1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000
1001
1002
1003
1004
1005
1006
1007
1008
1009
1010
1011
1012
1013
1014
1015
1016
1017
1018
1019
1020
1021
1022
1023
1024
1025
1026
1027
1028
1029
1030
1031
1032
1033
1034
1035
1036
1037
1038
1039
1040
1041
1042
1043
1044
1045
1046
1047
1048
1049
1050
1051
1052
1053
1054
1055
1056
1057
1058
1059
1060
1061
1062
1063
1064
1065
1066
1067
1068
1069
1070
1071
1072
1073
1074
1075
1076
1077
1078
1079
1080
1081
1082
1083
1084
1085
1086
1087
1088
1089
1090
1091
1092
1093
1094
1095
1096
1097
1098
1099
1100
1101
1102
1103
1104
1105
1106
1107
1108
1109
1110
1111
1112
1113
1114
1115
1116
1117
1118
1119
1120
1121
1122
1123
1124
1125
1126
1127
1128
1129
1130
1131
1132
1133
1134
1135
1136
1137
1138
1139
1140
1141
1142
1143
1144
1145
1146
1147
1148
1149
1150
1151
1152
1153
1154
1155
1156
1157
1158
1159
1160
1161
1162
1163
1164
1165
1166
1167
1168
1169
1170
1171
1172
1173
1174
1175
1176
1177
1178
1179
1180
1181
1182
1183
1184
1185
1186
1187
1188
1189
1190
1191
1192
1193
1194
1195
1196
1197
1198
1199
1200
1201
1202
1203
1204
1205
1206
1207
1208
1209
1210
1211
1212
1213
1214
1215
1216
1217
1218
1219
1220
1221
1222
1223
1224
1225
1226
1227
1228
1229
1230
1231
1232
1233
1234
1235
1236
1237
1238
1239
1240
1241
1242
1243
1244
1245
1246
1247
1248
1249
1250
1251
1252
1253
1254
1255
1256
1257
1258
1259
1260
1261
1262
1263
1264
1265
1266
1267
1268
1269
1270
1271
1272
1273
1274
1275
1276
1277
1278
1279
1280
1281
1282
1283
1284
1285
1286
1287
1288
1289
1290
1291
1292
1293
1294
1295
1296
1297
1298
1299
1300
1301
1302
1303
1304
1305
1306
1307
1308
1309
1310
1311
1312
1313
1314
1315
1316
1317
1318
1319
1320
1321
1322
1323
1324
1325
1326
1327
1328
1329
1330
1331
1332
1333
1334
1335
1336
1337
1338
1339
1340
1341
1342
1343
1344
1345
1346
1347
1348
1349
1350
1351
1352
1353
1354
1355
1356
1357
1358
1359
1360
1361
1362
1363
1364
1365
1366
1367
1368
1369
1370
1371
1372
1373
1374
1375
1376
1377
1378
1379
1380
1381
1382
1383
1384
1385
1386
1387
1388
1389
1390
1391
1392
1393
1394
1395
1396
1397
1398
1399
1400
1401
1402
1403
1404
1405
1406
1407
1408
1409
1410
1411
1412
1413
1414
1415
1416
1417
1418
1419
1420
1421
1422
1423
1424
1425
1426
1427
1428
1429
1430
1431
1432
1433
1434
1435
1436
1437
1438
1439
1440
1441
1442
1443
1444
1445
1446
1447
1448
1449
1450
1451
1452
1453
1454
1455
1456
1457
1458
1459
1460
1461
1462
1463
1464
1465
1466
1467
1468
1469
1470
1471
1472
1473
1474
1475
1476
1477
1478
1479
1480
1481
1482
1483
1484
1485
1486
1487
1488
1489
1490
1491
1492
1493
1494
1495
1496
1497
1498
1499
1500
1501
1502
1503
1504
1505
1506
1507
1508
1509
1510
1511
1512
1513
1514
1515
1516
1517
1518
1519
1520
1521
1522
1523
1524
1525
1526
1527
1528
1529
1530
1531
1532
1533
1534
1535
1536
1537
1538
1539
1540
1541
1542
1543
1544
1545
1546
1547
1548
1549
1550
1551
1552
1553
1554
1555
1556
1557
1558
1559
1560
1561
1562
1563
1564
1565
1566
1567
1568
1569
1570
1571
1572
1573
1574
1575
1576
1577
1578
1579
1580
1581
1582
1583
1584
1585
1586
1587
1588
1589
1590
1591
1592
1593
1594
1595
1596
1597
1598
1599
1600
1601
1602
1603
1604
1605
1606
1607
1608
1609
1610
1611
1612
1613
1614
1615
1616
1617
1618
1619
1620
1621
1622
1623
1624
1625
1626
1627
1628
1629
1630
1631
1632
1633
1634
1635
1636
1637
1638
1639
1640
1641
1642
1643
1644
1645
1646
1647
1648
1649
1650
1651
1652
1653
1654
1655
1656
1657
1658
1659
1660
1661
1662
1663
1664
1665
1666
1667
1668
1669
1670
1671
1672
1673
1674
1675
1676
1677
1678
1679
1680
1681
1682
1683
1684
1685
1686
1687
1688
1689
1690
1691
1692
1693
1694
1695
1696
1697
1698
1699
1700
1701
1702
1703
1704
1705
1706
1707
1708
1709
1710
1711
1712
1713
1714
1715
1716
1717
1718
1719
1720
1721
1722
1723
1724
1725
1726
1727
1728
1729
1730
1731
1732
1733
1734
1735
1736
1737
1738
1739
1740
1741
1742
1743
1744
1745
1746
1747
1748
1749
1750
1751
1752
1753
1754
1755
1756
1757
1758
1759
1760
1761
1762
1763
1764
1765
1766
1767
1768
1769
1770
1771
1772
1773
1774
1775
1776
1777
1778
1779
1780
1781
1782
1783
1784
1785
1786
1787
1788
1789
1790
1791
1792
1793
1794
1795
1796
1797
1798
1799
1800
1801
1802
1803
1804
1805
1806
1807
1808
1809
1810
1811
1812
1813
1814
1815
1816
1817
1818
1819
1820
1821
1822
1823
1824
1825
1826
1827
1828
1829
1830
1831
1832
1833
1834
1835
1836
1837
1838
1839
1840
1841
1842
1843
1844
1845
1846
1847
1848
1849
1850
1851
1852
1853
1854
1855
1856
1857
1858
1859
1860
1861
1862
1863
1864
1865
1866
1867
1868
1869
1870
1871
1872
1873
1874
1875
1876
1877
1878
1879
1880
1881
1882
1883
1884
1885
1886
1887
1888
1889
1890
1891
1892
1893
1894
1895
1896
1897
1898
1899
1900
1901
1902
1903
1904
1905
1906
1907
1908
1909
1910
1911
1912
1913
1914
1915
1916
1917
1918
1919
1920
1921
1922
1923
1924
1925
1926
1927
1928
1929
1930
1931
1932
1933
1934
1935
1936
1937
1938
1939
1940
1941
1942
1943
1944
1945
1946
1947
1948
1949
1950
1951
1952
1953
1954
1955
1956
1957
1958
1959
1960
1961
1962
1963
1964
1965
1966
1967
1968
1969
1970
1971
1972
1973
1974
1975
1976
1977
1978
1979
1980
1981
1982
1983
1984
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
2016
2017
2018
2019
2020
2021
2022
2023
2024
2025
2026
2027
2028
2029
2030
2031
2032
2033
2034
2035
2036
2037
2038
2039
2040
2041
2042
2043
2044
2045
2046
2047
2048
2049
2050
2051
2052
2053
2054
2055
2056
2057
2058
2059
2060
2061
2062
2063
2064
2065
2066
2067
2068
2069
2070
2071
2072
2073
2074
2075
2076
2077
2078
2079
2080
2081
2082
2083
2084
2085
2086
2087
2088
2089
2090
2091
2092
2093
2094
2095
2096
2097
2098
2099
2100
2101
2102
2103
2104
2105
2106
2107
2108
2109
2110
2111
2112
2113
2114
2115
2116
2117
2118
2119
2120
2121
2122
2123
2124
2125
2126
2127
2128
2129
2130
2131
2132
2133
2134
2135
2136
2137
2138
2139
2140
2141
2142
2143
2144
2145
2146
2147
2148
2149
2150
2151
2152
2153
2154
2155
2156
2157
2158
2159
2160
2161
2162
2163
2164
2165
2166
2167
2168
2169
2170
2171
2172
2173
2174
2175
2176
2177
2178
2179
2180
2181
2182
2183
2184
2185
2186
2187
2188
2189
2190
2191
2192
2193
2194
2195
2196
2197
2198
2199
2200
2201
2202
2203
2204
2205
2206
2207
2208
2209
2210
2211
2212
2213
2214
2215
2216
2217
2218
2219
2220
2221
2222
2223
2224
2225
2226
2227
2228
2229
2230
2231
2232
2233
2234
2235
2236
2237
2238
2239
2240
2241
2242
2243
2244
2245
2246
2247
2248
2249
2250
2251
2252
2253
2254
2255
2256
2257
2258
2259
2260
2261
2262
2263
2264
2265
2266
2267
2268
2269
2270
2271
2272
2273
2274
2275
2276
2277
2278
2279
2280
2281
2282
2283
2284
2285
2286
2287
2288
2289
2290
2291
2292
2293
2294
2295
2296
2297
2298
2299
2300
2301
2302
2303
2304
2305
2306
2307
2308
2309
2310
2311
2312
2313
2314
2315
2316
2317
2318
2319
2320
2321
2322
2323
2324
2325
2326
2327
2328
2329
2330
2331
2332
2333
2334
2335
2336
2337
2338
2339
2340
2341
2342
2343
2344
2345
2346
2347
2348
2349
2350
2351
2352
2353
2354
2355
2356
2357
2358
2359
2360
2361
2362
2363
2364
2365
2366
2367
2368
2369
2370
2371
2372
2373
2374
2375
2376
2377
2378
2379
2380
2381
2382
2383
2384
2385
2386
2387
2388
2389
2390
2391
2392
2393
2394
2395
2396
2397
2398
2399
2400
2401
2402
2403
2404
2405
2406
2407
2408
2409
2410
2411
2412
2413
2414
2415
2416
2417
2418
2419
2420
2421
2422
2423
2424
2425
2426
2427
2428
2429
2430
2431
2432
2433
2434
2435
2436
2437
2438
2439
2440
2441
2442
2443
2444
2445
2446
2447
2448
2449
2450
2451
2452
2453
2454
2455
2456
2457
2458
2459
2460
2461
2462
2463
2464
2465
2466
2467
2468
2469
2470
2471
2472
2473
2474
2475
2476
2477
2478
2479
2480
2481
2482
2483
2484
2485
2486
2487
2488
2489
2490
2491
2492
2493
2494
2495
2496
2497
2498
2499
2500
2501
2502
2503
2504
2505
2506
2507
2508
2509
2510
2511
2512
2513
2514
2515
2516
2517
2518
2519
2520
2521
2522
2523
2524
2525
2526
2527
2528
2529
2530
2531
2532
2533
2534
2535
2536
2537
2538
2539
2540
2541
2542
2543
2544
2545
2546
2547
2548
2549
2550
2551
2552
2553
2554
2555
2556
2557
2558
2559
2560
2561
2562
2563
2564
2565
2566
2567
2568
2569
2570
2571
2572
2573
2574
2575
2576
2577
2578
2579
2580
2581
2582
2583
2584
2585
2586
2587
2588
2589
2590
2591
2592
2593
2594
2595
2596
2597
2598
2599
2600
2601
2602
2603
2604
2605
2606
2607
2608
2609
2610
2611
2612
2613
2614
2615
2616
2617
2618
2619
2620
2621
2622
2623
2624
2625
2626
2627
2628
2629
2630
2631
2632
2633
2634
2635
2636
2637
2638
2639
2640
2641
2642
2643
2644
2645
2646
2647
2648
2649
2650
2651
2652
2653
2654
2655
2656
2657
2658
2659
2660
2661
2662
2663
2664
2665
2666
2667
2668
2669
2670
2671
2672
2673
2674
2675
2676
2677
2678
2679
2680
2681
2682
2683
2684
2685
2686
2687
2688
2689
2690
2691
2692
2693
2694
2695
2696
2697
2698
2699
2700
2701
2702
2703
2704
2705
2706
2707
2708
2709
2710
2711
2712
2713
2714
2715
2716
2717
2718
2719
2720
2721
2722
2723
2724
2725
2726
2727
2728
2729
2730
2731
2732
2733
2734
2735
2736
2737
2738
2739
2740
2741
2742
2743
2744
2745
2746
2747
2748
2749
2750
2751
2752
2753
2754
2755
2756
2757
2758
2759
2760
2761
2762
2763
2764
2765
2766
2767
2768
2769
2770
2771
2772
2773
2774
2775
2776
2777
2778
2779
2780
2781
2782
2783
2784
2785
2786
2787
2788
2789
2790
2791
2792
2793
2794
2795
2796
2797
2798
2799
2800
2801
2802
2803
2804
2805
2806
2807
2808
2809
2810
2811
2812
2813
2814
2815
2816
2817
2818
2819
2820
2821
2822
2823
2824
2825
2826
2827
2828
2829
2830
2831
2832
2833
2834
2835
2836
2837
2838
2839
2840
2841
2842
2843
2844
2845
2846
2847
2848
2849
2850
2851
2852
2853
2854
2855
2856
2857
2858
2859
2860
2861
2862
2863
2864
2865
2866
2867
2868
2869
2870
2871
2872
2873
2874
2875
2876
2877
2878
2879
2880
2881
2882
2883
2884
2885
2886
2887
2888
2889
2890
2891
2892
2893
2894
2895
2896
2897
2898
2899
2900
2901
2902
2903
2904
2905
2906
2907
2908
2909
2910
2911
2912
2913
2914
2915
2916
2917
2918
2919
2920
2921
2922
2923
2924
2925
2926
2927
2928
2929
2930
2931
2932
2933
2934
2935
2936
2937
2938
2939
2940
2941
2942
2943
2944
2945
2946
2947
2948
2949
2950
2951
2952
2953
2954
2955
2956
2957
2958
2959
2960
2961
2962
2963
2964
2965
2966
2967
2968
2969
2970
2971
2972
2973
2974
2975
2976
2977
2978
2979
2980
2981
2982
2983
2984
2985
2986
2987
2988
2989
2990
2991
2992
2993
2994
2995
2996
2997
2998
2999
3000
3001
3002
3003
3004
3005
3006
3007
3008
3009
3010
3011
3012
3013
3014
3015
3016
3017
3018
3019
3020
3021
3022
3023
3024
3025
3026
3027
3028
3029
3030
3031
3032
3033
3034
3035
3036
3037
3038
3039
3040
3041
3042
3043
3044
3045
3046
3047
3048
3049
3050
3051
3052
3053
3054
3055
3056
3057
3058
3059
3060
3061
3062
3063
3064
3065
3066
3067
3068
3069
3070
3071
3072
3073
3074
3075
3076
3077
3078
3079
3080
3081
3082
3083
3084
3085
3086
3087
3088
3089
3090
3091
3092
3093
3094
3095
3096
3097
3098
3099
3100
3101
3102
3103
3104
3105
3106
3107
3108
3109
3110
3111
3112
3113
3114
3115
3116
3117
3118
3119
3120
3121
3122
3123
3124
3125
3126
3127
3128
3129
3130
3131
3132
3133
3134
3135
3136
3137
3138
3139
3140
3141
3142
3143
3144
3145
3146
3147
3148
3149
3150
3151
3152
3153
3154
3155
3156
3157
3158
3159
3160
3161
3162
3163
3164
3165
3166
3167
3168
3169
3170
3171
3172
3173
3174
3175
3176
3177
3178
3179
3180
3181
3182
3183
3184
3185
3186
3187
3188
3189
3190
3191
3192
3193
3194
3195
3196
3197
3198
3199
3200
3201
3202
3203
3204
3205
3206
3207
3208
3209
3210
3211
3212
3213
3214
3215
3216
3217
3218
3219
3220
3221
3222
3223
3224
3225
3226
3227
3228
3229
3230
3231
3232
3233
3234
3235
3236
3237
3238
3239
3240
3241
3242
3243
3244
3245
3246
3247
3248
3249
3250
3251
3252
3253
3254
3255
3256
3257
3258
3259
3260
3261
3262
3263
3264
3265
3266
3267
3268
3269
3270
3271
3272
3273
3274
3275
3276
3277
3278
3279
3280
3281
3282
3283
3284
3285
3286
3287
3288
3289
3290
3291
3292
3293
3294
3295
3296
3297
3298
3299
3300
3301
3302
3303
3304
3305
3306
3307
3308
3309
3310
3311
3312
3313
3314
3315
3316
3317
3318
3319
3320
3321
3322
3323
3324
3325
3326
3327
3328
3329
3330
3331
3332
3333
3334
3335
3336
3337
3338
3339
3340
3341
3342
3343
3344
3345
3346
3347
3348
3349
3350
3351
3352
3353
3354
3355
3356
3357
3358
3359
3360
3361
3362
3363
3364
3365
3366
3367
3368
3369
3370
3371
3372
3373
3374
3375
3376
3377
3378
3379
3380
3381
3382
3383
3384
3385
3386
3387
3388
3389
3390
3391
3392
3393
3394
3395
3396
3397
3398
3399
3400
3401
3402
3403
3404
3405
3406
3407
3408
3409
3410
3411
3412
3413
3414
3415
3416
3417
3418
3419
3420
3421
3422
3423
3424
3425
3426
3427
3428
3429
3430
3431
3432
3433
3434
3435
3436
3437
3438
3439
3440
3441
3442
3443
3444
3445
3446
3447
3448
3449
3450
3451
3452
3453
3454
3455
3456
3457
3458
3459
3460
3461
3462
3463
3464
3465
3466
3467
3468
3469
3470
3471
3472
3473
3474
3475
3476
3477
3478
3479
3480
3481
3482
3483
3484
3485
3486
3487
3488
3489
3490
3491
3492
3493
3494
3495
3496
3497
3498
3499
3500
3501
3502
3503
3504
3505
3506
3507
3508
3509
3510
3511
3512
3513
3514
3515
3516
3517
3518
3519
3520
3521
3522
3523
3524
3525
3526
3527
3528
3529
3530
3531
3532
3533
3534
3535
3536
3537
3538
3539
3540
3541
3542
3543
3544
3545
3546
3547
3548
3549
3550
3551
3552
3553
3554
3555
3556
3557
3558
3559
3560
3561
3562
3563
3564
3565
3566
3567
3568
3569
3570
3571
3572
3573
3574
3575
3576
3577
3578
3579
3580
3581
3582
3583
3584
3585
3586
3587
3588
3589
3590
3591
3592
3593
3594
3595
3596
3597
3598
3599
3600
3601
3602
3603
3604
3605
3606
3607
3608
3609
3610
3611
3612
3613
3614
3615
3616
3617
3618
3619
3620
3621
3622
3623
3624
3625
3626
3627
3628
3629
3630
3631
3632
3633
3634
3635
3636
3637
3638
3639
3640
3641
3642
3643
3644
3645
3646
3647
3648
3649
3650
3651
3652
3653
3654
3655
3656
3657
3658
3659
3660
3661
3662
3663
3664
3665
3666
3667
3668
3669
3670
3671
3672
3673
3674
3675
3676
3677
3678
3679
3680
3681
3682
3683
3684
3685
3686
3687
3688
3689
3690
3691
3692
3693
3694
3695
3696
3697
3698
3699
3700
3701
3702
3703
3704
3705
3706
3707
3708
3709
3710
3711
3712
3713
3714
3715
3716
3717
3718
3719
3720
3721
3722
3723
3724
3725
3726
3727
3728
3729
3730
3731
3732
3733
3734
3735
3736
3737
3738
3739
3740
3741
3742
3743
3744
3745
3746
3747
3748
3749
3750
3751
3752
3753
3754
3755
3756
3757
3758
3759
3760
3761
3762
3763
3764
3765
3766
3767
3768
3769
3770
3771
3772
3773
3774
3775
3776
3777
3778
3779
3780
3781
3782
3783
3784
3785
3786
3787
3788
3789
3790
3791
3792
3793
3794
3795
3796
3797
3798
3799
3800
3801
3802
3803
3804
3805
3806
3807
3808
3809
3810
3811
3812
3813
3814
3815
3816
3817
3818
3819
3820
3821
3822
3823
3824
3825
3826
3827
3828
3829
3830
3831
3832
3833
3834
3835
3836
3837
3838
3839
3840
3841
3842
3843
3844
3845
3846
3847
3848
3849
3850
3851
3852
3853
3854
3855
3856
3857
3858
3859
3860
3861
3862
3863
3864
3865
3866
3867
3868
3869
3870
3871
3872
3873
3874
3875
3876
3877
3878
3879
3880
3881
3882
3883
3884
3885
3886
3887
3888
3889
3890
3891
3892
3893
3894
3895
3896
3897
3898
3899
3900
3901
3902
3903
3904
3905
3906
3907
3908
3909
3910
3911
3912
3913
3914
3915
3916
3917
3918
3919
3920
3921
3922
3923
3924
3925
3926
3927
3928
3929
3930
3931
3932
3933
3934
3935
3936
3937
3938
3939
3940
3941
3942
3943
3944
3945
3946
3947
3948
3949
3950
3951
3952
3953
3954
3955
3956
3957
3958
3959
3960
3961
3962
3963
3964
3965
3966
3967
3968
3969
3970
3971
3972
3973
3974
3975
3976
3977
3978
3979
3980
3981
3982
3983
3984
3985
3986
3987
3988
3989
3990
3991
3992
3993
3994
3995
3996
3997
3998
3999
4000
4001
4002
4003
4004
4005
4006
4007
4008
4009
4010
4011
4012
4013
4014
4015
4016
4017
4018
4019
4020
4021
4022
4023
4024
4025
4026
4027
4028
4029
4030
4031
4032
4033
4034
4035
4036
4037
4038
4039
4040
4041
4042
4043
4044
4045
4046
4047
4048
4049
4050
4051
4052
4053
4054
4055
4056
4057
4058
4059
4060
4061
4062
4063
4064
4065
4066
4067
4068
4069
4070
4071
4072
4073
4074
4075
4076
4077
4078
4079
4080
4081
4082
4083
4084
4085
4086
4087
4088
4089
4090
4091
4092
4093
4094
4095
4096
4097
4098
4099
4100
4101
4102
4103
4104
4105
4106
|
<HTML> <HEAD>
<TITLE>Sequin help documentation</TITLE>
<!-- if you use the following meta tags, uncomment them.
