible editing sites +elementNROWS(hyper.rep) + +# genes with reproducible sites +sapply(hyper.rep, function(hyper) length(unique(hyper$gene.id))) + +elementNROWS(hyper.rep) +``` + +```{r} +# Venn diaram +venn.list = lapply(hyper.rep, function(hyper){ + return(hyper$id) +}) + +# FigS6A and B + +ggvenn(venn.list, names(venn.list), fill_color = c(khd4.col, ada.col)) + + ggtitle("Overlap between Khd4.Ada and Ada.ctrl") + + +``` + +# Studying the editing events + +```{r} +# distribution of % editing +pf = data.frame( + perc.editing = c(khd4.tab$perc.editing.rep1, khd4.tab$perc.editing.rep2, ctrl.tab$perc.editing.rep1, ctrl.tab$perc.editing.rep2), + sample = rep(c("khd4.rep1", "khd4.rep2", "ctrl.rep1", "ctrl.rep2"), rep(c(nrow(khd4.tab), nrow(ctrl.tab)), each=2)), + condition = rep(c("khd4", "khd4", "ctrl", "ctrl"), rep(c(nrow(khd4.tab), nrow(ctrl.tab)), each=2))) + + +# khd4-ada +#FigS6C +pl1 = ggplot(pf, aes(x=perc.editing, fill=sample)) + + geom_density(adjust = 1/5, alpha=0.5) + + scale_fill_manual(values=c(khd4.rep1 = lighten(khd4.col, 0.2), khd4.rep2 = darken(khd4.col, 0.2), ctrl.rep1 = lighten(ada.col, 0.2), ctrl.rep2 = darken(ada.col, 0.2)))+ + xlab("% editing")+ + ylab("density")+ + myTheme1 + +pl1 +``` + +Note that I used coefficient determination to check the editing reproducibility because the R2 indicates the distance of points between 1:1 line, on the other hand magnitude. Therefore, I did not use pearson (the analysis is independent of magnitude, in our case, editing percentage) and spearman (as the editing relationship is not monotonic). + +```{r} +# Note: used coefficient determination because the R2 indicates the distance of +# points between 1:1 line, on the other hand magnitude; didnt use pearson (as it +# is is independent of magnitude, in our case, independent of editing percentage) +# no spearman (as the editing relationship is not monotonic) + +# scatter plots of % editing in overlapping sites +# Khd4-Ada-Gfp +pl3 = ggplot(khd4.tab, aes(x=perc.editing.rep1, y=perc.editing.rep2)) + + geom_point(adjust=0.1, color="grey") + + ggtitle("Reproducible editing sites in Khd4-Ada-Gfp") + + theme_bw() + + labs(x="Khd4-Ada-Gfp #rep1 (editing %)", + y = "Khd4-Ada-Gfp #rep2 (editing %)")+ + theme(aspect.ratio = 1) + + expand_limits(x=c(0,100), y=c(0,100))+geom_smooth(method = "lm", size=1)+ + stat_regline_equation(label.y = 95, aes(label = ..eq.label..)) + + stat_regline_equation(label.y = 85, aes(label = ..rr.label..))+ myTheme1 +pl3 + +# Control-Ada +pl4 = ggplot(ctrl.tab, aes(x=perc.editing.rep1, y=perc.editing.rep2))+ + geom_point(adjust=0.1, color="grey")+ + ggtitle("Reproducible editing sites in control-Ada") + + labs(x="Control-Ada #rep1 (editing %)", + y = "Control-Ada #rep2 (editing %)")+ + theme_bw() + + theme(aspect.ratio = 1) + + expand_limits(x=c(0,100), y=c(0,100))+geom_smooth(method = "lm", size=1)+ + stat_regline_equation(label.y = 95, aes(label = ..eq.label..)) + + stat_regline_equation(label.y = 85, aes(label = ..rr.label..))