<meta name="author" content="sequindoc">
<META NAME="keywords" CONTENT="national center for biotechnology information, ncbi, national library of medicine, nlm, national institutes of health, nih, database, archive, bookshelf, pubmed, pubmed central, bioinformatics, biomedicine, sequence submission, sequin, bankit, submitting sequences">
<META NAME="description" CONTENT="Sequin is a stand-alone software tool developed by the National Center for Biotechnology Information (NCBI) for submitting and updating entries to the GenBank, EMBL, or DDBJ sequence databases. "> -->
<link rel="stylesheet" href="ncbi_sequin.css">
</HEAD>
<body bgcolor="#FFFFFF" text="#000000" link="#0033CC" vlink="#0033CC">
<!-- change the link and vlink colors from the original orange (link="#CC6600" vlink="#CC6600") -->
<!-- the header -->
<table border="0" width="600" cellspacing="0" cellpadding="0">
<tr>
<td width="140"><a href="http://www.ncbi.nlm.nih.gov"> <img src="http://www.ncbi.nlm.nih.gov/corehtml/left.GIF" width="130" height="45" border="0"></a></td>
<td width="360" class="head1" valign="BOTTOM"> <span class="H1">Sequin Help Documentation</span></td>
<!-- <td width="100" valign="BOTTOM">Your Logo</td> -->
</tr>
</table>
<!-- the quicklinks bar -->
<table CLASS="TEXT" border="0" width="600" cellspacing="0" cellpadding="3" bgcolor="#000000">
<tr CLASS="TEXT" align="CENTER">
<td width="100"><a href="index.html" class="BAR">Sequin</a></td>
<td width="100"><a href="http://www.ncbi.nlm.nih.gov/Entrez/" class="BAR">Entrez</a></td>
<td width="100"><a href="http://www.ncbi.nlm.nih.gov/BLAST/" class="BAR">BLAST</a></td>
<td width="100"><a href="http://www.ncbi.nlm.nih.gov/omim/" class="BAR">OMIM</a></td>
<td width="100"><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html" class="BAR">Taxonomy</a></td>
<td width="100"><a href="http://www.ncbi.nlm.nih.gov/Structure/" class="BAR">Structure</a></td>
</tr>
</table>
<!-- the contents -->
<P> 
<H2>Table of Contents</H2>
<HR>
>Introduction
#Sequin is a program designed to aid in the submission of sequences to
the GenBank, EMBL, and DDBJ sequence databases. It was written at the
National Center for Biotechnology Information, part of the National
Library of Medicine at the National Institutes of Health. This section
of the help document provides a basic overview of how to submit
sequences using the Sequin forms. Subsequent sections provide detailed
instructions for entering information on each form.
*The Help Documentation
#The Sequin help documentation is available in both on-line and World
Wide Web (http://www.ncbi.nlm.nih.gov/Sequin/sequin.hlp.html) formats.
The text of the on-line version scrolls as you progress through the
Sequin forms. Specific words or phrases can be identified with the
"find" command at the top of the window. The on-line document can also
be saved as a text file, or printed directly to a printer. Click on the
window that contains the help documentation. Under the Sequin File
menu, choose Export Help... to save the documentation as a text file.
To print the documentation without saving it first, click on the help
window, and choose Print from the Sequin File menu.
*Organization of Forms
#Information is entered into Sequin on a number of different forms. Each
form is made up of pages, which are indicated by folder tabs at the top
of the form. You can move to the desired page by clicking on the
appropriate folder tab. You can also move between pages of a form by
clicking on the "Next page" or "Prev page" buttons at the bottom of the
screen. You can move to the previous form or the next form by clicking
on the "Prev form" or "Next form" buttons on the first or last pages of
a form, respectively.
#There are numerous ways to enter information onto a page of a form,
including text fields, radio buttons, check boxes, scrolling boxes,
pop-up menus and spreadsheets.
#You may also use tables to import annotation of source information.
The formatting of these tables will be discussed below.
#When entering information, you must use ASCII characters. Non-ASCII
characters, such as those with an accent or umlaut, can not be displayed
properly. If you enter a non-ASCII character, you will be prompted to select
an alternative in a pop-up box.
*Overview of Sequin
#If you are using Sequin for the first time, you will be prompted to
fill out four forms: the Welcome to Sequin form, the Submitting
Authors Form, the Sequence Format form, and the Organism and Sequences
Form. After you have filled out these forms, a window will appear that
contains the Sequin record viewer. This viewer allows you to access
many other forms in which you can edit fields filled out in the three
initial forms, as well as add additional information. Detailed
instructions on how to fill out the forms and use the record viewer are
presented below.
>Welcome to Sequin Form
#First, indicate with one of the three radio buttons whether you are
submitting the sequence to the GenBank, EMBL, or DDBJ database. If you
are working on a sequence submission for the first time, click on
"Start New Submission". If you are modifying an existing submission
record, click on "Read Existing Record". If you would like to quit from
Sequin, click on "Quit Program".
#You can also "Read Existing Record" to read in a FASTA-formatted
sequence file for analysis purposes. The sequence will be displayed in Sequin and
can be analyzed with tools such as CDD Search, but it should not be
submitted because it does not have the appropriate annotations.
#If you are running Sequin in its network-aware mode, you will see
another button labeled "Download from Entrez". This option allows you
to update an existing database record using Sequin. The record will be
downloaded from GenBank into Sequin using NCBI's Entrez retrieval
system. The contents of the record will appear in Sequin, and you can
edit them by updating the sequence or the annotations, as necessary. If
you do not see the button labeled "Download from Entrez" on the Welcome
to Sequin form, you are not running Sequin in its network-aware mode.
To make Sequin network-aware, see the
<A HREF="#NetConfigure">
instructions
</A>
later in the help documentation.
#You can update only those records that you have submitted, not those
submitted by others. To update an existing record, first select which
of the databases you will be sending the update to. This should be the
database to which the original record was submitted. If you do not
know which database to use, send the record to GenBank and the NCBI
staff will forward it to the appropriate database. Next, click on the
button "Download from Entrez". Enter the nucleotide Accession number or
GI of the sequence on the first form. Then enter "yes" if you are
planning to submit the record as an update to one of the databases.
Fill out the Submitting Authors form.
<A HREF="#EditSubmitterInfo">
Instructions
</A>
for this form are found in the Sequin help documentation under "Edit
Submitter Info" under the Sequin File menu. The record will then open
in the record viewer. Explanations of how to add annotations or update
sequences are presented in the documentation entitled
<A HREF="#EditingtheRecord">
"Editing the record"
</A>
and
<A HREF="#SequenceEditor">
Sequence Editor
</A>
respectively. You will not see the Submitting Authors Form, the
Sequence Format Form, or the Organism and Sequences Form. Note that
updates, as well as new records, must be emailed to the appropriate
database. Sequin does not support direct submission of records over the
Internet.
#Additional configuration options are available under the Misc menu.
You can toggle between the stand-alone and network-aware modes of
Sequin. The default mode of Sequin, which is sufficient for most
sequence submissions, is stand-alone. In its network-aware mode, Sequin
can exchange data with NCBI and, for example, retrieve sequences
from Entrez and perform Taxonomy searches. The network-aware mode of
Sequin is described in detail in the
<A HREF="#NetConfigure">
Net Configure
</A>
section below. You can also start the NCBI DeskTop, which is for
advanced Sequin users only.
>Submitting Authors Form
#Information from this form will be used as a citation for the sequence
entry itself. It can contain the same information found in citations
associated with the formal publication of the sequence.
#On the bottom of each form are two buttons. Click "Prev form" (first
page in a form) or "Prev page" (subsequent pages in a form) to go to the
previous form or page. Click "Next Form" (last page on a form) or "Next
Page" (earlier pages on a form) to move to the next form or page.
#Form pages can also be saved individually by using the "Export" function
under the File menu. If you are processing multiple submissions, you
can use the "Import" function under the File menu to paste previously
entered information directly on the page.
#The Contact, Authors, and Affiliation pages can be saved as a block so
that you can use this information for your next submission. For your
first Sequin submission, fill in the requested information on the
Submitting Authors form and proceed with the preparation of the
submission. Choose Export Submitter Info under the File menu to export
this to a file. You can then import this information in subsequent
submissions using the Import Submitter Info in the File menu. You will
need to fill in the manuscript title for each submission however.
*Submission Page
**When May We Release Your Sequence Record?
#Please select one of the two radio buttons. If you select
#"Immediately After Processing", the
entry will be released to the public after the database staff has added
it to the database. If you select "Release Date", fields will appear in
which you can indicate the date on which the sequences should be
released to the public. The submission will then be held back until
formal publication of the sequence or GenBank Accession number, or
until the release date, whichever comes first. The maximum hold
time is five years.
**Tentative Title for Manuscript
#Please enter a title that appropriately describes the sequence entry.
Later in the submission process, you will have the
opportunity to change this information and add details for published
or in press references.
*Contact Page
#Please enter the name, telephone and fax numbers, and email address of
the person who is submitting the sequence. This is the person who will
be contacted regarding the sequence submission. The phone, fax, and
email address will not be visible in the database record, but are
essential for contact by the database staff.
*Authors Page
#Please enter the names of the people who should receive scientific
credit for the generation of sequences in this entry. The person on
the Contact page is automatically listed as the first author. This
information can be changed if necessary. The author names should be
entered in the order first name, middle initial, surname. You can add
as many authors to this page as you wish. After you type in the name
of the third author, the box becomes a spreadsheet, and you can scroll
down to the next line by using the space bar. The consortium box
should only be used for consortium names, not institute or department
names.
*Affiliation Page
#Please enter information about the principal institution where the
sequencing was performed. This is not necessarily the same as the
workplace of the person described on the Contact page. This information
will show up in the reference section of the record, with the title
Direct Submission.
>Sequence Format Form
#Use this form to indicate the type, format and category of sequence
you are submitting.
#Sequin can process single nucleotide sequences, gapped sequences and
sets of related sequences. If the sequences are related in terms of
coming from the same publication, or the same organism, they may be
candidates for a Batch submission. Biologically related sequences may
be classified as environmental samples, population, phylogenetic,
mutation, or segmented sets as appropriate. Segmented sets consist of
a collection of non-overlapping sequences covering a specific genetic
region. In all cases, although the sequences are handled as a single
submission, each sequence in a set will receive its own database
Accession number and can be annotated independently.
#Sequin can display the alignments of sequences that are submitted as
part of an aligned phylogenetic, population, mutation set, or
environmental samples. Such sequences can be submitted in FASTA,
Contiguous (FASTA+GAP, NEXUS, MACAW), or Interleaved (PHYLIP, NEXUS)
formats. If the sequences are in FASTA format, Sequin can generate an
alignment. If the sequences have already been aligned in FASTA+GAP,
PHYLIP, MACAW, or NEXUS, Sequin will not change the alignment. If one
of the sequences in your alignment is already present in the
GenBank/EMBL/DDBJ database, you must mark that sequence so that it does
not receive a new Accession number. Instead of supplying that sequence
with a new Sequence Identifier, give it the identifier accU12345, where
U12345 is the Accession number of the sequence.
#Single sequences, gapped sequences, segmented sequences, and batch
submissions must be submitted in FASTA format.
*Submission Type
#Use the radio buttons to indicate which of the following types of
submissions you are creating:
#-Single sequence: a single mRNA or genomic DNA sequence. If you are
submitting multiple sequences from the same publication, consider a
Batch Submission. If you decide to submit multiple Sequin files, each
with one or more sequences, please send each file in a separate email
message.
#-Segmented sequence: a collection of non-overlapping, non-contiguous
sequences that cover a specified genetic region. A standard example is
a set of genomic DNA sequences that encode exons from a gene along with
fragments of their flanking introns. If the segmented set is part of
an alignment, however, select the appropriate Population, Phylogenetic,
or Mutation study button.
#-Gapped sequence: a single, non-contiguous mRNA or genomic DNA sequence.
A gapped sequence contains specified gaps of know or unknown length
where the exact nucleotide sequence has not been determined. The FASTA
format for gapped sequences is slightly different and is explained
below.
#-Population study: a set of sequences that were derived by sequencing
the same gene from different isolates of the same organism.
#-Phylogenetic study: a set of sequences that were derived by sequencing
the same gene from different organisms.
#-Mutation study: a set of sequences that were derived by sequencing
multiple mutations of a single gene.
#-Environmental samples: a set of sequences that were derived by
sequencing the same gene from a population of unclassified or unknown
organisms.
#-Batch submission: a set of related sequences that are not part of a
population, mutation, or phylogenetic study. The sequences should be
related in some way, such as coming from the same publication or
organism. You should plan that all sequences will be released to the
public on the same date.
*Sequence Data Format
#If you are submitting a single, gapped, or segmented sequence, or a
batch submission, your sequence must be in FASTA format, described
below. If you are submitting a set of sequences as part of a
population, phylogenetic, or mutation study, you have a choice of
sequence formats. You may submit the set as individual sequences in
FASTA format. Alternatively, you can submit the sequences as part of
an alignment. Sequin currently accepts the alignment formats
FASTA+GAP, PHYLIP, MACAW, NEXUS Interleaved, and NEXUS Contiguous.
*Submission Category
#Use the radio buttons to indicate whether your sequence corresponds to
an original submission or a third-party annotation submission. If you
have directly sequenced the nucleotide sequence in your laboratory,
your submission would be considered an original submission.
#If you have downloaded the sequence from GenBank and added to it your
own annotations, your entry may be eligible for submission to the
Third-Party Annotation Database
<A HREF="http://www.ncbi.nlm.nih.gov/Genbank/tpa.html">
(TPA)
</A>
.
#In order to be released into the TPA database, the sequence must appear
in a peer-reviewed publication in a biological journal. If you select
this option, a pop-up box will appear upon the completion of the
Sequence Format form. You must provide some description of the
biological experiments used as evidence for the annotation of your TPA
submission in this box.
#You will be asked later in the submission process to provide the GenBank
Accession number(s) of the primary sequence(s) from which your TPA
submission was derived.
>Organism and Sequences Form
#This form is made up of four pages. If your sequences are imported as
properly formatted FASTA files, there will be minimum input necessary
in these pages.
>FASTA Format for Nucleotide Sequences
#In FASTA format the line before the nucleotide sequence, called the
FASTA definition line, must begin with a carat (">"), followed by a
unique SeqID (sequence identifier). The SeqID must be unique for each
nucleotide sequence and should not contain any spaces. Use of brackets
("[]") in the SeqID is also prohibited. The identifier will be
replaced with an Accession number by the database staff when your
submission is processed.
#Information about the source organism from which the sequence was
obtained follows the SeqID and must be in the format [modifier=text].
Do not put spaces around the "=". At minimum, the scientific name of
the organism should be included. Optional modifiers can be added to
provide additional information. A complete list of available source
<A HREF="http://www.ncbi.nlm.nih.gov/Sequin/modifiers.html">
modifiers
</A>
and their format is available.
#The final optional component of the FASTA definition line is the
sequence title, which will be used as the DEFINITION field in the final
flatfile. The title should contain a brief description of the
sequence. There is a preferred format for nucleotide and protein
titles and Sequin can generate them automatically using the Generate
Definition Line function under the Annotate menu in the record viewer.
#Note in all cases, the FASTA definition line must not contain any hard
returns. All information must be on a single line of text. If you
have trouble importing your FASTA sequences, please double check that
no returns were added to the FASTA definition line by your editing
software.
#Examples of properly formatted FASTA definition lines for nucleotide
sequences are:
<KBD><PRE>>Seq1 [organism=Mus musculus] [strain=C57BL/6] Mus musculus neuropilin 1 (Nrp1) mRNA, complete cds.
</KBD></PRE>
<KBD><PRE>>ABCD [organism=Plasmodium falciparum] [isolate=ABCD] Plasmodium falciparum isolate ABCD merozoite surface protein 2 (msp2) gene, partial cds.
</KBD></PRE>
<KBD><PRE>>DNA.new [organism=Homo sapiens] [chromosome=17] [map=17q21] [moltype=mRNA] Homo sapiens breast and ovarian cancer susceptibility protein (BRCA1) mRNA, complete cds.
</KBD></PRE>
#The line after the FASTA definition line begins the nucleotide
sequence. Unlike the FASTA definition line, the nucleotide sequence
itself can contain returns. It is recommended that each line of
sequence be no longer than 80 characters. Please only use IUPAC
symbols within the nucleotide sequence. For sequences that are not
contained within an alignment, do not use "?" or "-" characters. These
will be stripped from the sequence. Use the IUPAC approved symbol "N"
for ambiguous characters instead.
#A single file containing multiple FASTA sequences can be imported into
Sequin in order to create a
<A HREF="#SubmissionType">
Batch Submission
</A>
. Make sure that the FASTA definition line for each sequence is
formatted as above.
#If the FASTA definition line is not properly formatted a pop-up box
will appear upon importing the nucleotide FASTA. The top box in this
pop-up will list any errors in the FASTA definition lines, including
missing SeqIDs, duplicate SeqIDs for different sequences, or improperly
formatted modifiers. You can add or edit this information in the
spreadsheet provided. The toggle at the bottom of the pop-up allows
you to select whether all sequences or only those with errors are
listed in the spreadsheet above. After making changes, click on Refresh
Error List to ensure that all errors have been corrected. You must
correct any errors involving the SeqID in order to proceed with your
submission.
*FASTA Format for Segmented Sequence
#Each segment of a segmented sequence must have its own SeqID, but the
organism name and other modifiers are only indicated in the FASTA
definition line of the first segment. Square brackets are used to
delimit the members of the segmented set. For example,
![
!>A-0V-1-Apart1 [organism=Gallus gallus] [clone=C]
!TCACTCTTTGGCAAC
!>A-0V-1-Apart2
!GACCCGTCGTCATAATAAAGATAGAGGGGCAACTAAAGGAAGCTCTA
!TTAGATACAGGAGCAGATGATACAGTATTAGAAGAAAT
!]
*FASTA Format for Gapped Sequence
#The FASTA definition line for a gapped sequence follows the same format
as above. To indicate a gap within the sequence, enter a hard return
within the sequence at the point of the gap, then insert an extra line
starting with a carat (">") and a question mark ("?"). If the gap size
is unknown, enter "unk100" after the question mark. If the gap size is
known, enter the length of the gap after the question mark. For
example,
!>Dobi [organism=Canis familiaris] [breed=Doberman pinscher]
!AAATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTTAGCCCAGAAGTAATACCCATGTTTTCAGCATTA
!GGAAAAAGGGCTGTTG
!>?unk100
!TGGATGACAGAAACCTTGTTGGTCCAAAATGCAAACCCAGATKGTAAGACCATTTTAAAAGCATTGGGTC
!TTAGAAATAGGGCAACACAGAACAAAAAT
!>?234
!AAAAATAAAAGCATTAGTAGAAATTTGTACAGAACTGGAAAAGGAAGGAAAAATTTCAAAAATTGGGCCT
!GAAAACCCATACAATACTCCGGG
will generate a sequence containing two gaps. The first gap is of
unknown length, the second is 234 nucleotides long.
*FASTA+GAP Format for Aligned Nucleotide Sequences
#A number of programs output sets of aligned sequences in FASTA format.
Frequently, to align these sequences, gaps must be inserted. The
default alignment settings should correctl interpret gap and ambiguous
characters in most cases. If Sequin can not read your alignment, you
may need to change these settings using the Optional Alignment Settings
button on the
<A HREF="#NucleotidePage">
Nucleotide Page
</A>
form. Each sequence, including gaps, must be the same length. The
gaps will only show up in the alignment, not in the individual sequence
in the database.
#Sequences in FASTA+GAP format resemble FASTA sequences. The previous
section on
<A HREF="#FASTAFormatforNucleotideSequences">
FASTA Format for Nucleotide Sequences
</A>
has instructions for formatting FASTA sequences. If one of the
sequences in your alignment is already present in the GenBank/EMBL/DDBJ
database, you must mark that sequence so that it does not receive a new
Accession number. To do this, use a SeqID in the format accU12345,
where U12345 is the Accession number of the pre-existing sequence. All
sequences in FASTA+GAP format should be in the same file.
#The following is an example of FASTA+GAP format:
!>A-0V-1-A [organism=Gallus gallus] [clone=C]
!TCACTCTTTGGCAACGACCCGTCGTCATAATAAAGATAGAGGGGCAACTAAAGGAAGCTCTA
!TTAGATACAGGAGCAGATGATACAGTATTAGAAGAAAT
!
!>A-0V-2-A [organism=Drosophila melanogaster] [strain=D]
!TCACTCTTTGGCAAC---GCGTCGTCACAATAAAGATAGAGGGGCAACTAAAGGAAGCTCTA
!TTAGATACAGGAGCAGATGATACAGTATTAGAAGAAAT
!
!>A-0V-3-A [organism=Caenorhabditis elegans] [strain=E]
!TCACTCTTTGGCAAC---GCGTCGTCACAATAAAGATAGAGGGGCAACTAAAGGAAGCTCTA
!TTAGATACAGGAGCAGATGATACAGTATTAGAAGAAAT
!
!>A-0V-4-A [organism=Rattus norvegicus] [strain=F]
!TCACTCTTTGGCAACGACCCGTCGTCACAATAAAGATAGAGGGGCAACTAAAGGAAGCTCTA
!TTAGATACAGGAGCAGATGATACAGTATTAGAAGAAAT
!
!>A-0V-7-A [organism=Aspergillus nidulans] [strain=G]
!TCACTCTTTGGCAACGACCAGTCGTCACAATAAAGATAGAGGGGCAACTAAAGGAAGCTCTA
!TTAGATACAGGAGCAGATGATACAGTATTAGAAGAAAT
*PHYLIP Format for Aligned Nucleotide Sequences
#A number of programs output sets of aligned sequences in PHYLIP format.
#The following is an example of PHYLIP format.
! 5 100
!A-0V-1-A TCACTCTTTG GCAACGACCC GTCGTCATAA TAAAGATAGA GGGGCAACTA
!A-0V-2-A TCACTCTTTG GCAAC---GC GTCGTCACAA TAAAGATAGA GGGGCAACTA
!A-0V-3-A TCACTCTTTG GCAAC---GC GTCGTCACAA TAAAGATAGA GGGGCAACTA
!A-0V-4-A TCACTCTTTG GCAACGACCC GTCGTCACAA TAAAGATAGA GGGGCAACTA
!A-0V-7-A TCACTCTTTG GCAACGACCA GTCGTCACAA TAAAGATAGA GGGGCAACTA
!
!
! AAGGAAGCTC TATTAGATAC AGGAGCAGAT GATACAGTAT TAGAAGAAAT
! AAGGAAGCTC TATTAGATAC AGGAGCAGAT GATACAGTAT TAGAAGAAAT
! AAGGAAGCTC TATTAGATAC AGGAGCAGAT GATACAGTAT TAGAAGAAAT
! AAGGAAGCTC TATTAGATAC AGGAGCAGAT GATACAGTAT TAGAAGAAAT
! AAGGAAGCTC TATTAGATAC AGGAGCAGAT GATACAGTAT TAGAAGAAAT
#In this example, the first line indicates that there are 5 sequences,
each with 100 nt of sequence. The following five lines contain the
Sequence IDs, followed by the sequences. Specifically, the sequence
identifier for the first sequence is A-0V-1-A. Note that subsequent
blocks of sequence do not contain the Sequence ID. If one of the
sequences in your alignment is already present in the GenBank/EMBL/DDBJ
database, you must mark that sequence so that it does not receive a new
Accession number. To do this, use a SeqID in the format accU12345,
where U12345 is the Accession number of the pre-existing sequence.
#The default alignment settings should correctly interpret gap and
ambiguous characters in most cases. If Sequin can not read your
alignment, you may need to change these settings using the Optional
Alignment Settings button on the
<A HREF="#NucleotidePage">
Nucleotide Page
</A>
form.
#You can modify the PHYLIP format so that Sequin can
determine the correct organism and any other modifiers for each
sequence. An example of such modifications are below in the section on
<A HREF="#SourceModifiersforPHYLIPandNEXUS">
Source Modifiers for PHYLIP and NEXUS
</A>
.
#Alternatively, you can leave your sequence alignment in
standard PHYLIP format and enter the organism, strain, chromosome, etc.
information on the following
<A HREF="#SourceModifiersForm">
Source Modifers form
</A>
.
*NEXUS Format for Aligned Nucleotide Sequences
#A number of programs output sets of aligned sequences in one of two
NEXUS formats, NEXUS Interleaved and NEXUS Contiguous.
#NEXUS files can contain ? for "missing" at the 5' and 3' ends of
sequences, as long as this parameter is properly defined within the
header of the NEXUS file.
#The following is an example of NEXUS Interleaved format.
!#NEXUS
!
!begin data;
! dimensions ntax=5 nchar=100;
! format datatype=dna missing=? gap=- interleave;
! matrix
!
!A-0V-1-A TCACTCTTTG GCAACGACCC GTCGTCATAA TAAAGATAGA GGGGCAACTA
!A-0V-2-A TCACTCTTTG GCAAC---GC GTCGTCACAA TAAAGATAGA GGGGCAACTA
!A-0V-3-A TCACTCTTTG GCAAC---GC GTCGTCACAA TAAAGATAGA GGGGCAACTA
!A-0V-4-A TCACTCTTTG GCAACGACCC GTCGTCACAA T????ATAGA GGGGCAACTA
!A-0V-7-A TCACTCTTTG GCAACGACCA GTCGTCACAA TAAAGATAGA GGGGCAACTA
!
!
!A-0V-1-A AAGGAAGCTC TATTAGATAC AGGAGCAGAT GATACAGTAT TAGAAGAAAT
!A-0V-2-A AAGGAAGCTC TATTAGATAC AGGAGCAGAT GATACAGTAT TAGAAGAAAT
!A-0V-3-A AAGGAAGCTC TATTAGATAC AGGAGCAGAT GATACAGTAT TAGAAGAAAT
!A-0V-4-A AAGGAAGCTC TATTAGATAC AGGAGCAGAT GATACAGTAT TAGAAGAAAT
!A-0V-7-A AAGGAAGCTC TATTAGATAC AGGAGCAGAT GATACAGTAT TAGAAGAAAT
#In this example, the first few lines provide information about the data
in the sequence alignment. The following five lines contain the
Sequence IDs, followed by the sequences. Specifically, the sequence
identifier for the first sequence is A-0V-1-A. Note that subsequent
blocks of sequence also contain the Sequence ID. If one of the
sequences in your alignment is already present in the GenBank/EMBL/DDBJ
database, you must mark that sequence so that it does not receive a new
Accession number. To do this, use a SeqID in the format accU12345,
where U12345 is the Accession number of the pre-existing sequence.
Also, Sequin will replace the "?" characters in the sequences with "N"s
since they are defined as "missing" data in the header. The default
alignment settings should correctly interpret gap and ambiguous
characters in most cases. If Sequin can not read your alignment, you
may need to change these settings using the Optional Alignment Settings
button on the
<A HREF="#NucleotidePage">
Nucleotide Page
</A>
form.
#You can modify either NEXUS format so that Sequin can
determine the correct organism and any other modifiers for each
sequence. An example of such modifications are below in the section on
<A HREF="#SourceModifiersforPHYLIPandNEXUS">
Source Modifiers for PHYLIP and NEXUS
</A>
.
#Alternatively, you can leave your sequence alignment in
standard NEXUS format and enter the organism, strain, chromosome, etc.
information on the following
<A HREF="#SourceModifiersForm">
Source Modifers form
</A>
.
#The following is an example of NEXUS Contiguous format.
!#NEXUS
!BEGIN DATA;
!DIMENSIONS NTAX=5 NCHAR=100;
!FORMAT MISSING=? GAP=- DATATYPE=DNA ;
!MATRIX
!
!A-0V-1-A
!TCACTCTTTGGCAACGACCCGTCGTCATAATAAAGATAGAGGGGCAACTAAAGGAAGCTCTA
!TTAGATACAGGAGCAGATGATACAGTATTAGAAGAAAT
!
!A-0V-2-A
!TCACTCTTTGGCAAC---GCGTCGTCACAATAAAGATAGAGGGGCAACTAAAGGAAGCTCTA
!TTAGATACAGGAGCAGATGATACAGTATTAGAAGAAAT
!
!A-0V-3-A
!TCACTCTTTGGCAAC---GCGTCGTCACAATAAAGATAGAGGGGCAACTAAAGGAAGCTCTA
!TTAGATACAGGAGCAGATGATACAGTATTAGAAGAAAT
!
!A-0V-4-A
!TCACTCTTTGGCAACGACCCGTCGTCACAATAAAGATAGAGGGGCAACTAAAGGAAGCTCTA
!TTAGATACAGGAGCAGATGATACAGTATTAGAAGAAAT
!
!A-0V-7-A
!TCACTCTTTGGCAACGACCAGTCGTCACAATAAAGATAGAGGGGCAACTAAAGGAAGCTCTA
!TTAGATACAGGAGCAGATGATACAGTATTAGAAGAAAT
#In this example, the first few lines provide information about the data
in the sequence alignment. The following five lines contain the
Sequence IDs, followed by the sequences. Specifically, the sequence
identifier for the first sequence is A-0V-1-A. Note that subsequent
blocks of sequence also contain the Sequence ID. If one of the
sequences in your alignment is already present in the GenBank/EMBL/D
DBJ database, you must mark that sequence so that it does not receive a
new Accession number. To do this, use a SeqID in the format accU12345,
where U12345 is the Accession number of the pre-existing sequence.
#You can modify either NEXUS format so that Sequin can
determine the correct organism and any other modifiers for each
sequence. An example of such modifications are below in the section on
<A HREF="#SourceModifiersforPHYLIPandNEXUS">
Source Modifiers for PHYLIP and NEXUS
</A>
.
#Alternatively, you can leave your sequence alignment in
standard NEXUS format and enter the organism, strain, chromosome, etc.
information on the following
<A HREF="#SourceModifiersForm">
Source Modifers form
</A>
.
**Source Modifiers for PHYLIP and NEXUS
#You can modify the PHYLIP or NEXUS formats so that Sequin can determine
the correct organism and any other modifiers for each sequence by
adding lines at the end of the file. The first line applies to the
first sequence, the second line to the second sequence, and so on. You
must have one line for each sequence. These inserted lines contain
modifiers formatted like in the FASTA definition line, but do not begin
with a SeqID. Instead, the SeqID is present at the beginning of the
sequence lines as shown above.
#Each of the initial lines starts with the character ">". The
scientific organism name follows in brackets. Optional modifiers also
follow in brackets. For further information on the data that can go in
the lines preceding the sequences, see the instructions entitled "FASTA
Format for Nucleotide Sequences",
<A HREF="#FASTAFormatforNucleotideSequences">
above.
</A>
#The following lines indicating the organisms and strain of each sequence
would follow immediately after the sequence in the PHYLIP and NEXUS
examples, above.
!;
!END;
!
!begin ncbi;
!sequin
!>[organism=Gallus gallus] [clone=C]
!>[organism=Drosophila melanogaster] [strain=D]
!>[organism=Caenorhabditis elegans] [strain=E]
!>[organism=Rattus norvegicus] [strain=F]
!>[organism=Aspergillus nidulans] [strain=G]
!;
!end;
#The number of lines of source information must exactly match the number
of sequences provided. Complete examples can be found in the
<A HREF="http://www.ncbi.nlm.nih.gov/Sequin/QuickGuide/sequin.htm#AlignmentFormats">
Alignment Formats
</A>
section of the Sequin Quick Guide.
#Alternatively, you can leave your sequence alignment in
standard NEXUS or PHYLIP format and enter the organism, strain, chromosome, etc.
information on the following
<A HREF="#OrganismPage">
Organism Page
</A>
.
*Importing Aligned Sets of Segmented Sequences
#Sequin can also read segmented sets that are part of an alignment if
the sequences are in FASTA or FASTA+GAP format. Each segment should
have its own Sequence ID, but organism name and source modifiers should
only be indicated for the first segment from each sequence. Square
brackets are used to delimit the members of a set. For example,
![
!>A-0V-1-Apart1 [organism=Gallus gallus] [strain=C]
!TCACTCTTTGGCAAC
!>A-0V-1-Apart2
!GACCCGTCGTCATAATAAAGATAGAGGGGCAACTAAAGGAAGCTCTA
!TTAGATACAGGAGCAGATGATACAGTATTAGAAGAAAT
!]
![
!>A-0V-2-Apart1 [organism=Drosophila melanogaster] [strain=D]
!TCACTCTTTGGCAAC
!>A-0V-2-Apart2
!GAAGCGTCGTCACAATAAAGATAGAGGGGCAACTAAAGGAAGCTCTA
!TTAGATACAGGAGCAGATGATACAGTATTAGAAGAAAT
!]