+ myTheme1 + +pl4 +``` + +## De novo motif discovery analysis on sequences carrying unique control-Ada editing sites. + +XSTREME analysis was done using the random genome sequences of lenght 501-nt as a background and unique-Control-Ada editing sites (501-nt) as an input + +```{r} +# unique to control-Ada +Motif.frame.con = data.frame( Motif = c("GCGCGUG","CGABGACGAVGAVG","RVCAAGAWGRRCAAG","GUGAYUCGUGAYUGU","AGCAGCAGCAG","CGACUUGCG","WSVYCAAGMWGMUCR","ACCGCCAUCU","CAGCUCWAGCC","AWUCAYGAAUS"), +absolute = c(43.7,40.9,21.7,4.9,30.8,72.5,1.7,19.9,76.7,26.3), +EnR = c(1.25,1.17,1.20,1.58,1.09,1.03,1.44,1.01,1.00,0.97), +set=rep("ctrl",10)) + +# make a ggplot +pl.con = ggplot(Motif.frame.con, aes(x=absolute, y=EnR, color=set))+ + geom_point(alpha=1, size=2)+ + scale_color_manual(values = ada.col)+ + coord_cartesian(ylim=c(0,10))+ + labs(subtitle= "control-Ada editing sites", + x="sequences with motif (%)", + y = "motif enrichment ratio")+ + theme_paper()+ + theme(legend.position = "none") + +pl.con +``` + + +```{r} +sessionInfo() +``` diff --git a/assays/S5_A2_HyperTRIBE_motif_analysis/HyperTRIBE_identifies_highly_specific_mRNA_Targets_of_Khd4.html b/assays/S5_A2_HyperTRIBE_motif_analysis/HyperTRIBE_identifies_highly_specific_mRNA_Targets_of_Khd4.html new file mode 100644 index 0000000000000000000000000000000000000000..ebaa4e7358fb446ea838d25fe7446c9940cb8a2d --- /dev/null +++ b/assays/S5_A2_HyperTRIBE_motif_analysis/HyperTRIBE_identifies_highly_specific_mRNA_Targets_of_Khd4.html @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:e23c284e386f20611dda6c05c75bc7ba87a7c4aa9946400f67a5342fc542b8f9 +size 1480451 diff --git a/assays/S5_A2_HyperTRIBE_motif_analysis/HyperTRIBE_identifies_highly_specific_mRNA_Targets_of_Khd4.qmd b/assays/S5_A2_HyperTRIBE_motif_analysis/HyperTRIBE_identifies_highly_specific_mRNA_Targets_of_Khd4.qmd new file mode 100644 index 0000000000000000000000000000000000000000..3ffdd0ad4ae092f0b699c74c0a5e76e4559de9c9 --- /dev/null +++ b/assays/S5_A2_HyperTRIBE_motif_analysis/HyperTRIBE_identifies_highly_specific_mRNA_Targets_of_Khd4.qmd @@ -0,0 +1,508 @@ +--- +title: "HyperTRIBE_identifies_highly_specific_targets_of_Khd4" +date: last-modified +author: + - name: "Srimeenakshi Sankaranarayanan" + +title-block-banner: true +format: + html: + theme: flatly + self-contained: true + code-fold: true + code-tools: true + code-summary: "Show the code" + toc: true + number-sections: true + anchor-sections: true +editor: visual +execute: + echo: true + error: false + warning: false + message: false +--- + +```{r} +# library +library(BSgenome.Umaydis.ENSMBL.UM1) # forged ustilago genome +library(ggplot2) +library(BindingSiteFinder) +library(rtracklayer) +library(ComplexHeatmap) +library(GenomicFeatures) +library(forcats) +library(tidyr) +library(dplyr) +library(tidyverse) +library(GenomicRanges) +library(magick) +library(magrittr) +library(gridExtra) +library(IRanges) +library(Biostrings) +library(ggpp) +library(gginnards) +library(ggrepel) +library(ggpubr) +library(ggforce) +library(ggrastr) +library(viridis) +library(reshape2) +library(gprofiler2) +library(ggsci) +library(ggh4x) +library(ggplotify) +library(gridExtra) +library(circlize) +library(EnrichedHeatmap) +library(UpSetR) +library(kableExtra) +library(cowplot) +library(rstatix) +library(beeswarm) +library(clusterProfiler) +library(ggseqlogo) +library(tidyHeatmap) +library(paletteer) +library(ggvenn) +library(colorspace) +library(ggpointdensity) +library(lookup) +library(rstatix) +library(ggrepel) + +``` + +```{r} +load("C:/Users/Sri/Documents/Khd4/DGE_analysis_SM_PhD/Kathis_lab/hyperTRIBE/hyperTRIBE.rds") +``` + +```{r} +# reproducible editing sites in Khd4-Ada-Gfp and control-Ada +hyper.rep +``` + + +```{r} +# Venn diaram +venn.list = lapply(hyper.rep, function(hyper){ + return(hyper$id) +}) + +names(venn.list) = c("Khd4-Ada-Gfp", "control-Ada") +# FigS6A and B + +ggvenn(venn.list, names(venn.list), + fill_color = c("dodgerblue3", ada.col), + set_name_size = 6) + + ggtitle("Overlap between Khd4-Ada-Gfp and control-Ada")+ + theme(plot.title = element_text(size = 20, hjust = 0.5, vjust = 3)) + +``` + + + +```{r} +# Identifying the RNA sequence motif + +################################################################################ +#AUACCC +################################################################################ + +# get gene sequences +gene.seqs = getSeq(Umaydis, genes.GR) + +# find AUACCC motifs +motif.auaccc = DNAString("ATACCC") + +# find all AUACCC motifs in the genome +auaccc.gr = vmatchPattern(motif.auaccc, Umaydis) + +# count motifs in genes +genes.GR$AUACCC = countOverlaps(genes.GR, auaccc.gr) +#table(genes.GR$AUACCC) + +# assign motifs to genes +ov = findOverlaps(auaccc.gr, genes.GR) + +# check whether motifs overlap with more than one gene +table(duplicated(from(ov))) + +# keep only motifs in genes & duplicate motifs that overlap with 2 genes, so they count for both genes +auaccc.gr = auaccc.gr[from(ov)] +auaccc.gr$gene.id = genes.GR$gene_id[to(ov)] + +# count motifs in transcript regions +#findOverlaps(hits, tx.regions) %>% to(.) %>% table(.) +#findOverlaps(auaccc.gr, tx.regions, select="first") %>% names(tx.regions)[.] %>% table(.) + + +################################################################################ +#AGAUCU +################################################################################ + +# find AGAUCU motifs +motif.ctrl = DNAString("AGATCT") +agaucu.gr = vmatchPattern(motif.ctrl, Umaydis) +genes.GR$AGAUCU = countOverlaps(genes.GR, agaucu.gr) +table(genes.GR$AGAUCU) + +# assign motifs to genes +ov = findOverlaps(agaucu.gr, genes.GR) + +# check whether motifs overlap with more than one gene +#table(duplicated(from(ov))) + +# keep only motifs in genes & duplicate motifs that overlap with 2 genes, so they count for both genes +agaucu.gr = agaucu.gr[from(ov)] +agaucu.gr$gene.id = genes.GR$gene_id[to(ov)] + +# count motifs in transcript regions +#findOverlaps(hits, tx.regions) %>% to(.) %>% table(.) +#findOverlaps(agaucu.gr, tx.regions, select="first") %>% names(tx.regions)[.] %>% table(.) +``` + +## De novo motif discovery analysis on sequences carrying unique Khd4–Ada–Gfp editing sites. + +XSTREME analysis was done using the random genome sequences of lenght 501-nt as a background and unique-Khd4-Ada-Gfp editing sites (501-nt) as an input. + +First, the Khd4-Ada-Gfp editing sites were extended on each side by 250nt to make a 501-nt window. + +```{r eval=FALSE} +# keep only id column +hyper.spe = lapply(hyper.rep, function (x){ + mcols(x) = x[,c(1)] + names(x) = x$id + return(x) +}) + +# split the reproducible editing sites that are unique to Khd4 and Control , and shared +hyper.specific = list( + Khd4.specific = hyper.spe$khd4[!(hyper.spe$khd4$id %in% hyper.spe$ctrl$id)], + shared = hyper.spe$khd4[hyper.spe$khd4$id %in% hyper.spe$ctrl$id], + ctrl.specific = hyper.spe$ctrl[!(hyper.spe$ctrl$id %in% hyper.spe$khd4$id)] +) + +# as a GRanges list +hyper.specific = as(hyper.specific, "GRangesList") + +# extend the window by 250 bp on both sides +hyper_250 = lapply(hyper.specific, function(x){ + x = x+250 # window 250 bp +}) + +# get fastafiles +# get sequence +khd4.specific = Biostrings::getSeq(Umaydis, hyper_250$Khd4.specific) +khd4.ctrl.shared = Biostrings::getSeq(Umaydis, hyper_250$shared) +ctrl.specific = Biostrings::getSeq(Umaydis, hyper_250$ctrl.specific) + +# export the fasta files +output = "C:/Users/Sri/Documents/Khd4/DGE_analysis_SM_PhD/TRIBE_data/Editing_events_all/Khd4/bedgraphs/MEME/fasta/" + +writeXStringSet(khd4.specific, filepath = paste0(output,"khd4.specific_250.fasta")) +writeXStringSet(khd4.ctrl.shared, filepath = paste0(output,"khd4.ctrl.shared_250.fasta")) +writeXStringSet(ctrl.specific, filepath = paste0(output,"ctrl.specific_250.fasta")) + +``` + +Scatter plot comparing the relative enrichment ratio of enriched motifs with the percentage of each motif in the tested sequences. + +```{r} +# making a scatter plot +# unique to Khd4-Ada-Gfp +Motif.frame.khd4 = data.frame(Motif = +c("HMHHKAUACCC","HMKAUACCCVH","UGSAGGCGCURSWGS","UHCCUCUYCHCCMUC","CAGCARCARCADCAG","CCUCKAAACSNCGC","CYAAKSMUGCUYCUR","CYCCCW","CUUGAYUUGUWWYCC","YYUUGUAYUUGUA","UYGCUGCUCUC", "ACMUCUCCAAGVYCA","CUCCUGAUCCUGCCU", "AGCGSUCRUUUGASC", "CCUCUKU", "CAACGCUCCAC","CCAGCAACAGCARSM", "GCAGAUGRUAYCCAK", "RASAAGMVSAWCAAG", "AVUGGUGCUGU"), +absolute = c(60.9, 59.3, 16.0, 65.8, 13.6, 37.4, 11.1, 37.9, 2.5, 6.2, 18.5, 6.2, 5.3, 3.7, 16.0, 50.6, 29.2, 3.7, 38.3, 11.1), +EnR = c(9.40,9.60,8.35,1.46,4.01,1.80,3.30,1.60,37.98,4.82,2.08, 4.34, 5.06, 4.93, 1.81, 1.27, 1.43, 3.88 , 1.10, 1.05), +set=rep("khd4",20)) + +pl.khd4 = ggplot(Motif.frame.khd4, aes(x=absolute, y=EnR, color=set))+ + geom_point(alpha=1, size=2)+ + scale_color_manual(values = khd4.col)+ + coord_cartesian(ylim=c(0,10))+ + labs(subtitle= "Khd4-Ada-Gfp", + x="sequences with motif (%)", + y = "motif enrichment ratio")+ + theme_paper()+ + theme(legend.position = "none") + +pl.khd4 +``` + +Make the sequence logo