>Nucleotide Page
#The options on this page will vary depending on the
<A HREF="#SubmissionType">
Submission Type
</A>
and
<A HREF="#SequenceDataFormat">
Sequence Data Format
</A>
selected earlier. Segmented sets and gapped sequences mut be imported
as properly formatted FASTA files. Details about importing alignment
files are
<A HREF="#NucleotidePageforAlignedDataFormats">
below
</A>
.
*Nucleotide Page for FASTA Data Format
**Create Alignment
#If you have selected a Population study, Phylogenetic study, Mutation
study, or Environmental samples set as a
<A HREF="#SubmissionType">
Submission Type
</A>
a check box will appear at the top of the Nucleotide Page. If you
check 'Create Alignment', Sequin will attempt to generate an alignment
of the seqeunces within your submission.
**Import Nucleotide FASTA
#Use this button to import your properly formatted
<A HREF="#FASTAFormatforNucleotideSequences">
FASTA file
</A>
. You will see a window containing information about the imported
sequence(s). Please check the number of sequences, Sequence IDs
(SeqIDs) and length of each sequence to make sure they are correct. If
you have included source information within the FASTA definition line,
this will also be listed.
**Add/Modify Sequences
#This option allows you to add or modify sequences without using a
previously formatted FASTA file, but is not available if you have
selected a Segmented sequence or Gapped sequence as a
<A HREF="#SubmissionType">
Submission Type
</A>
. On the Specify Sequences box you can either import a nucleotide FASTA
or add a new sequence. If you choose Add New Sequence, a new box will
pop-up where you can either import an existing sequence file or
directly paste or type the nucleotide sequence.
#If you add a sequence where the FASTA definition line is not properly
formatted a pop-up box will appear. The top box in this pop-up will
list any errors in the FASTA definition lines, including missing
SeqIDs, duplicate SeqIDs for different sequences, or improperly
formatted modifiers. You can add or edit this information in the
spreadsheet provided. The toggle at the bottom of the pop-up allows
you to select whether all sequences or only those with errors are
listed in the spreadsheet above. After making changes, click on
Refresh Error List to ensure that all errors have been corrected. You
must correct any errors involving the SeqID in order to proceed with
your submission. Click on Accept to save your sequences and return to
the Specify Sequences box.
#In the Specify Sequences box, you can choose to add another sequence or
select a sequence from the list and choose to edit or delete it. You
can also delete all sequences at this point. You will need to click on
Done to save your sequences and return to the Nucleotide Page.
**Clear Sequences
#This option will remove all imported nucleotide sequences.
**Specify Molecule
#A database sequence can represent one of several different molecule
types. The default molecule is genomic DNA. If the sequence was not
derived from genomic DNA, you can edit that information here. If you
are submitting multiple sequences you can apply one molecule type to
all sequences or apply the molecule type to each sequence individually.
Enter in the Molecule pop-up menu the type of molecule that was
sequenced.
#-Genomic DNA: Sequence derived directly from the DNA of an organism.
Note: The DNA sequence of an rRNA gene has this molecule type, as does
that from a naturally-occurring plasmid.
#-Genomic RNA: Sequence derived directly from the genomic RNA of certain
organisms, such as viruses.
#-Precursor RNA: An RNA transcript before it is processed into mRNA,
rRNA, tRNA, or other cellular RNA species.
#-mRNA[cDNA]: A cDNA sequence derived from mRNA.
#-Ribosomal RNA: A sequence derived from the RNA in ribosomes. This
should only be selected if the RNA itself was isolated and sequenced.
If the gene for the ribosomal RNA was sequence, select Genomic DNA.
#-Transfer RNA: A sequence derived from the RNA in a transfer RNA, for
example, the sequence of a cDNA derived from tRNA.
#-Small nuclear RNA: A sequence derived from small nuclear RNA, for
example, the sequence of a cDNA derived from snRNA.
#-Small cytoplasmic RNA: A sequence derived from small cytoplasmic RNA,
for example, the sequence of a cDNA derived from small cytoplasmic RNA.
#-Other-Genetic: A synthetically derived sequence including cloning
vectors and tagged fusion constructs.
#-cRNA: A sequence derived from complementary RNA transcribed from DNA,
mainly used for viral submissions.
#-Small nucleolar RNA: A sequence derived from small nucleolar RNA, for
example, the sequence of a cDNA derived from snoRNA.
**Specify Topology
#Most sequences have a Linear topology and this is the default. You
should change this setting to Circular only if the sequence is complete
and it has a circular topology. For example, a complete plasmid or a
complete mitochondrial genome would have a Circular topology, but a
single gene from a plasmid or mitochondrion would have a Linear
topology. If you are submitting multiple sequences you can apply one
topology to all sequences or set the topology for each sequence
individually.
*Nucleotide Page for Aligned Data Formats
**Sequence Characters
#If you are submitting a set of aligned sequences, you can specify sequence
characters used in your alignment here. Sequin requires that you
define any non-IUPAC nucleotide characters in your alignment file. The
five types of variable characters are listed under Sequence Characters.
#Every sequence within an alignment file must contain the same number of
characters (nucleotides + gaps). Gap characters are used to represent the
spaces between contiguous nucleotides in an alignment. Gaps that appear at
the beginning or end of a sequence are treated differently than gaps that
appear between nucleotides and each must be defined. GenBank prefers to
use a hyphen (-) to represent gaps. If you use a different character to
represent a gap, you will need to add this character to the list in the
Beginning Gap, Middle Gap, or End Gap boxes.
#Ambiguous characters represent nucleotides that are known to exist, but
whose identity has not been experimentally validated. GenBank prefers to
use 'n' to represent any ambiguous nucleotides. If you are using a
different character to represent an ambiguous base, you will need to add
this character to the list in the Ambiguous/Unknown box. Sequin will
convert these characters to 'n's when your file is imported.
#Match characters denote nucleotides that are identical in every member of
an alignment. GenBank prefers the use of a colon (:) to represent match
characters. If you are using a different character to represent a match
character, you will need to add this character to the list in the Match box.
**Import Nucleotide Alignment
#Once you have imported the alignment using the Import Nucleotide
Alignment button, you can edit the molecule information using the
<A HREF="#SpecifyMolecule">
Specify Molecule
</A>
and
<A HREF="#SpecifyTopology">
Specify Topology
</A>
buttons explained above. Note that you can not access the
<A HREF="#Add/ModifySequences">
Add/Modify Sequences
</A>
dialog for submissions of aligned sequences.
>Organism Page
#Information about the organism from which the sequence was derived
should be entered or edited on this page. If there are any potential
problems with the organism information previously provided in either
the
<A HREF="#FASTAFormatforNucleotideSequences">
FASTA definition line
</A>
or entered in the
<A HREF="#Add/ModifySequences">
Add/Modify Sequences
</A>
dialog, a window listing these problems will appear at the top of the
form. Please review these problems and edit using the
<A HREF="#AddSourceModifiers">
</A>
Add Source Modifiers button as necessary. At minimum, you must supply
the scientific name of the organism from which the sequence was
obtained in order to proceed with your submission.
#The second window is a summary of the organism information provided so
far. Double clicking on a line of text within this window will launch a
modifier-specific editing window. In each of these windows, you can
edit the available information for the specific modifier. In most
cases, you have the choice to edit the modifier for each sequence
separately, or to enter text and select Apply above value to all
sequences. These changes will be reflected in the windows of the
Organism page immediately upon closing the modifier-specific editor.
*Add Organisms, Locations, and Genetic Codes
#If you have not added organism information using either the
<A HREF="#FASTAFormatforNucleotideSequences">
FASTA definition line
</A>
or the
<A HREF="#Add/ModifySequences">
Add/Modify Sequences
</A>
dialog, you can use the Add Organisms, Locations, and Genetic Codes to
do so at this point. This button will launch the Multiple Organism
Editor pop-up where you may add or edit existing information concerning
the
<A HREF="#Organism">
Organism
</A>
name,
<A HREF="#Location">
Location
</A>
and
<A HREF="#GeneticCode">
Genetic Code
</A>
. The SeqID of each sequence is listed at the left of the spreadsheet
format. You can change the information in the spreadsheet individually
or globally for all sequences.
**Organism
#The scrollable list at the top of the pop-up contains the scientific
names of many organisms. To reach a name on the list, type the first
few letters of the scientific name into the box above the list or the
appropriate box in the spreadsheet. The list will scroll to the names
beginning with those letters, and you can select the organism within
the list itself. You can then use the arrow button to copy this name
into the appropriate box in the spreadsheet.
#To apply the same scientific name to all sequences in the submission,
click on the Organism button in the spreadsheet column header. A
separate pop-up box will appear with the same organism list. You can
select a name from this list and choose Accept to apply this name to
all sequences.
#If you have any questions about the scientific name of an organism, see
the NCBI
<A HREF="http://www.ncbi.nlm.nih.gov/Taxonomy/tax.html">
Taxonomy Browser
</A>
http://www.ncbi.nlm.nih.gov/Taxonomy/tax.html
#If the name of the organism is not on the list, type it in directly. If
you do not know the scientific name, please be as specific as you can
and include a unique identifier, such as a clone, isolate, strain or
voucher number, or cultivar name, e.g.; Nostoc ATCC29106, uncultured
spirochete Im403, Lauraceae sp. V�squez 25230 (MO), Rosa hybrid
cultivar 'Kazanlik'. Also, if applicable, indicate if the name is
unpublished as of the time of submission. Additional information such
as strain, isolate, or serotype can be entered later in the submission
process.
**Location
#The default Location for all seqeunces is "Genomic". If the sequence
is not genomic, select the alternative location (ie, organelle) from
the pull-down list. You can change the location of all sequences
globally by clicking on the Location button in the spreadsheet header.
The following is a brief description of the choices in this list:
#-Genomic: chromosome. This category includes
mitochondrial and chloroplast proteins that are encoded by the nuclear
genome.
#-Chloroplast: a chlorophyllous plastid.
#-Chromoplast: a non-chlorophyllous, pigmented plastid, found in
fruits and flowers.
#-Kinetoplast: a specialized type of mitochondrion found exclusively
in Kinetoplastida (e.g., Leishmania). NOTE: kinetoplast should
be applied ONLY to members of the Kinetoplastida (trypanosomes and
bodonids).
#-Mitochondrion: a semi-autonomous, self-reproducing organelle that
occurs in the cytoplasm of most eukaryotic cells.
#-Plastid: any of a class of double membrane-bound, light-harvesting
organelles (or derived from same). NOTE: plastid should be used
ONLY when a more precise term, e.g., chloroplast, is not
applicable.
#-Macronuclear: a specialized type of nucleus found exclusively in the
ciliated protists (e.g., Tetrahymena). NOTE: macronucleus
should be applied ONLY to members of the Ciliophora.
#-Extrachromosomal: other extrachromosomal elements not listed here,
such as a B chromosome or an F factor.
#-Plasmid: extrachromosomal genetic element found in bacterial species.
Note this does not include the cloning vector used to propagate
the sequence of interest.
#-Cyanelle: a specialized type of plastid found exclusively in
glaucocystophytes (e.g., Cyanophora). NOTE: cyanelle should be
applied ONLY to members of the Glaucocystophyceae.
#-Proviral: a virus that is integrated into a host cell chromosome.
#-Virion: a completed virus particle.
#-Nucleomorph: a reduced nuclear remnant found in Chlorarachniophyceae
(e.g., Chlorarachnion) and Cryptophyta (e.g, Cryptomonas). NOTE:
nucleomorph should be applied ONLY to members of the
Chlorarachniophyceae or Cryptophyta.
#-Apicoplast: a reduced plastid characteristic of apicomplexans
(e.g., Plasmodium). NOTE: apicoplast should be applied ONLY to
members of the Apicomplexa.
#-Leucoplast: a plastid lacking pigments of any type.
#-Proplastid: an immature plastid.
#-Endogenous_virus: a virus that has integrated permanently into the
host genome, and which is inherited vertically through the
germ-line of the host.
#-Hydrogenosome: an organelle that produces hydrogen and ATP and is
found mainly in ciliates, fungi and trichomonads. Hydrogenosomes may
be reduced mitochondria
**Genetic Code
#If you selected a scientific organism name from the scrollable list
described above, this field will be filled out automatically. However,
if the organism is not on the list, this field will default to the
"Standard" genetic code. If this is incorrect, you can select the
correct genetic code from the pull-down list. To globally change the
genetic code for all sequences which are not automatically filled out,
click on the Genetic Code button in the spreadsheet header.
#For more information regarding the genetic codes available, see the NCBI
<A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wprintgc?mode=c">
Taxonomy page
</A>.
http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wprintgc?mode=c
*Import Source Modifiers
#Using this button allows you to import a tab-delimited table of source
modifiers. The first column in the table must contain the Sequince
Identifiers (SeqIDs) used earlier in the submission and each subsequent
column must contain a different source modifier. The first row in the
table must contain the labels for each column. The label for the
Sequence Identifiers column should be in the format "Seq_ID". A list
of available
<A HREF="http://www.ncbi.nlm.nih.gov/Sequin/modifiers.html">
modifiers
</A>
in the format to be used in the column headers can be found at
http://www.ncbi.nlm.nih.gov/Sequin/modifiers.html .
*Add Source Modifiers
#Using this button will launch the Specify Source Modifiers pop-up box
where you can add or edit any source modifier. You can also import a
source modifier table or export the existing source modifiers in table
format from this page.
#The Select Modifier dialog allows you to select a modifier from the
pull-down list and edit the value of this modifier for each sequence or
globally add a value to all sequences.
#The two windows in this pop-up provide information about the current
source modifiers for the sequences in your submission. The top window
provides a summary of these modifiers and the lower window lists the
values of each modifier for each sequence. If any sequences have
missing organism names or have source information that is identical to
another sequence, the SeqIDs will be shown in red in this window.
Double-clicking on a modifier value in this window will launch a pop-up
where you can edit this value. Double-clicking on the modifier name
used in the header will launch a modifier-specific pop-up where you can
globally edit the modifier value for all sequences or change the value
for individual sequences.
*Clear All Source Modifiers
#This button will clear all modifiers previously entered in either the
FASTA definition lines or the submission dialogs. This includes the
organism name which is required for submission.
>Protein Page
#This page allows you to provide the protein sequence translated from
the nucleotide sequence that you just entered. If the nucleotide
sequence is alternatively spliced or contains multiple open reading
frames, enter all of the protein sequences on this page. Each protein
sequence will appear in the database record as a coding sequence (CDS)
feature. Sequin will automatically determine which nucleotide
sequences code for the protein and indicate the nucleotide sequence
interval on the database record. Sequin also provides tools that allow
you to view a graphical representation of all the open reading frames
in your nucleotide sequence and to convert these reading frames into
CDS features. These tools are described later in the help
documentation under the
<A HREF="#ORFFinder">
ORF Finder.
</A>
*Conceptual Translation Confirmed by Peptide Sequencing
#Most protein entries are computer-generated conceptual translations of
a nucleic acid sequence. If you have confirmed this translation by
direct sequencing either of the entire protein or of peptides derived
from the protein, please check this box.
*Incomplete at NH3 end/Incomplete at COOH end
#If the sequence is lacking amino acids at the amino- or
carboxy-terminal end of the protein, please check the appropriate box.
*Create Initial mRNA with CDS Intervals
#If you check this box, Sequin will make an mRNA feature with the same
initial intervals (i.e., range of sequence) as the CDS feature. After
the record has been assembled, you should edit the mRNA feature location
to add the 5' UTR and 3' UTR intervals. This may be done either in the
mRNA editor or in the sequence editor.
*Import Protein FASTA
#You can import a single or multiple protein sequences contained within
a previously generated protein FASTA file.
**FASTA Format for Protein Sequences
#The basic FASTA format is the same as that used for
<A HREF="#FASTAFormatforNucleotideSequences">
nucleotide sequences
</A>
, with a FASTA definition line followed by the sequence itself.
#In order to match the protein sequence to the correct nucleotide
sequence, you must use the same Sequence Identifier (SeqID) that you
used to identify the nucleotide sequence. Thus in cases of
alternatively spliced genes, a single protein FASTA file can contain
two unique sequences that have the same SeqID. Both coding regions
will be added to the same nucleotide sequence.
#The available modifiers for use in a protein FASTA definition line are
different than those for a nucleotide FASTA definition line and are
limited to information about the protein or gene itself and are
contained within the examples below. The format remains [modifer=text].
#Note in all cases, the FASTA definition line must not contain any hard
returns. All information must be on a single line of text.
#Examples of properly formatted protein FASTA definition lines are:
<KBD><PRE>>Seq1 [protein=neuropilin 1] [gene=Nrp1]</KBD></PRE>
<KBD><PRE>>ABCD [protein=merozoite surface protein 2] [gene=msp2] [protein_desc=MSP2]</KBD></PRE>
<KBD><PRE>>DNA.new [protein=breast and ovarian cancer susceptibility protein] [gene=BRCA1] [note=breast cancer 1, early onset]</KBD></PRE>
#The protein name should be included in the entry; all other fields are
optional.
#The line after the FASTA definition line begins the amino acid
sequence. It is recommended that each line of sequence be no longer
than 80 characters. Please only use IUPAC symbols within the amino
acid sequence. Non-IUPAC amino acid symbols will be stripped from the
sequence.
#After you import your sequence, a window will appear with information
about the sequence. The first line will describe the number of protein
sequences imported and the total length in amino acids of
all sequences. Each sequence is numbered, and its length,
unique identifier (SeqID), Gene symbol, Protein name, and title
(Definition line) as supplied in the FASTA definition line are listed.
>Annotation Page
#Note: This page will not be available if you have selected a segmented
or gapped sequence as the
<A HREF="#SubmissionType">
Submission Type
</A>
.
#On this page, you can add a
<A HREF="#gene">
gene
</A>
,
<A HREF="#rRNA">
ribosomal RNA
</A>
or
<A HREF="#CDS">
CDS
</A>
feature across the entire span of each sequence you are submitting.
You can not specify locations within each sequence using this page.
More options are available under the
<A HREF="#AnnotateMenu">
Annotate Menu
</A>
in the record viewer.
#If the feature should be partial at one or both ends, check the
appropriate box and then fill in the text boxes for the relevant
feature.
#You may add a title to all sequences if this was not included in the
FASTA definition line. This will be used as the DEFINITION field in
the final flatfile. The title should contain a brief description of
the sequence. There is a preferred format for nucleotide and protein
titles and Sequin can generate them automatically using the Generate
Definition Line function under the Annotate menu in the record viewer.
>Assembly Tracking
#You will only see this form if you had previously indicated that the
entry is a Third-Party Annotation submission. You must provide the
GenBank Accession number(s) of the primary sequence used to assemble
your TPA sequence. We can not accept primary sequences corresponding
to Reference Sequences or those from proprietary databases. More
information about this can be found on the
<A HREF="http://www.ncbi.nlm.nih.gov/Genbank/tpa.html">
TPA
</A>
home page.
#If a proper GenBank Accession is entered in the first column of the
Assembly Tracking form, the GenBank staff can map the coordinates for
you. You do not need to fill out the 'from' and 'to' columns. Note
that multiple accessions may be entered to provide full coverage of the
assembled sequence.
#If the accession entered is not recognized as a GenBank Accession
number, a pop-up box is generated requesting that you edit the numbers
listed. Sequences from the trace archive can be used primary sequence
data for TPA records but must be entered in the format "TI123456789".
#You may also generate an Assembly Tracking form in the record viewer
under the Annotate menu. Select Descriptors and TPA Assembly from the
pull-down menu in order to generate the Assembly Tracking form.
>Editing the Record
*Overview
#After you finish the Organism and Sequences Form, Sequin will process
your entry based on the information you have entered. The window you
see now is called the record viewer. This is also the window you will
see if you are submitting an update to an existing record. The
instructions after this point are the same whether you are submitting a
new record or an update.
#In the default window of the record viewer, you will see your entry
approximately as it would appear in the database. Most of the
information that you entered earlier in the submission process is
present in the viewer; other information, such as the contact, is still
present in the record but will not be visible in the database entry. If
you have provided a conceptual translation of the nucleotide sequence,
the translation will be listed as a CDS Feature. Sequin automatically
determines which nucleotides encode for the protein, and lists them,
even if the nucleotide sequence contains introns and exons.
#You can save the entry to a file by selecting Save or Save As under the
File menu. This is not the same as saving the entry for submission to
the database. It is a good idea to save the file at this point so that
if you make any unwanted changes during the editing process you can
revert to the original copy. If you wish to edit the entry later, click
on "Read Existing Record" on the Welcome to Sequin form and choose
the file.
#It is likely that the entry could be processed now for submission to
the database. However, you may wish to add information to
the entry. This information may be in the form of Descriptors or
Features. Descriptors are annotations that apply to an
entire sequence, or an entire set of sequences, and Features are
annotations that apply to a specific sequence interval. For example,
you may want to change the Reference Descriptor to add a published
manuscript, or to annotate the sequence by adding features such as a
signal peptide or polyA signal.
#Information in the record viewer can be edited in different ways. One
way to modify information is to double click within the block of
information you wish to edit. Many blocks, such as "Definition",
"Source", "Reference", or "Features" can be edited.
#To add information, create a new descriptor
or feature by selecting the appropriate form from the Misc or Features
menus. These options are described later in this help document.
#Finally, you may need to edit the sequence itself.
<A HREF="#SequenceEditor">
Instructions
</A>
for working with the sequence are presented in the documentation for the
Sequence Editor.
*Submitting the Finished Record to the Database
#Once you are satisfied that you have added all the appropriate
information, you must process your entry for submission to the database.
Select "Validate" under the Search menu. This function detects
discrepancies between the format of your submission and that required by
the database selected for entry.
#If Sequin detects problems with the format of your record, you will see a
screen listing the validation errors as well as suggestions for how to fix the
discrepancies. Single clicking on an error message scrolls the record viewer
to the feature that is causing the error. Double clicking on the error
message launches the relevant feature editor on which you can correct the
problem. If you are annotating a set of multiple sequences, shift-click to
scroll to the target sequence and feature. When you think you have corrected
all the problems, click on "Revalidate". You can submit files with errors,
but it is strongly recommended that you correct as many errors as possible
prior to submission.
#Message: Select Verbose, Normal, Terse, or Table. Verbose gives a more
detailed explanation of the problem.
#Filter: Select the error messages you wish to see. You can select
ALL, SEQ_INST (errors regarding the sequence itself, its type, or
length), SEQ_DESCR (descriptor errors), SEQ_FEAT (feature errors), or
errors specific to your record.
#Severity: Select the types of error messages you wish to see. You
will see the type of message selected, as well as any messages warning
of more serious problems.
#There are four types of error messages, Info, Warning, Error, and
Reject. Info is the least severe, and Reject is the most severe. You
may submit the record even if it does contain errors. However, we
encourage you to fix as many problems as possible. Note that some
messages may be merely suggestions, not discrepancies. A possible
Warning message is that a splice site does not match the consensus.
This may be a legitimate result, but you may wish to recheck the
sequence. A possible Error message is that the conceptual translation
of the sequence that you supplied does not encode an open reading
frame. In this case, you should check that you translated the sequence
in the correct reading frame. A possible Reject message is that you
neglected to include the name of the organism from which the sequence
was derived. The name of the organism is absolutely required for a
database entry.
#If Sequin does not detect any problems with the format of your record,
you will see a message that "Validation test succeeded".
#To prepare the submission, click the "Done" button on the record
viewer, or select "Prepare Submission" under the File menu. You will be
prompted to save the file. Email this file to the database at the
address shown. You MUST email the file; Sequin does not submit the
file automatically over the network. The email addresses for the
databases are:
!-GenBank: gb-sub@ncbi.nlm.nih.gov
!-EMBL: datasubs@ebi.ac.uk
!-DDBJ: ddbjsub@ddbj.nig.ac.jp
#After your entry is complete, close the record viewer. You will be
returned to the Welcome to Sequin form and can begin another entry.
>The Record Viewer
*Target Sequence
#This pop-up menu shows a list of SeqIDs of all nucleotide and protein
sequences associated with the Sequin entry. Use the menu to select the
sequences displayed in the record viewer, as well as the sequences you
want to "target", that is, the sequences to which you want to apply a
descriptor (see
<A HREF="#Descriptors">
Descriptors
</A>
in the Sequin help documentation). You may select either an individual
sequence by name or a set of sequences, such as All Sequences, or
SEG_dna if you have a segmented nucleotide set. You may change the
selection at any time.
*Display Format
#You may change the display format of the record viewer to any of the
formats described below. Editing a field in one display format will
change that field in all formats. Subsequent pop-up menus will appear
depending on which format is selected.
**GenBank
#This display format allows you to see the submission as it would appear
as a GenBank or DDBJ entry. It is the default format.
#The Mode pop-up default setting is Sequin. Release mode shows certain
qualifiers and db_xrefs in RefSeq entries which are non-collaborative.
Entrez mode is used fro web display and can show new elements that have
not yet finished their four month quarentine period. Dump mode requires
that the accession slot be populated. In most cases, there is no need
to change from the default Sequin mode.
#The Style pop-up allows different views of segmented records. The
default is Normal. Segment style is the traditional representation of
segmented sequences, while Contig style displays a CONTIG line with a
join of accessions instead of raw sequence. Master style shows
features mapped to the segmented sequence coordinates instead of the
coordinates of the individual parts.
**Graphic
#This display format shows the entry in a graphical view. The top bar
represents the nucleotide sequence. Lower arrows or bars represent
different features on the sequence. Double click on an arrow or bar to
launch the appropriate editing window. Any sequence highlighted in the
Sequence Editor will be boxed on the graphical view of the sequence.
To see a graphical representation of a segmented set (see
<A HREF="#Submissiontype">
Submission type
</A>,
above), the Target Sequence must be set to
SEG_dna.
#The Style pop-up menu allows you to see the display in different styles
and colors.
#The Scale pop-up menu allows you to see the display in different sizes.
The smaller the number, the larger the display.
**Sequence
#This display format shows the nucleotide sequence in the record along
with any annotated features (such as CDS or mRNA). You can only view a
single sequence at a time with this option. You can use the Features
pop-up menu to change the display of the features. With the numbering
pop-up menu, select where you want the sequence numbers to be
indicated, at the side of the sindow, at the top of each sequence line,
or not at all.
**Alignment
#This display format shows sets of aligned sequences, such as those
imported as part of a population, phylogenetic, mutation, or
environmental samples set. When toggled to All Sequences in the Target
Sequence pop-up, the alignment of all entries will be displayed. To
more closely analyze similarities, you can select a single entry in the
Target Sequence pop-up. The complete sequence of the entry selected
will be displayed. Any nucleotides in the other sequences that differ
from that selected will be displayed, while identical nucleotides will
be displayed as a period. You can also display features annotated on
the selected target sequence or all sequences using the Feature display
toggle. To launch the alignment editor, select
<A HREF="#AlignmentAssistant">
Alignment Assistant
</A>
from the record viewer Edit menu.
**EMBL
#This display format allows you to see the submission as it would appear
as an EMBL entry.
**Table
#This display format shows the annotation in a five-column, tab-delimited
<A HREF="table.html">table</A>
format. This format can be imported to add annotation to a record that
has none.
**FASTA
#This display shows the sequence and Definition line only, without any
annotations, in a format called the FASTA format. This is a format used
by many molecular biology analysis programs. You cannot edit in this
display mode.
**Quality
#This display format shows quality score data ifit has been included in
the submission.
**ASN.1
#This display shows the entry in Abstract Syntax Notation 1, a data
description language used by the NCBI. You cannot edit in this display
mode.
**XML
#This display format shows the entry in XML language, sometimes used by
various databases. You cannot edit in this display mode.
**INSDSeq
#This display format shows the entry in the XML format used by the INSD.
You cannot edit in this display mode.
**Desktop
#The NCBI DeskTop displays the internal
structure of the record being viewed in Sequin. The
<A HREF="#NCBIDeskTop">
DeskTop
</A>
is explained under the Misc menu.
*Done
#This button allows you to validate the entry when you are finished with
the submission. See
<A HREF="#SubmittingtheFinishedRecordtotheDatabase">
Submitting the Finished Record to the Database
</A>
in the Sequin help documentation.
*Controls for Downloaded Entries
#If you have downloaded a sequence from Entrez, you will see an
additional button labeled PubMed. This button will launch a web
browser containing the target sequence as it appears in Entrez. From
here, you can access any Entrez-supported Links, including related
sequences and associated references in PubMed.
>Descriptors
*Overview
#Descriptors are annotations that apply to an entire sequence, or an
entire set of sequences, in a given entry. They do not have a specific
location on a sequence, as they apply to the entire sequence. They can
be contrasted to
<A HREF="#Features">
Features,
</A>
which apply to a specific interval of the sequence.
#You may edit descriptors in one of two ways.
#(1) In the record viewer, double click within the text of the
descriptor to bring up a form on which information can be added.
#(2) Choose the option Descriptors from the Annotate menu.
*Annotate Menu
#This menu allows you either to create new descriptors or to modify
existing ones. Select the descriptor that you wish to modify.
#When you first select a descriptor, you will see a window called
"Descriptor Target Control". Using the target control pop-up menu,
select the sequences you wish this descriptor to cover. The name(s)
listed correspond to the SeqID(s) given to the nucleotide or amino acid
sequences when when they were imported into Sequin. The default
selection for this menu is set in the Target Sequence pop-up menu on
the record viewer. You may choose to have the descriptor cover just
one sequence, or a set of sequences in your entry. If you are creating
a new descriptor, select "Create New". If you wish to modify a
previous descriptor, select "Edit Old".
#The following is a list of some of the descriptors that can be added.
Two additional descriptors, those for
<A HREF="#Publications">
Publications
</A>
and
<A HREF="#BiologicalSourceDescriptororFeature">
Biological Source,
</A>
are described in other sections.
**TPA Assembly
#If you indicated that your sequence is a TPA submission, a
<A HREF="#AssemblyTracking">
TPA Assembly
</A>
was created from the information regarding primary accession numbers.
This Assembly information can be edited here. Note that it is not
necessary to enter nucleotide location in the "from" and "to" columns.
**Update Date
#This is for database staff use only. Please do not modify the date.
**Create Date
#This is for database staff use only. Please do not modify the date.
**Region
#This descriptor provides general information about the genetic context
of the sequence. For example, if your nucleotide sequence is cloned
from the region surrounding the Huntington's Disease gene, you could
enter that information here. Providing information for this descriptor
is optional.
**Name
#Alternative place for a descriptive name for the sequence. This
information will not appear in the flatfile view, but will be
maintained in the ASN1.
**Comment
#This descriptor is used to list any additional information that you
wish to provide about the sequence. Use of this descriptor is optional.
Most information can be better annotated using the appropriate
features and qualifiers rather than a generic comment descriptor.
**Title
#This descriptor contains the information that will go on the Definition
line of the database entry. If you supplied a title for your
nucleotide sequence when you imported it into Sequin, that information
is here. If you wish to change the Definition line, or if you did not
supply a title when you submitted the sequence, edit this Descriptor.
For more information on creating proper Definition lines, please see
the Sequin help documentation for the
<A HREF="#NucleotideDefinitionLine(Title)">
Nucleotide Definition Line (Title)
</A>.
**Molecule Description
#This descriptor indicates the characteristics of the molecule from
which the sequence was derived. The information that you have already
entered can be edited here. In most cases, the molecule and class are
the only choices which should be edited from the default values.
***Molecule
#A GenBank sequence can represent one of several different molecule
types. Enter in the Molecule pop-up menu the type of molecule that was
sequenced. A brief description of the choices in this pop-up menu were
listed previously.
***Completedness
Choose the appropriate option from the pop-up menu.
#-Complete: Use this designation when a complete molecule, such as a
complete mitochondrial genome, is being submitted.
#-Partial: Use this designation when an incomplete unit, such as the
partial coding sequence of a gene, is being submitted.
#-No left: Use this designation when an incomplete unit, such as the
partial coding sequence of a gene, or a partial protein sequence, is
being submitted. The sequence has no left if it is incomplete on the
5', or amino-terminal, end.
#-No right: Use this designation when an incomplete unit, such as the
partial coding sequence of a gene, or a partial protein sequence, is
being submitted. The sequence has no right if it is incomplete on the
3', or carboxy-terminal, end.
#-No ends: Use this designation when an incomplete unit, such as the
partial coding sequence of a gene, or a partial protein sequence, is
being submitted, The sequence has no ends if it is incomplete at both
the 5' and 3', or amino- and carboxy- terminal, ends.
#-Other: Use this designation when none of the above descriptions apply.
***Technique
#From the pop-up menu, select the technique that was used to generate the
sequence.
#-Standard: standard sequencing technique.
#-EST:
<A HREF="http://www.ncbi.nlm.nih.gov/dbEST/index.html">
Expressed Sequence Tag
</A>
: single-pass, low-quality mRNA sequences
derived from cDNAs. These sequences will appear in the EST division.
#-STS:
<A HREF="http://www.ncbi.nlm.nih.gov/dbSTS/index.html">
Sequence Tagged Site
</A>
: short sequences that are operationally
unique in a genome and that define a specific position on the physical
map. These sequences will appear in the STS division.
#-Survey:
<A HREF="http://www.ncbi.nlm.nih.gov/dbGSS/index.html">
single-pass genomic sequence
</A>
. These sequences will appear in
the Genome Survey Sequence (GSS) division.
#-Genetic Map: Genetic map information, for example, in the Genomes division.
#-Physical Map: Physical map information, for example in the Genomes division.
#-Derived: A sequence assembled into a contig from shorter sequences.
#-Concept-trans: A protein translation generated with the appropriate
genetic code.
#-Seq-pept: Protein sequence was generated by direct sequencing of a
peptide.
#-Both: Protein sequence was generated by conceptual translation and
confirmed by peptide sequencing.
#-Seq-pept-Overlap: Protein sequence was generated by sequencing
multiple peptides, and the order of peptides was determined by overlap
in their sequences.
#-Seq-pept-Homol: Protein sequence was generated by sequencing
multiple peptides, and the order of peptides was determined by homology
with another protein.
#-Concept-Trans-A: Conceptual translation of the nucleotide sequence
provided by the author of the entry.
#-HTGS 0:
<A HREF="http://www.ncbi.nlm.nih.gov/HTGS/">
High Throughput Genome Sequence
</A>
, Phase 0. These sequences
are produced by high-throughput sequencing projects and will be in the
HTG division.
#-HTGS 1:
<A HREF="http://www.ncbi.nlm.nih.gov/HTGS/">
High Throughput Genome Sequence
</A>
, Phase 1. These sequences
are produced by high-throughput sequencing projects and will be in the
HTG division.
#-HTGS 2:
<A HREF="http://www.ncbi.nlm.nih.gov/HTGS/">
High Throughput Genome Sequence
</A>
, Phase 2. These sequences
are produced by high-throughput sequencing projects and will be in the
HTG division.
#-HTGS 3:
<A HREF="http://www.ncbi.nlm.nih.gov/HTGS/">
High Throughput Genome Sequence
</A>
, Phase 3. These sequences
are produced by high-throughput sequencing projects and will be in the
HTG division.
#-FLI_cDNA: Full Length Insert cDNA. Sequence corresponds to entire cDNA but
not necessarily entire transcript. These sequences are produced by large
sequencing projects.
#-HTC: High Throughput cDNA. These sequences are produced by large sequencing
projects.
#-WGS:
<A HREF="http://www.ncbi.nlm.nih.gov/Genbank/wgs.html">
Whole Genome Shotgun
</A>
. These sequences are produced by large sequencing projets and follow a
separate submission process.
#-Barcode: Nucleotide sequence is part of Barcodes of Life project. This
selection should only be used by members of the Consortium for the
Barcodes of Life.
#-Other: Do not use this designation.
***Class
#From the pop-up menu, select the type of molecule that was sequenced.
#-DNA: DNA
#-RNA: RNA
#-Protein: Protein
#-Nucleotide: Do not select this item
#-Other: Do not select this item
***Topology
#From the pop-up menu, select the topology of the sequenced molecule.
#-Linear: Linear molecule (most sequences).
#-Circular: Circular molecule (such as a complete plasmid or mitochondrion).
#-Tandem: Do not select this item.
#-Other: Do not select this item.
***Strand
#From the pop-up menu, select whether the sequence was derived from an
organism with a single- or double-stranded genome. This is used primarily for
viral submissions.
#-Single: The organism contains only a single-stranded genome, for
example, ssRNA viruses.
#-Double: The organism contains only a double-stranded genome, for
example, dsDNA viruses.
#-Mixed: Do not select this item.