of the most enriched two motifs + +```{r} +################################################################################ +# De novo discoevered motif on Khd4-Ada-Gfp edited transcripts +################################################################################ + +# motif logo with u in it +# Motif-1 TP% = 60.9 ENR-Ratio = 9.40 HMHHKAUACCC +A= c(0.220238,0.380952,0.369048,0.392857,0.041667,1000000,0.000000,1000000,0.000000,0.000000,0.059524 +) +C= c(0.440476,0.321429,0.351190,0.261905,0.107143,0.000000,0.000000,0.000000,0.976190,1000000,0.940476 +) +G = c(0.107143,0.113095,0.029762,0.119048,0.333333,0.000000,0.000000,0.000000,0.000000,0.000000,0.000000 +) +U = c(0.232143,0.184524,0.250000,0.226190,0.517857,0.000000,1000000,0.000000,0.023810,0.000000,0.000000 +) + +pwDF1bU = rbind(A,C,G,U) + + +# MoUif-2 UP% = 59.3 ENR-RaUio = 9.60 HMKAUACCCVH + +A= c(0.366890,0.432382,0.026357,0.999729,0.000082,0.999729,0.019779,0.000082,0.050456,0.234389,0.213404 +) +C= c( 0.304485,0.245517,0.122745,0.000103,0.000103,0.000103,0.927513,0.999751,0.949376,0.374600,0.306852 +) +G = c(0.032919,0.116138,0.316355,0.000090,0.006656,0.000090,0.006656,0.000090,0.000090,0.211350,0.159894 +) +U = c(0.295706,0.205963,0.534542,0.000078,0.993160,0.000078,0.046052,0.000078,0.000078,0.179661,0.319849 +) + +pwDF2bU = rbind(A,C,G,U) + +# make ggseqlogo +m1 = ggseqlogo(pwDF1bU, col_scheme="nucleotide", font="helvetica_bold") + +m2 = ggseqlogo(pwDF2bU, col_scheme="nucleotide", font="helvetica_bold") + +pl = ggarrange(m1,m2, + labels = LETTERS[1:2], + nrow = 1) + +pl + +``` + + +Next I want to select mRNA targets that are specific to Khd4-Ada-Gfp and control-Ada data + +```{r} + +# Khd4.spe +Khd4.spe= hyper.rep$khd4[!(hyper.rep$khd4$gene.id %in% hyper.rep$ctrl$gene.id)] + +# mRNAs specific to control +ctrl.spe.tx = hyper.rep$ctrl[!(hyper.rep$ctrl$gene.id %in% hyper.rep$khd4$gene.id)] + +# make a list with mRNAs specific to each data set +# list +hyper.spe = list( + Khd4_Ada_Gfp = Khd4.spe, + control_Ada = ctrl.spe.tx +) %>% as(., "GRangesList") + + +``` + +## Distribution of editing sites from the nearest AUACCC motif + +Only editing sites on mRNAs specific to each dataset were considered + +```{r} +pf = lapply(names(hyper.spe), function(i){ + #i = "khd4" + hyper = hyper.spe[[i]] + head(hyper) + + # calculate distance to nearest AUACCC motif + dists.auacc = distanceToNearest(hyper, auaccc.gr) + + # keep only pairs on same genes + sel = hyper$gene.id[from(dists.auacc)] == auaccc.gr$gene.id[to(dists.auacc)] + table(sel) + dists.auacc= subset(dists.auacc, sel) + mcols(dists.auacc)$motif = "AUACCC" + + # calculate distance to nearest AGAUCU motif + dists.agaucu = distanceToNearest(hyper, agaucu.gr) + + # keep only pairs on same genes + sel = hyper$gene.id[from(dists.agaucu)] == agaucu.gr$gene.id[to(dists.agaucu)] + table(sel) + dists.agaucu= subset(dists.agaucu, sel) + mcols(dists.agaucu)$motif = "AGAUCU" + + # get stats of distances + summary(mcols(dists.auacc)$distance) + summary(mcols(dists.agaucu)$distance) + + pf = rbind(mcols(dists.auacc), mcols(dists.agaucu)) %>% as.data.frame() + pf$set = i + + return(pf) +}) + +pf = do.call(rbind, pf) + +# FigS7B + +# Histogram +pl4 = ggplot(pf, aes(x=distance, fill=set)) + + geom_histogram( binwidth = 150, aes(y = ..density..), position="dodge") + + coord_cartesian(xlim=c(0, 2500)) + + scale_fill_manual(values=c(Khd4_Ada_Gfp = "dodgerblue4", control_Ada = "grey60")) + + labs(x="distance of editing sites to the nearest motif (nt)", + y="motif density")+ + theme_bw() + + facet_wrap(~factor(motif, levels=c("AUACCC", "AGAUCU")))+myTheme1 + +pl4 + +``` + + +```{r} +# median distance between editing sites and the motif +#Khd4-Ada-Gfp +Khd4_auaccc= filter(pf, set == "khd4", motif == "AUACCC") +med_K_auaccc = median(Khd4_auaccc$distance) + + +#Control-Ada +Ctrl_auaccc= filter(pf, set == "ctrl", motif == "AUACCC") +med_C_auaccc = median(Ctrl_auaccc$distance) + + +data.frame(Khd4_Ada_Gfp = med_K_auaccc, + control_Ada = med_C_auaccc) %>% + kbl(caption = "median distance between auaccc and editing events",html_font = "Cambria", bold = T, color="black") %>% + kable_styling(bootstrap_options = "striped", "hover", full_width = F, html_font = "Cambria") %>% + column_spec(1, bold = T) +``` + + +## Studying the enrichment of motifs in mRNAs specific to each datasets + +Here, we consider only the editing events on Khd4-bound mRNAs and on mRNAs specific to control-Ada. +Khd4-bound mRNAs- All mRNAs that carry reproducible Khd4-Ada-Gfp editing sites with atleast one AUACCC motif. +Control-Ada mRNAs- mRNAs that have reproducible control-Ada editing sites but no Khd4-Ada-Gfp sites + +```{r} +# Khd4-bound mRNAs +Khd4_boundRNA= hyper.rep$khd4[(hyper.rep$khd4$gene.id %in% auaccc.gr$gene.id)] + +# mRNAs specific to control +ctrl.spe.tx = hyper.rep$ctrl[!(hyper.rep$ctrl$gene.id %in% hyper.rep$khd4$gene.id)] + +#check if the numbers are similar to the venn diagram +#n_distinct(khd4.spe.tx$gene.id) + +# make a list with mRNAs specific to each data set +Khd4.spe= hyper.rep$khd4[!(hyper.rep$khd4$gene.id %in% hyper.rep$ctrl$gene.id)] + + +# list +hyper.rep.txspe = list( + khd4 = Khd4_boundRNA, + ctrl = ctrl.spe.tx +) %>% as(., "GRangesList") + + +``` + + + + +# Analyis of the high-confidence targets + +```{r} +# only Khd4 +khd4.rep.edit=hyper.rep$khd4 +motifs=genes.GR +khd4.rep.edit$AUACCC=lookup(khd4.rep.edit$gene.id, motifs$gene_id,motifs$AUACCC) + +#granges of high confidence Khd4 +high.conf.khd4=subset(khd4.rep.edit,AUACCC>0) + +high.conf = high.conf.khd4 +``` + + +## Transcript region enrichment of motif + +Since Khd4 binds AUACCC specifically, we first want to check the transcript region that the AUACCC motif is enriched in high-confidence mRNA targets. Besides using the AGAUCU motif as a control, I also want to use the all hyphae expressed AUACCC-containing mRNAs as a putative targets + +Furthermore, I will use the already available AUACCC and AGAUCU motif position in *U. maydis* genome, as these already include