#-Mixed Rev: Do not select this item.
#-Other: Do not select this item.
**Biological Source
#The Biological Source descriptor is described in more detail
<A HREF="#BiologicalSourceDescriptororFeature">
below.
</A>
>Features
*Overview
#Features are annotations which apply to one or more intervals on a
sequence. They can be contrasted to
<A HREF="#Descriptors">
Descriptors,
</A>
that apply to an entire sequence or an entire set of sequences.
Features will be added to the Target Sequence selected in the record
viewer pop-up menu.
#You may add or modify features in one of three ways.
#(1) In the record viewer, double click on the text of an existing
feature to bring up a form on which information can be added or edited.
#(2) Choose the feature from the Annotate menu to add a new feature.
#(3) Choose the feature from the Sequence Editor Features menu to add a
new feature.
#The features listed in the Annotate menu and the Sequence Editor
Features menu are identical, and the instructions for adding them are
the same, with one exception. If you annotate them in the Annotate
menu, you must provide the nucleotide sequence location of the feature.
However, if you add features from the Sequence Editor, you can
highlight the sequence that the feature covers, and the location of the
sequence will be automatically entered in the feature location box.
*Annotate Menu
#This menu allows you to add or modify features on the sequence selected
in the Target Sequence pop-up menu of the record viewer. Features are
grouped into six categories. Select the feature that you would like to
mark on your sequence. A new form will appear.
#Feature forms share a common design. The first page is specific to the
particular feature, e.g., Coding Region or Gene. The second page lists
Properties of the Feature. The third page describes the Location of the
feature. Details about the common second and third pages are provided
below.
**Properties Page
***General Subpage
#Enter general comments about the feature here.
#Select any of the flags if necessary. If this sequence contains only a
partial representation of the feature you are describing, check the
"Partial" box. Check the "Exception" box if the feature annotates a
post-transcriptional modification of the nucleotide sequence, such as
ribosomal slippage or RNA editing. This is generally used only on CDS
features. The evidence dialogs will only be editable if information
has been entered in the Evidence subpage.
#If a gene feature overlaps the feature you are editing, the gene symbol
will appear in the pull-down menu. If you want to add the name of a
new gene, select new, and enter its name and optional description. By
default, mapping between the feature and the gene is done by overlap,
that is, the gene associated with the feature is the gene whose
location overlaps with the location of the feature. Under some
circumstances, for example, if the sequences of two genes overlap, you
may wish the feature to apply to a different gene. In this case,
select cross-reference, and select the name of the new gene in the
pop-up menu. If you do not want the feature to map to any existing
gene, select suppress. You may also edit information on the Gene
feature form by clicking on Edit Gene Feature.
***Comment Subpage
#Add any comments about the feature here, especially if you checked the
"Exception" box on the General Subpage.
***Citations Subpage
#This page is used to list any citations that specifically apply to the
feature you are annotating. The citation must have already been entered
into the record (see
<A HREF="#Publications">
Publications
</A>)
in the Sequin help documentation. Click on Edit Citations, and
place a check mark in box next to the publication you want to cite.
However, we discourage the use of citations on features.
***Cross-Refs Subpage
#This is a read-only page used to cross-reference this entry to entries
in external databases (databases other than GenBank, EMBL/EBI, and
DDBJ), such as dbEST or FLYBASE. For more information on this topic,
see the International Nucleotide Sequence Database Collaboration
<A HREF="http://www.ncbi.nlm.nih.gov/collab/db_xref.html">
page
</A>.
http://www.ncbi.nlm.nih.gov/collab/db_xref.html
***Evidence Subpage
#This page is primarily used by large sequencing centers to explain
annotation prediction methods and its use is optional. More details
about these qualifiers can be found in the
<A HREF="http://www.ncbi.nlm.nih.gov/GenBank/evidence.html">
genome submission guidelines
</A>.
The two choices of evidence are Experiment or Inference.
#Wet-bench, experimental evidence can be entered as free text in the
Experiment section. Please be as brief as possible.
#The Inference section allows for information to be added in cases where
the feature is annotated based solely on sequence similarity or
prediction software. In order to fill in text, you must select one of
the options from the Category pull-down menu. Different pull-down and
text boxes will appear depending on the selection you choose from the
Category menu. If you select one of the 'similar to' categories, you
must include the name of the database and the corresponding accession
number of the sequence used as the basis for the annotation. If you
choose one of the prediction categories, you must include the name and
version of the prediction program used as the basis for the annotation.
#For example, if your annotation of a coding region was based on
similarity to the sequence and annotation in GenBank Accession number
AY411252, you would select "similar to DNA sequence" from the pull-down
menu and then select "INSD" in the Database pull-down. You would then
type "AY411252.1" in the Accession text box. If the annotation is
based on the Genscan prediction algorithm, you would select "ab initio
prediction" from the pull-down menu, select "Genscan" in the Program
pull-down and enter 2.0 in the Program Version text box. If the
database or program used is not listed in the appropriate pull-down
list, select Other from the list. A new text box will appear where you
can enter the name of the database or program used. You still must
include the appropriate accession number or version in the subsequent
text box.
***Identifiers Subpage
#This is a read-only page used by the database staff for tracking
features within the record.
**Location Page
#This page allows you to select the location of the feature you are
citing. Each feature must have a sequence interval associated with it.
In most cases, Sequin will limit the option to the nucleic acid or
protein sequence as appropriate.
#Check the 5' Partial or 3' Partial box if the feature in your nucleic
acid sequence is missing residues at the 5' or 3' ends, respectively.
Check the NH2 Partial or COOH Partial if the feature in your amino acid
sequence is missing residues at the amino- or carboxy-terminal ends,
respectively. If you checked "Partial" on the Properties page, you
must check either the 5' and/or 3' partial boxes.
#Enter the sequence range of the feature. The numbers should correspond
to the nucleotide sequence interval if the SeqID is set to a nucleotide
sequence, and to an amino acid sequence interval if the SeqID is set to
a protein sequence. If the feature spans multiple, non-continuous
intervals on the sequence, indicate the beginning and end points of each
interval. If each interval is separate, and should not be joined with
the others to describe the feature, check the Intersperse intervals with
gaps box (for example, when annotating multiple primer binding sites).
If the feature is composed of several intervals that should all be
joined together, do not check the box (for example, when annotating mRNA
on a genomic DNA sequence).
#For nucleic acid Features only: From the pop-up menu, select the
strand on which the feature is found.
#-Plus: Plus strand, or coding strand.
#-Minus: Minus strand, or non-coding strand.
#-Both: Both strands.
#-Reverse: Do not select this item.
#-Other: Do not select this item.
#Use the pop-up menu to select the SeqID of the sequence you are
describing by the location. Clicking on the X button to the left will clear
location spans, strand, and SeqID from that row.
#If you are working on a set of sequences which contain an alignment,
you will see a toggle at the bottom of the Location Page where you can
select to add or view the location of the feature using the Sequence
Coordinates of the target sequence or the Alignment Coordinates. In
either case, the feature will only be added to the target sequence. If
you want to add features to all members of the set using the alignment
coordinates, you must use the
<A HREF="http://www.ncbi.nlm.nih.gov/Sequin/sequin.hlp.html#Workingwithsetsofalignedsequences">
Alignment Assistant
</A>
.
#A brief description of the available features follows. A detailed
explanation of how to use the coding region (CDS) feature is included.
The DDBJ/EMBL/GenBank feature table definition
<A HREF="http://www.ncbi.nlm.nih.gov/collab/FT/index.html">
page
</A>
http://www.ncbi.nlm.nih.gov/collab/FT/index.html
provides detailed information about other features.
*attenuator
#1) region of DNA at which regulation of termination of transcription
occurs, which controls the expression of some bacterial operons; 2)
sequence segment located between the promoter and the first structural
gene that causes partial termination of transcription.
*C_region
#Constant region of immunoglobulin light and heavy chains, and T-cell
receptor alpha, beta, and gamma chains. Includes one or more exons,
depending on the particular chain.
*CAAT_signal
#CAAT box; part of a conserved sequence located about 75 bp upstream of
the start point of eukaryotic transcription units that may be involved
in RNA polymerase binding; consensus=GG(C or T)CAATCT.
*CDS
#coding sequence; sequence of nucleotides that corresponds with the
sequence of amino acids in a protein (location includes stop codon).
Feature includes amino acid conceptual translation.
**Coding Region Page
#Most users add a coding region to their sequence when they fill out the
Organism and Sequences form. However, you may need to edit the coding
region, or add additional ones. Choose CDS under the Coding Regions
and Transcripts submenu of the Features menu, or to edit an existing
CDS, double click on the record viewer. If you appended the partial
sequence of a coding region to the Organism and Sequences form, you will
probably need to edit the Coding Region feature to avoid validation
error messages about the location of the coding region.
***General (Product) Subpage
#Choose the genetic code that should be used to translate the
nucleotide sequence. For more information, and for the translation
tables themselves, see the NCBI Taxonomy
<A HREF="http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c">
page
</A>.
If the genetic code is already populated from the taxonomy database, do
not change this selection.
#Choose the reading frame in which to translate the sequence. Do not
fill in the Protein Product or SeqID selections.
#Supply additional information about the protein by clicking on Edit
Protein Information to launch the Protein feature forms. The protein
name must have already been filled out on the Protein subpage.
#Checking retranslate on accept will translate the nucleotide sequence
according to the interval(s) indicated on the Locations page when you
click on Accept to exit the editor. This new translation will replace
any earlier translations you have supplied. This should not be a
problem if the interval was indicated appropriately.
#If the coding sequence that you supply is a partial sequence and you
have checked a Partial box on the Location subpage, it is a good idea to
check the Synchronize Partials box. In this case, Sequin will ensure
that all other appropriate features (such as protein) are also marked as
partial.
#When editing existing CDS features, choose the sequence you want to
view by selecting its name uder the Product pop-up menu. You may also
import a new protein sequence by selecting Import Protein FASTA under
the file menu. The sequence should be formatted as described above on
the Organism and Sequences form.
#After you have imported a protein sequence, click on Predict Interval.
This function will predict the interval on the nucleotide sequence to
which the coding region applies. If you do not select this function,
the interval will likely be wrong, and you will get an error message
when you attempt to validate the record. If your sequence is a 5' or 3'
partial, you must first indicate this manually on the Location Page.
#You may also have Sequin generate the protein sequence from the
nucleotide sequence by clicking on Translate Product. However, you must
first indicate the location and partialness of the coding region on the
Location page in order to obtain the correct translation.
#The Edit Protein Sequence button will launch an amino acid
<A HREF="#SequenceEditor">
Sequence Editor
</A>
as discussed below.
#The Adjust for Stop Codon button will truncate a displayed translation
at the first stop codon. If no stop codon is present in the current
translation, this function will extend the translation to the first stop
codon or to the end of the sequence. In both cases, the spans of the
coding region will be automatically updated on the Location Page to
reflect the new translation.
***Protein Subpage
#Use this page to enter or edit a name or descriptionof the protein
product. For a new sequence, enter information directly into the
boxes. You can edit descriptions of an existing sequence by clicking
on Edit Protein Feature which will bring up the Protein feature form.
The Launch Product Viewer displays the flatfile view of ht eprotein
record generated from the information in the CDS feature.
***Exceptions Subpage
#Exceptions describe places where there is a posttranslational
modification. Enter the amino acid position at which the modification
occurs, and select the amino acid that is actually represented in the
protein from the pop-up list. Sequin will change the amino acid number
to a nucleotide interval. Please provide some explanation for the
exception in a comment.
*conflict
#Independent determinations of the "same" sequence differ at this site
or region.
*D-loop
#Displacement loop; a region within mitochondrial DNA in which a short
stretch of RNA is paired with one strand of DNA, displacing the
original partner DNA strand in this region; also used to describe the
displacement of a region of one strand of duplex DNA by a single
stranded invader in the reaction catalyzed by RecA protein.
*D_segment
#Diversity segment of immunoglobulin heavy chain, and T-cell receptor
beta chain.
*enhancer
#A cis-acting sequence that increases the utilization of (some)
eukaryotic promoters and can function in either orientation and in any
location (upstream or downstream) relative to the promoter.
*exon
#Region of genome that codes for portion of spliced mRNA; may contain
5' UTR, all CDSs, and 3' UTR.
*gap
#Gap in the sequence, only applied to gaps of unknown length. The
location span of the gap feature is 100 base pairs, indicated by 100 "n"s
in the sequence. The qualifier /estimated_length=unknown is mandatory.
*GC_signal
#GC box; a conserved GC-rich region located upstream of the start point
of eukaryotic transcription units that may occur in multiple copies or
in either orientation; consensus=GGGCGG.
*gene
#Region of biological interest identified as a gene and for which a name
has been assigned.
*iDNA
#Intervening DNA; DNA which is eliminated through any of several kinds
of recombination.
*intron
#A segment of DNA that is transcribed, but removed from within the
transcript, by splicing together the sequences (exons) on either side of
it.
*J_segment
#Joining segment of immunoglobulin light and heavy chains, and T-cell
receptor alpha, beta, and gamma chains.
*LTR
#Long terminal repeat, a sequence directly repeated at both ends of a
defined sequence, of the sort typically found in retroviruses.
*mat_peptide
#Mature peptide or protein coding sequence; coding sequence for the
mature or final peptide or protein product following post-translational
modification. The location does not include the stop codon (unlike the
corresponding CDS).
*misc_binding
#Site in nucleic acid that covalently or non-covalently binds another
moiety that cannot be described by any other Binding key (primer_bind or
protein_bind).
*misc_difference
#Feature sequence is different from that presented in the entry and
cannot be described by any other Difference key (conflict, unsure,
mutation, variation, allele, or modified_base).
*misc_feature
#Region of biological interest which cannot be described by any other
feature key.
*misc_recomb
#Site of any generalized, site-specific, or replicative recombination
event where there is a breakage and reunion of duplex DNA that cannot be
described by other recombination keys (iDNA and virion) or qualifiers of
source key (/proviral).
*misc_RNA
#Any transcript or RNA product that cannot be defined by other RNA keys
(prim_transcript, precursor_RNA, mRNA, 5'UTR, 3'UTR,
exon, transit_peptide, polyA_site, rRNA, tRNA, and ncRNA).
*misc_signal
#Any region containing a signal controlling or altering gene function or
expression that cannot be described by other Signal keys (promoter,
CAAT_signal, TATA_signal, -35_signal, -10_signal, GC_signal, RBS,
polyA_signal, enhancer, attenuator, terminator, and rep_origin).
*misc_structure
#Any secondary or tertiary structure or conformation that cannot be
described by other Structure keys (stem_loop and D-loop).
*modified_base
#The indicated nucleotide is a modified nucleotide and should be
substituted for by the indicated molecule (given in the mod_base
qualifier value).
*mRNA
#messenger RNA; includes 5' untranslated region (5' UTR), coding sequences
(CDS, exon) and 3' untranslated region (3' UTR).
*ncRNA
#non-coding RNA; a non-protein-coding transcript other than ribosomal RNA and
transfer RNA, including antisense RNA, guide RNA, scRNA, siRNA, miRNA, piRNA,
snoRNA, and snRNA. The specific type of ncRNA must be specified in the
/ncRNA_class qualifier.
*N_region
#Extra nucleotides inserted between rearranged immunoglobulin segments.
*operon
#Region containing polycistronic transcript under the control of the same
regulatory sequences.
*oriT
Origin of transfer; region of DNA where transfer is initiated during the
process of conjugation or mobilization.
*polyA_signal
#Recognition region necessary for endonuclease cleavage of an RNA
transcript that is followed by polyadenylation; consensus=AATAAA.
*polyA_site
#Site on an RNA transcript to which will be added adenine residues by
post-transcriptional polyadenylation.
*precursor_RNA
#Any RNA species that is not yet the mature RNA product; may include 5'
clipped region (5' clip), 5' untranslated region (5' UTR), coding
sequences (CDS, exon), intervening sequences (intron), 3' untranslated
region (3' UTR), and 3' clipped region (3' clip).
*prim_transcript
#Primary (initial, unprocessed) transcript; includes 5' clipped region
(5' clip), 5' untranslated region (5' UTR), coding sequences (CDS, exon),
intervening sequences (intron), 3' untranslated region (3' UTR), and 3'
clipped region (3' clip).
*primer_bind
#Non-covalent primer binding site for initiation of replication,
transcription, or reverse transcription. Includes site(s) for synthetic
e.g., PCR primer elements.
*promoter
#Region on a DNA molecule involved in RNA polymerase binding to initiate
transcription.
*protein_bind
#Non-covalent protein binding site on nucleic acid.
*RBS
#Ribosome binding site.
*repeat_region
#Region of genome containing repeating units. Some qualifiers such as
rpt_type and mobile_element have controlled vocabularies. These
qualifiers have check boxes or pull-down menus to ensure that the
correct format is used.
*rep_origin
#Origin of replication; starting site for duplication of nucleic acid to
give two identical copies.
*rRNA
#Mature ribosomal RNA ; the RNA component of the ribonucleoprotein
particle (ribosome) that assembles amino acids into proteins.
*S_region
#Switch region of immunoglobulin heavy chains. Involved in the
rearrangement of heavy chain DNA leading to the expression of a
different immunoglobulin class from the same B-cell.
*satellite
#Many tandem repeats (identical or related) of a short basic repeating
unit; many have a base composition or other property different from the
genome average that allows them to be separated from the bulk (main
band) genomic DNA.
*sig_peptide
#Signal peptide coding sequence; coding sequence for an N-terminal
domain of a secreted protein; this domain is involved in attaching
nascent polypeptide to the membrane; leader sequence.
*source
#Identifies the biological source of the specified span of the sequence.
This key is mandatory. Every entry will have, as a minimum, a single
source key spanning the entire sequence. More than one source key per
sequence is permittable.
*stem_loop
#Hairpin; a double-helical region formed by base-pairing between
adjacent (inverted) complementary sequences in a single strand of RNA or
DNA.
*STS
#Sequence Tagged Site. Short, single-copy DNA sequence that
characterizes a mapping landmark on the genome and can be detected by
PCR. A region of the genome can be mapped by determining the order of a
series of STSs.
*TATA_signal
#TATA box; Goldberg-Hogness box; a conserved AT-rich septamer found
about 25 bp before the start point of each eukaryotic RNA polymerase II
transcript unit that may be involved in positioning the enzyme for
correct initiation; consensus=TATA(A or T)A(A or T).