the transcript region locations of the motif. + +```{r} +# load DGE data to identify hyphae expressed genes +# Use the lookup function to identify geneIDs overlapping with the high confident targets +Fi_wtvskd_DEG$high.conf_targets=lookup(Fi_wtvskd_DEG$gene.ID,high.conf$gene.id, high.conf$gene.id ) + +# auaccc motif +Fi_wtvskd_DEG$auaccc=lookup(Fi_wtvskd_DEG$gene.ID,auaccc.gr$gene.id, auaccc.gr$gene.id ) + +# agaucu motif +Fi_wtvskd_DEG$agaucu=lookup(Fi_wtvskd_DEG$gene.ID,agaucu.gr$gene.id, agaucu.gr$gene.id ) + +# only expressed genes +Fi_wtvskd_BM = Fi_wtvskd_DEG[Fi_wtvskd_DEG$baseMean>10,] +``` + +```{r} +auaccc_DGE = Fi_wtvskd_BM[!is.na(Fi_wtvskd_BM$auaccc),] + +agaucu_DGE = Fi_wtvskd_BM[!is.na(Fi_wtvskd_BM$agaucu),] + +``` + +```{r} +# AUACCC +auaccc.mot=read.csv("C:/Users/Sri/Documents/Khd4/DGE_analysis_SM_PhD/TRIBE_data/Editing_events_all/Khd4/bedgraphs/AUACCCmotif.csv") + +auaccc.mot$high.conf.targets = lookup::lookup(auaccc.mot$Gene_ID,high.conf$gene.id,high.conf$gene.id) + +auaccc.mot$put.targets = lookup::lookup(auaccc.mot$Gene_ID, auaccc_DGE$gene.ID, auaccc_DGE$gene.ID) + +# AGAUCU +agaucu.mot=read.csv("C:/Users/Sri/Documents/Khd4/DGE_analysis_SM_PhD/TRIBE_data/Editing_events_all/Khd4/bedgraphs/AGAUCUmotif.csv") + +agaucu.mot$high.conf.targets = lookup::lookup(agaucu.mot$GeneID,high.conf$gene.id,high.conf$gene.id) + +agaucu.mot$put.targets = lookup::lookup(agaucu.mot$GeneID, auaccc_DGE$gene.ID, auaccc_DGE$gene.ID) +``` + + +```{r} +# highconfident targets (auaccc motif) +df_high_conf_auaccc = auaccc.mot[!is.na(auaccc.mot$high.conf.targets),] %>% .[,"Position"] %>% table(.) %>% as.data.frame %>% set_colnames(.,c("variable", "Freq")) %>% mutate(motif= "AUACCC", set = "Khd4_bound_mRNA") + +# highconfident targets (agaucu motif) +df_high_conf_agaucuc = agaucu.mot[!is.na(agaucu.mot$high.conf.targets),] %>% .[,"Position"] %>% table(.) %>% as.data.frame %>% set_colnames(.,c("variable", "Freq")) %>% mutate(motif= "AGAUCU", set = "Khd4_bound_mRNA") + +# putative targets (auaccc motif) +df_put_auaccc = auaccc.mot[!is.na(auaccc.mot$put.targets),] %>% .[,"Position"] %>% table(.) %>% as.data.frame %>% set_colnames(.,c("variable", "Freq")) %>% mutate(motif= "AUACCC", set = "putative_targets") + +# putative targets (agaucu motif) +df_put_agaucuc = agaucu.mot[!is.na(agaucu.mot$put.targets),] %>% .[,"Position"] %>% table(.) %>% as.data.frame %>% set_colnames(.,c("variable", "Freq")) %>% mutate(motif= "AGAUCU", set = "putative_targets") + +df = rbind(df_high_conf_auaccc,df_high_conf_agaucuc,df_put_auaccc,df_put_agaucuc) + +ggplot(df, aes(x=factor(set, levels=c("Khd4_bound_mRNA", "putative_targets")), y = Freq, fill = factor(variable, levels=c("5UTR", "ORF", "3UTR"))))+ + geom_bar(position = position_fill(), stat = "identity")+ + scale_fill_brewer(palette = "Greens")+ + facet_wrap(~factor(motif, levels=c("AUACCC", "AGAUCU")))+ + labs(x="", + y = "% with motif", + fill= "transcript region")+ + myTheme1 + + +``` + +```{r} +sessionInfo() +``` + + + + + + + +