*terminator
#Sequence of DNA located either at the end of the transcript or adjacent
to a promoter region that causes RNA polymerase to terminate
transcription; may also be site of binding of repressor protein.
*tmRNA
#Transfer messenger RNA; acts as a tRNA first, then an mRNA that encodes a
peptide tag.
*transit_peptide
#Transit peptide coding sequence; coding sequence for an N-terminal
domain of a nuclear-encoded organellar protein; this domain is involved
in post- translational import of the protein into the organelle.
*tRNA
#Mature transfer RNA, a small RNA molecule (75-85 bases long) that
mediates the translation of a nucleic acid sequence into an amino acid
sequence.
*unsure
#Author is unsure of exact sequence in this region.
*V_region
#Variable region of immunoglobulin light and heavy chains, and T-cell
receptor alpha, beta, and gamma chains. Codes for the variable amino
terminal portion. Can be made up from V_segments, D_segments,
N_regions, and J_segments.
*V_segment
#Variable segment of immunoglobulin light and heavy chains, and T-cell
receptor alpha, beta, and gamma chains. Codes for most of the variable
region (V_region) and the last few amino acids of the leader peptide.
*variation
#A related strain contains stable mutations from the same gene (e.g.,
RFLPs, polymorphisms, etc.) that differ from the presented sequence at
this location (and possibly others).
*3'UTR
#Region near or at the 3' end of a mature transcript (usually following
the stop codon) that is not translated into a protein; trailer.
*5'UTR
#Region near or at the 5' end of a mature transcript (usually preceding
the initiation codon) that is not translated into a protein; leader.
* -10_signal
#Pribnow box; a conserved region about 10 bp upstream of the start point
of bacterial transcription units that may be involved in binding RNA
polymerase; consensus=TAtAaT.
* -35_signal
#A conserved hexamer about 35 bp upstream of the start point of
bacterial transcription units; consensus = TTGACa or TGTTGACA.
>Biological Source Descriptor or Feature
#This annotation is very important, as an entry cannot be processed by
the databases unless it includes some basic information about the
organism from which the sequence was derived. This basic information was
entered previously in the submission, in the Organism and Sequences
Form. The more detailed Organism Information form allows you to alter
or add to the data you entered earlier.
*Overview: Descriptor or Feature?
#Sequin allows two types of biological source information to be entered,
Biological Source Descriptors and Biological Source Features. Biological
Source Descriptors, like other descriptors, provide organism information
about an entire sequence, or an entire set of sequences, in an entry.
Biological Source Features, like other features, provide organism
information about a specific interval on a given sequence.
#In most cases, you will want to use a Biological Source Descriptor, because
all the sequences in the entry will derive from the same source. However, if
you have sequenced a transgenic molecule, for example, one that is part plant
and part bacterial, you would use Biological Source Features to annotate which
sequence was derived from plant and which from bacteria.
#To add a Biological Source Descriptor, select Biological Source under
the Descriptor section of the Annotate menu. To add a Biological
Source Feature, select Biological Source under the Bibliographic and
Comments section of the Annotate menu.
#Annotating a Biological Source Descriptor or Feature is similar to
annotating any descriptor or feature. For help in creating descriptors
and features, see the appropriate section of the help documentation.
The following are instructions for filling out Biological
Source-specific forms.
*Organism Page
**Names Subpage
#The scrollable list contains the scientific names of many organisms.
To reach a name on the list, either type the first few letters of the
scientific name, or use the thumb bar. Click on a name from the list to
fill out the scientific name field. If there is a common name for the
organism, that field will be filled out automatically. You may also
directly type in the scientific name. If you have any questions about
the scientific or common name of an organism, see the NCBI
<A HREF="http://www.ncbi.nlm.nih.gov/Taxonomy/tax.html">
taxonomy browser
</A>
http://www.ncbi.nlm.nih.gov/Taxonomy/tax.html
**Location Subpage
***Location of Sequence
#From the selection list, please enter the location of the genome that
contains your sequence. Most entries will have a "Genomic" location.
A brief description of the choices in this pop-up menu were listed
previously.
***Origin of Sequence
#This menu is for the use of database personnel. Please leave this
field empty. The Biological focus box should be checked in rare cases
where multiple source features are annotated.
**Genetic Codes Subpage
#Please use these fields to select the nuclear and mitochondrial genetic
code that should be used to translate the nucleic acid sequence. The
genetic code for a eukaryotic organism is "Standard". If you selected
an organism name from the scrollable list described above, this field
was filled out automatically. Do not change these fields if they have
been filled out automatically.
#For more information regarding the translation tables available, see
the NCBI Taxonomy
<A HREF="http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c">
page
</A>.
**Lineage Subpage
#This information is normally entered by the database staff. They will
use the
<A HREF="http://www.ncbi.nlm.nih.gov/Taxonomy/tax.html">
Taxonomy database
http://www.ncbi.nlm.nih.gov/Taxonomy/tax.html
</A>
maintained by the NCBI/GenBank.
#If you disagree with the lineage supplied please notify the database
staff.
#If you are running Sequin in its
<A HREF="#NetConfigure">
network-aware
</A>
mode, you will see a button labeled "Lookup Taxonomy". Click on this
button to perform an automatic look-up of the taxonomic lineage of the
organism. Sequin will perform the look-up by accessing the Taxonomy
database and will fill out the Taxonomic Lineage and
Division fields.
#If you have any comments about the taxonomic lineage determined by
Sequin, please submit these comments with your entry. Under the Sequin
File menu, select Edit Submitter Info. Enter your comments in the box
entitled "Special Instructions to Database Staff", on the Submission
page.
*Modifiers Page
#This page allows you to enter additional information about the source
and/or organism. Entering information is optional.
**Source Subpage
#Choose a modifier from the pull-down menu on the left side of the page
and type the appropriate name on the right side of the page. If you do
not find appropriate modifiers in the scroll down list, you can enter
additional source information as text in the field at the bottom of the
page. You may add multiple modifiers to describe the source organism.
#Clicking on the X button to the right of the text box will remove the
text and clear the modifier from the pull-down in that line.
#The following is a description of the available modifiers:
#-Cell-line: Cell line from which sequence derives.
#-Cell-type: Type of cell from which sequence derives.
#-Chromosome: Chromosome to which the gene maps.
#-Clone: Name of clone from which sequence was obtained.
#-Clone-lib: Name of library from which sequence was obtained.
#-Collected-by: Name of person who collected sample. Do not use
accented or non-ASCII characters.
#-Collection-date: Date sample was collected. Must use format
23-Mar-2005, Mar-2005, or 2005.
#-Country: The country of origin of DNA samples used for epidemiological
or population studies.
#-Dev-stage: Developmental stage of organism.
#-Endogenous-virus-name: Name of inactive virus that is integrated into
the chromosome of its host cell and can therefore exhibit vertical
transmission.
#-Environmental-sample: Identifies sequence derived by direct molecular
isolation from an unidentified organism. You cannot include extra text when
using this modifier; the text box will change to TRUE upon selection of this
modifier from the pull-down list
#-Frequency: Frequency of occurrence of a feature.
#-Fwd-PCR-primer-name: Name or designation of forward primer used for
amplification.
#-Fwd-PCR-primer-seq: Sequence of forward primer used for amplification.
#-Genotype: Genotype of the organism.
#-Germline: If the sequence shown is DNA and a member of the
immunoglobulin family, this qualifier is used to denote that the sequence
is from unrearranged DNA. You cannot include extra text when using this
modifier; the text box will change to TRUE upon selection of this modifier
from the pull-down list.
#-Haplotype: Haplotype of the organism.
#-Identified-by: Name of person who identified sample. Do not use
accented or non-ASCII characters.
#-Isolation-source: Describes the local geographical source of the organism
from which the sequence was derived
#-Lab-host: Laboratory host used to propagate the organism from which
the sequence was derived.
#-Lat-Lon: Latitude and longitude of location where sample was
collected. Mandatory format is decimal degrees N/S E/W. Selecting this
modifier in the pull-down list will generate separate boxes for entering the
information in the mandatory format.
#-Map: Map location of the gene.
#-Metagenomic: Identifies sequence from a culture-independent genomic
analysis of an environmental sample submitted as part of a whole genome
shotgun project. You may not include extra text when using this modifier,
instead the text box will change to TRUE upon selection.
#-Plasmid-name: Name of plasmid from which the sequence was obtained.
#-Pop-variant: Name of the population variant from which the sequence was
obtained.
#-Rearranged: If the sequence shown is DNA and a member of the
immunoglobulin family, this qualifier is used to denote that the sequence
is from rearranged DNA. You cannot include extra text when using this
modifier; the text box will change to TRUE upon selection of this modifier
from the pull-down list.
#-Rev-PCR-primer-name: Name or description of reverse primer used for
amplification.
#-Rev-PCR-primer-seq: Sequence of reverse primer used for amplification.
#-Segment: Name of viral genome fragmented into two or more nucleic acid
molecules.
#-Sex: Sex of the organism from which the sequence derives.
#-Subclone: Name of subclone from which sequence was obtained.
#-Tissue-lib: Tissue library from which the sequence was obtained.
#-Tissue-type: Type of tissue from which sequence derives.
#-Transgenic: Identifies organism was the recipient of transgenic
DNA. You cannot include extra text when using this modifier; the text box
will change to TRUE upon selection of this modifier from the pull-down list.
**Organism Subpage
#Choose a modifier from the pull-down menu on the left side of the page
and type the appropriate name on the right side of the page. If you do
not find appropriate modifiers in the scroll down list, you can enter
additional organism information as text in the field at the bottom of
the page. You may add multiple modifiers to describe the source organism.
#Clicking on the X button to the right of the text box will remove the text
and clear the modifier from the pull-down in that line.
#The following is a description of the available modifiers:
#-Acronym: Standard synonym (usually of a virus) based on the initials
of the formal name. An example is HIV-1.
#-Anamorph: The scientific name applied to the asexual phase of a fungus.
#-Authority: The author or authors of the organism name from which sequence
was obtained.
#-Bio-material: An identifier of the stored biological material from which
the sequence was obtained. This qualifier should be used to cite collections
that are not appropriate in specimen-voucher or culture-collection. Examples
include stock centers and seed banks. Mandatory format is "institution
code:collection code:material_id". However, only material_id is required.
Selecting this modifier in the pull-down list will generate separate boxes for
entering the information in the correct format.
#-Biotype: See biovar.
#-Biovar: Variety of a species (usually a fungus, bacteria, or virus)
characterized by some specific biological property (often geographical,
ecological, or physiological). Same as biotype.
#-Breed: The named breed from which sequence was obtained (usually applied
to domesticated mammals).
#-Chemovar: Variety of a species (usually a fungus, bacteria, or virus)
characterized by its biochemical properties.
#-Common: Common name of the organism from which sequence was obtained.
#-Cultivar: Cultivated variety of plant from which sequence was obtained.
#-Culture-collection: Identifier and institution code of the microbial or
viral culture or stored cell-line from which the sequence was obtained. This
qualifier should be used to cite the collection where the author has deposited
the culture or from which the culture was obtained. Personal library
collections should be annotated in strain and not in culture-collection.
Mandatory format is "institution code:collection code:culture_id". However,
collection code is not required. Selecting this modifier in the pull-down
list will generate separate boxes for entering the information in the correct
format.
#-Ecotype: The named ecotype (population adapted to a local habitat) from
which sequence was obtained (customarily applied to populations of
Arabidopsis thaliana).
#-Forma: The forma (lowest taxonomic unit governed by the nomenclatural
codes) of organism from which sequence was obtained. This term is usually
applied to plants and fungi.
#-Forma-specialis: The physiologically distinct form from which sequence
was obtained (usually restricted to certain parasitic fungi).
#-Group: Do not select this item.
#-Isolate: Identification or description of the specific individual
from which this sequence was obtained. An example is Patient X14.
#-Metagenome-source: Used only for genome projects. Do not select this item.
#-Pathovar: Variety of a species (usually a fungus, bacteria or virus)
characterized by the biological target of the pathogen. Examples
include Pseudomonas syringae pathovar tomato and Pseudomonas syringae
pathovar tabaci.
#-Serogroup: See serotype.
#-Serotype: Variety of a species (usually a fungus, bacteria, or virus)
characterized by its antigenic properties. Same as serogroup and
serovar.
#-Serovar: See serotype.
#-Specific-host: When the sequence submission is from an organism that
exists in a symbiotic, parasititc, or other special relationship with
some second organism, use this modifier to identify the name of the
host species.
#-Specimen-voucher: Identifier of the physical specimen from which the
sequence was obtained. The qualifier is intended for use where the sample is
still available in a curated museum, herbarium, frozen tissue collection, or
personal collection. Mandatory format is "institution code:collection
code:specimen_id". However, only specimen_id is required. Selecting this
modifier in the pull-down list will generate separate boxes for entering the
information in the correct format.
#-Strain: Strain of organism from which sequence was obtained.
#-Subgroup: Do not select this item.
#-Sub-species: Subspecies of organism from which sequence was obtained.
#-Substrain: Sub-strain of organism from which sequence was obtained.
#-Subtype: Subtype of organism from which sequence was obtained.
#-Synonym: The synonym (alternate scientific name) of the organism name
from which sequence was obtained.
#-Teleomorph: The scientific name applied to the sexual phase of a fungus.
#-Type: Type of organism from which sequence was obtained.
#-Variety: Variety of organism from which sequence was obtained.
**GenBank Subpage
#Please do not use this form. This field is reserved for information from
NCBI's taxonomy database.
*Miscellaneous Page
**Synonyms Subpage
#If there are alternative names for the organism from which the sequence
was derived, enter them here. Please be aware that this is the
appropriate field only for alternative names for the organism, not for
alternative gene or protein names.
**Cross-Refs Subpage
#This page is for use by database staff only.
>Publications
*Overview: Descriptor or Feature?
#Sequin allows two types of publications to be entered, Publication
Descriptors and Publication Features. Publication Descriptors are
bibliographic references that, like other descriptors, cover an entire
sequence, or an entire set of sequences, in an entry. Publication
Features are bibliographic references that, like other features, cover
a specific interval on a given sequence.
#Publications are entered into the Reference field of the database
entry. References are citations of unpublished, in press, or published
works that are relevant to the submitted sequence. Publications
should provide information regarding the principle cloning and
determination of the sequence within the record.
#In general, there is one publication describing a sequence, and a
Publication Descriptor should be used. To enter a Publication
Descriptor, select Publications under the Annotate menu and click on
Publication Descriptor.
#However, if one publication describes the cloning of the 5' end of a
gene, and another publication describes the cloning of the 3' end of
the gene, Publication features may be used. To make a publication
feature, choose Publication Feature in the Publications section of the
Annotate menu. Enter the information about the publication, and then
enter the nucleotide interval to which the publication refers on the
Location page.
*Citation on Entry Form
**Status
#Using the radio buttons, select one of the three options:
#-Unpublished: Select this option if a manuscript has been written but
not yet submitted or has been submitted for publication but has not yet
been accepted.
#-In Press: The article has been accepted for publication but is not yet
in print.
#-Published: The article has been published.
**Class
#Using the radio buttons, select the type of publication in which the
sequence will appear.
#-Journal
#-Book Chapter
#-Book
#-Thesis/Monograph
#-Proceedings Chapter: Abstract from a meeting
#-Proceedings: A meeting
#-Patent
#-Online Publication: Used for journals which publish strictly online and
do not issue print copies.
#-Submission
**Scope
#Using the radio buttons, select one of the options.
#-Refers to the entire sequence: Most publications should be classified
as such.
#-Refers to part of the sequence: For use only when a publication
discusses only part of the presented sequence. You must enter the
locations in the location tab in later forms. This selection is only
valid when adding a Publication feature, not descriptor.
#-Cites a feature on the sequence: This selection should only be made in
limited cases. Its use must coincide with the use of the /citation
qualifier on the given feature.
#After you have filled out the Citation on Entry form, click on
"Proceed" to see the next form.
*Citation Information Form (General)
**Authors Page
***Names Subpage
#Please enter the names of the authors. Note that the first name of the
author is listed first. You can add as many authors to this page as
necessary. After you type in the name of the third author, the box
becomes a spreadsheet, and you can scroll down to the next line by
using the thumb bar. The suffix toggle allows the addition of common
suffixes to the author name. The consortium field should be used when
a consortium is responsible for the sequencing or publication of the
data. The consortium should not be the department or institute
affiliation of the authors. Individual authors may be listed along
with a consortium name.
***Affiliation Subpage
#Please enter information about the institution where the sequencing was
performed.
#Other pages in the Citation Information Form will be different,
depending on the Class of publication selected in the Citation on Entry
Form. Instructions for filling out the Citation Information Form for
Journals is included here.
*Citation Information Form (If Selected Class Was Journal)
**Title Page
#Enter title for manuscript in the box.
**Journal Page
#Fill in the appropriate Journal, Volume, Issue, Pages, Day, and Year
fields by typing information into the boxes. Select the month with the
pop-up menu. If necessary, choose an option from the Erratum pop-up
menu and explain the erratum.
#If you are running Sequin in its
<A HREF="#NetConfigure">
network-aware
</A>
mode, the program will look up the Title, Author, and Journal
information in the MEDLINE database if you supply it with some minimal
information. For example, if you know the MUID (MEDLINE Unique
Identifier) of the publication, enter it in the appropriate box and
select "Lookup By MUID." Sequin will automatically retrieve the rest
of the information. One way to find the MUID of the publication is to
look up the publication with the NCBI's
<A HREF="http://www.ncbi.nlm.nih.gov/Entrez">
Entrez
</A>
service. Alternatively, if you do not know the MUID, enter the Journal,
Volume, Pages, and Year. Then select "Lookup Article". Sequin will
retrieve the missing Title and Author information.
#The PubStatus toggle is used by database staff. If you have used the
"Lookup by MUID" or "Lookup by PMID" functions, this field may be
populated. Please do not edit the information.
**Remark Page
#This page is reserved for use by the database staff.
>File Menu
*About Sequin
#Details about the current version of Sequin.
*Help
#Launches the help documentation.
*Open
#Open an existing entry. This option will open a record that has been
previously saved in Sequin. Furthermore, for analysis purposes, it can also
open
a FASTA-formatted sequence file. The sequence will be displayed in Sequin and
can be analyzed with tools such as CDD Search, but it should not be submitted,
because it does not have the appropriate annotations.
*Close
#Close this entry.
*Export GenBank
#Exports the currently displayed format to a file. Do not use export
ASN1 for submission of sequences to the database.
*Duplicate View
#Duplicates the entry. You can then view the entry simultaneously in
different Display Formats.
*Save
#Saves the entry. Note: This merely saves the entry so you can go back
and edit it. It does not prepare the entry for submission to the
database, that is, it does not validate the entry.
*Save As
#See Save.
*Save as Binary Seq-entry
#Saves the file in a compressed format and should be used only when the
file is to be imported into other analysis programs. Do not use this
option to save files for submission directly to GenBank.
*Restore
#Replaces the displayed record with a previously saved version. This
feature is useful if you have made unwanted changes since you last saved
the record.
*Prepare Submission
#Prepares the entry for submission to the database. See
<A HREF="#SubmittingtheFinishedRecordtotheDatabase">
Submitting the Finished Record to the Database
</A>
in the Sequin help documentation.
*Print
#Prints the window that is currently selected. The selected window can
be one of the Sequin forms or pages, or the help documentation.
*Quit
#Exit from Sequin.
>Edit Menu
*Copy
#Copy the selected item.
*Clear
#Clear the selected item.
*Edit Sequence
#To edit a single sequence, select the sequence identifier in the Target
Sequence pop-up menu, and click on Edit sequence. The sequence editor
will be launched for that sequence. The
<A HREF="#SequenceEditor">
sequence editor
</A>
is discussed in more detail below.
*Alignment Assistant
#This option will launch the Alignment Assistant which is discussed in
more detail
<A HREF="#Workingwithsetsofalignedsequences">
below
</A>
.
*Edit Submitter Info
#Opens up the Submission Instructions form, which allows you to enter
additional information about the person submitting the record. Much of
this information was entered on the first form in Sequin, the Submitting
Authors form.
#You can also save the information from the Submitting Authors form
here, so that you can use it in subsequent Sequin submissions. Click
on "Edit Submitter Info" and, under the file menu in the resulting
Submission Instructions form, click on Export Submitter Info to save
the information to a file. For subsequent Sequin submissions, if you
have already saved the submittor information, click on Import Submitter
Info under the File menu on the Submission page of the Submitting
Authors form.
**Submission Page
#Indicate the type of submission. If it is a new submission, select
New. If you are updating an existing submission in order to resubmit it
to the databases, select Update. Check either the "Yes" or "No" radio
button to indicate if the record should be released before publication.
If you select "Yes", the entry will be released to the public after the
database staff has added it to the database. If you select "No", fields
will appear in which you can indicate the date on which the sequences
should be released to the public. The submission will then be held back
until formal publication of the sequence or
GenBank Accession number, or until the Release Date, whichever comes
first. If you have any special instructions, enter them in the box at
the bottom of the page.
**Contact Page
#Update the name, affiliation, or contact numbers of the person
submitting the record. Please supply a fax number to facilitate
communication with database staff.
**Citation Page
#Update the names and affiliation of the people who should receive
scientific credit for the generation of sequences in this entry. The
address should list the principal institution in which the sequencing
and/or analysis was carried out. If you are submitting the record as
an update to the databases, explain the reason for the update on the
Description subpage.
*Update Sequence
#This selection allows you to replace a sequence with another sequence,
merge two sequences that overlap at their ends, 'patch' a corrected
fragment of a sequence to the current sequence, or copy features from
one sequence to another.
#Use Single Sequence to import a sequence in FASTA or ASN.1 format (for
example, a sequence record that has already been saved in Sequin). If
you are running Sequin in
<A HREF="#NetConfigure">
Network Aware mode,
</A>
you can use Download Accession to import a record from Entrez. The
Multiple Sequences option allows you to update multiple sequences using
either FASTA or ASN.1 formats. In either format, each sequence
identifier must be identical in the new and old sequences.
#After you import the updated sequence, a new window will open that
displays two graphical views and the text of the alignment of the new
and old sequence. The first graphic displays the relative length of the
two sequences and the length of the overlapping region between
sequences. The second graphic represents any inserts, deletions, or
point changes within the aligned region between the new and old
sequences. Clicking on a region in this graphic will scroll to the
corresponding nucleotide sequence in the alignment text below.
#The Sequence Update box to the left shows the action that will be
performed upon updating the sequence, i.e., no change, replace, extend
5', extend 3', or patch. The patch function allows you to replace an
internal fragment of the sequence without affecting flanking regions.
You can also override the alignment between the new and old sequence
using the Ignore alignment checkbox to force a sequence change of
replace, Extend 5' or Extend 3'. This option allows you to append new
sequence to with no overlap.
#If the current sequence has annotation, you can use the Existing
Features box to determine whether the annotation should remain or be
removed upon updating the sequence. The Do not remove option is the
default. However, you may chose to remove annotated features only in
the aligned area, outside the aligned area, or to remove all currently
annotated features.
#When updating via Download Accession or an ASN.1 file, the Import
Features box allows you to specify whether features from the new file
should be imported to the existing record. The dialog offers
different options for cases where the features on the new file are
identical to those on the existing record.
#If you are using the Multiple Sequences option, you may choose to
review the sequences and update them one by one using the Update this
Sequence box at the bottom of the window. You may skip a sequence
update or choose to update all sequences at once without reviewing them
in the Update Sequence dialog.
#In any case, please carefully review the sequence and annotation in the
record viewer after using the Update Sequence function.
*Extend Sequence
#This selection functions similar to the
<A HREF="#UpdateSequence">
Update Sequence
</A>
function. However, you can only extend the existing sequence in either
the 5' or 3' direction in cases with no overlap between the existing
and new sequences.
*Feature Propagate
#This selection allows you to propagate any annotated feature from one
sequence in an aligned set to other sequences within the set. For
example, if one nucleotide sequence in the alignment contains a CDS
feature, you can annotate a similar CDS on the other nucleotide
sequences in the set.
#The default source of features to be propagated is the first member
of the set. If you would like to use a different entry as the source of
the features, scope to that entry in the Target Sequence menu before
selecting Feature Propagate from the Edit menu.
#The Feature Propagate window allows you to select which sequences
should receive the new annotation and which features will be
propagated. You can also select whether the features will be extended
or split at gaps in the alignment. The split at gaps selection will
produce two features, one on either side of the gap within the
alignment. If you are propagating a CDS feature, you may specify that
the translation end or extend through internal stop codons. You may
also extend the translation after the stop codon on the source entry by
chosing to translate the CDS after partial 3' boundary. If the CDS
that you are propagating to other records is partial on either end, you
should select the 'Cleanup CDS partials after propagation' check box.
This will retain the partial nature of the CDS features on all records.
The fuse adjacent propagated intervals function will create one
feature from two of the same type that contain abutting nucleotide
intervals due to the nature of the alignment used for propagation.
*Add Sequence
#This selection allows you to add a new sequence to an existing
population, mutation, phylogenetic, or environmental sample set.
You may import the new entry in FASTA format or ASN.1 format (for
example, a sequence record that has been saved in Sequin).
*Parse File to Source
#This selection allows you to add unique information for one source
qualifier for each of the records in a batch or set. The input file
must be in the format of a tab-delimited, two column table. The first
column should list the SeqID exactly as it was listed in the original
FASTA file. The second column should list the text value for the
desired source qualifier for each record. Once the file has been
imported, a pop-up box will appear with the source qualifiers listed in
the pull down menus. The qualifiers are separated into three menus:
one for taxonomic information, one for the Organism modifiers and one
for the Source modifiers. For example, in order to add the clone
designations 57 and 49 to the sequences labeled seq1 and seq2, the table
seq1 57
seq2 49
should be used and clone should be selected from the Source modifiers
pull-down menu.
>Search Menu
*Find ASN.1
#Under this command, you can find and replace strings of letters in
those fields of your submission that contain manually entered data.
The fields that can be altered are Locus, Definition, Accession,
Keywords, Source, Reference, and Features. To use this option, select
Find and fill the Find and Replace lines with the appropriate text.
Note that you cannot edit the sequence in this way.
*Find FlatFile
#Under this command, you can find strings of letters in all fields of
your submission. You can use the Find First and Find Next buttons to
identify the specified text sequentially through the flatfile.
*Find by Gene
#This option allows you to move quickly in the record viewer to a gene
feature containing the specified gene symbol.
*Find by Protein
#This option allows you to move quickly in the record viewer to a CDS
feature containing the specified product name.
*Find by Position
#This option allows you to move quickly in the record viewer to any
feature annotated at the specified nucleotide location.
*Validate
#This option detects discrepancies between the format of your submission
and that required by the database selected for entry. If discrepancies
are present, it suggests ways in which to correct them. See the topic on
<A HREF="#SubmittingtheFinishedRecordtotheDatabase">
Submitting the Finished Record to the Database
</A>
in the Sequin help documentation.
*CDD Search
#Performs a CDD BLAST search of the selected sequence against the
NCBI's
<A HREF="http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml">
Conserved Domain Database
</A>
. To do a CDD BLAST search, Sequin must be in its network aware mode.
#CDD currently contains domains derived from two popular collections,
Smart and Pfam, plus contributions from colleagues at NCBI. The source
databases also provide descriptions and links to citations. Since
conserved domains correspond to compact structural units, CDs contain
links to 3D-structure via Cn3D whenever possible.
#The results of the CDD search will be displayed in the record
viewer. These results are for your use only and should be removed
from the record before submission.
*Vector Screen
#This option allows you to run a BLAST search of your nucleotide
sequence(s) against NCBI's
<A HREF="http://www.ncbi.nlm.nih.gov/VecScreen/UniVec.html">
UniVec
</A>
database. We highly recommend that you run this analysis and remove
any vector contamination before submission. The UniVec database
contains only one copy of every unique sequence segment from a large
number of vectors. It also contains sequences for adapters, linkers
and primers commonly used.
#To run Vector screen on a submission containing multiple sequences,
scope to ALL SEQUENCES in the Target Sequence pull-down before running
the analysis. If there are many sequences, a status bar will appear
indicating the progress of the search. If no contamination is found, a
pop-up box will appear to notify you. If contamination is found, a
miscellaneous feature will be annotated on the flatfile with the
location of the contamination. Details will include the relative
strength of the BLAST hit. You must trim the nucleotide sequence to
remove this feature before submission.
*ORF Finder
#The ORF Finder shows a graphical representation of all open reading
frames (ORFs) in the nucleotide sequence. This tool allows you to
select ORFs and have them appear as coding sequence (CDS) features on
the sequence record.
#The ORFs, indicated by colored boxes, are defined as the longest sequence
containing a start codon and stop codon. All six reading frames are shown as
separate lines in the graphical view. The top three lines represent the plus
strands, and the bottom three lines the minus strands. In the default view,
the nucleotide sequence intervals of the ORFs are displayed in descending
length order on the right side of the window. Intervals on the complementary
(minus) strand are indicated by a 'c'. Selecting 'Order by Start' will
reorder the list based on the nucleotide location of the start codon.
#Clicking on the box labelled ORF changes the graphical display so that the
#potential start codons are indicated in white, and stop codons in red.
#The default settings display only those ORFs which contain an ATG start
codon. Selecting 'Alternative' in the 'Initiation Codon' box, will also
include ORFs beginning with all valid alternative start codons as determined
by the genetic code listed in the source feature. If the genetic code for the
source organism has not been specified, the default
<A HREF="http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c#SG1">
standard genetic code
</A>
will be used.
#The ORF length button sets the length of ORFs that are
displayed. For example, the default of 10 shows all ORFs that are
greater than 10 nucleotides in length.
#Checking the Show Partial ORFs box will display ORFs that extend to the
#end(s) of the nucleotide sequence but are 5' or 3' partial.
#ORFs can be selected by clicking on the graphical representation or on the
sequence interval. Once an ORF has been selected, its location and amino acid
sequence will automatically be fielded in the
<A HREF="#CDS">
CDS feature editor
</A>
accessed under the Annotate menu.
*Select Target
#This option changes the sequence that is selected in the Target
Sequence pop-up. Type the SeqID of the sequence in the box, and the
record viewer will be updated to display that sequence.
>Misc Menu
*Style Manager
#The Style manager allows you to choose between different formats in
which to view the Graphical Display Format. The graphical display is
selected by choosing the Graphic display format on the record viewer.
Using the Style Manager, you can also copy the style or modify it to
suit your needs.
*Net Configure
#As a default, Sequin is available as a stand-alone program. However,
the program can also be configured to exchange information with the NCBI
(GenBank) over the Internet. The network-aware mode of Sequin is
identical to the stand-alone mode, but it contains some additional
useful options.
#Sequin will only function in its network-aware mode if the computer on
which it resides has a direct Internet connection. Electronic mail
access to the Internet is insufficient. In general, if you can install
and use a WWW browser on your system, you should be able to install and
use network-aware Sequin. Check with your system administrator or
Internet provider if you are uncertain as to whether you have direct
Internet connectivity.
#There are two ways to change Sequin into its network-aware mode. If
you are still on the initial Welcome to Sequin form, select Net
Configure under the Misc menu. If you have already worked on a Sequin
submission and are looking at the record in the record viewer, select
the Net Configure option from the Misc menu.
#Most users will be able to use the default (Normal) settings on the
Network Configuration page; select Accept to complete the configuration
process.
#If a "Normal" Connection does not work, you may need to select the
Firewall Connection. Contact your system administrator to determine
what to enter into the Proxy and Port fields. If you do not have
access to the domain name server (DNS), uncheck this box.
#The Timeout pop-up selects the length of time that your local copy of
Sequin will wait for a reply from the NCBI server. You may need to set
this number higher (i.e., 60 seconds or 5 minutes) if you are outside
of the United States or have a bad internet connection.
#If you have problems setting up the network configuration, contact
<a href="mailto:info@ncbi.nlm.nih.gov">
info@ncbi.nlm.nih.gov.
</a>
#If you would like to change Sequin back to its stand-alone mode, select
Net Configure again from the Misc menu. Click on Connection: None.
#The network-aware mode of Sequin allows you to perform a number of
additional, important functions. These functions all appear as
additional menu items. A brief description of these functions follows.
Further descriptions are available as indicated elsewhere in the help
documentation.
**Updating Existing GenBank Records
#Using Sequin in its network-aware mode, you can download an existing
GenBank record from Entrez using the GenBank accession number or GI
identification number (NID). You can then use Sequin to make any
necessary changes to the record, and resubmit it to GenBank as a
sequence update.
<A HREF="#WelcometoSequinForm">
Instructions
</A>
for submitting sequence updates are presented under the Welcome to
Sequin Form. You can download any record from Entrez and look at it in
Sequin. However, you can only formally update those records which you
have submitted since submitters retain editorial control of their
records.
**Performing a PubMed Look-Up
#In its network-aware mode, Sequin can import the relevant sections of a
PubMed record directly into a sequence submission record. Rather than
typing in the entire citation, you can enter minimal information, such
as the PubMed Unique Identifier (PMID), or Journal name, volume, year,
and pages. The
<A HREF="#JournalPage">
PubMed lookup
</A>
is explained in the section of the documentation entitled Publications.
**Performing a Taxonomy Look-up
#In its network-aware mode, Sequin can look
up the taxonomic lineage of an organism from the NCBI's Taxonomy
database. This look-up is normally performed by the NCBI database staff
after the record has been submitted to GenBank. If you look up the
taxonomy before submitting the sequence, you can make a note in the
record of any disagreements. The
<A HREF="#LineageSubpage">
taxonomy lookup
</A>
is explained in the section of the documentation covering
Biological Source: Organism page: Lineage subpage.
**Accessing the NCBI DeskTop
#The NCBI DeskTop displays the internal
structure of the record being viewed in Sequin. The
<A HREF="#NCBIDeskTop">
DeskTop
</A>
is explained under the Misc menu.
*NCBI DeskTop
#This option is only available if you are running Sequin in its
<A HREF="#NetConfigure">
network-aware
</A>
mode.
#The NCBI DeskTop provides a view of the internal structure of the
Sequin record, the ASN.1. Its display resembles a Venn diagram and
represents all the structures represented in the ASN.1 data model.
#In addition, a number of undocumented software tools from the NCBI can
be accessed from the DeskTop. These tools are components of the NCBI
portable software Toolkit. You can also customize these functions using
the Toolkit with your own software tools. The Toolkit and its
documentation are available from the NCBI by anonymous
<A HREF="ftp://ftp.ncbi.nih.gov/toolbox/README">
FTP.
</A>
#The DeskTop should only be used by very seasoned users. At this time,
we are not providing any documentation for these specialized functions.
>Annotate Menu
#This menu allows you to enter features and descriptors on the sequence.
#The first six options, Genes and Named Regions, Coding Regions and
Transcripts, Structural RNAs, Bibliographic and Comments, Sites and
Bonds, and Remaining Features refer to types of Features that can be
added to the sequence. Features are described in more detail in the
above section entitled
<A HREF="#Features">
Features.
</A>
#If you are submitting a set of similar sequences, you can add the same
feature across the entire span of each by using the Batch Feature Apply
option. The feature must span the entire nucleotide sequence of each
member; you can not annotate specific nucleotide locations using this
option (for this, see
<A HREF="#FeaturePropagate">Feature Propagate</A>).
For each feature, you will be prompted to designate whether the feature
is 5' or 3' partial and whether is is on the plus or minus strand. You
may also add a comment or other qualifier to the feature. The Add
Qualifier option allows you to add a qualifier to an existing feature.
You must specify the feature and qualifier in the Add Qualifier pop-up
box. Source qualifiers can be added to all entries using the Add
Source Qualifier option. Qualifiers specific to the CDS and gene can
be added using Add CDS-Gene-Prot-mRNA and RNA qualifiers using Add RNA
Qual. In each case, a pop-up box appears with qualifier options
appropriate for that feature.
#The Batch Feature Edit function allows you to edit existing qualifiers.
For each menu choice, a pop-up box allows you to select the feature
containing the qualifier and the specific qualifier to be edited. You
can use the Find/Replace text boxes to edit the information contained
within the qualifier.
#The Publications option allows you to add a Publication Feature or
Publication Descriptor to the record. Publications are described in
more detail in the above section entitled
<A HREF="#Publications">
Publications.
</A>
#The Descriptors option allows you to add Descriptors to the
record. Descriptors are described in more detail in the section
entitled
<A HREF="#Descriptors">
Descriptors,
</A>
above.
#The Generate Definition Line option will generate a title for your
sequence based on the information provided in the record. This option
will work for single sequences as well as sets of sequences, and can
handle complex annotations with multiple features. The title will
follow GenBank conventions, but may be modified by the database staff
if it is not appropriate. The title you enter here will replace any
title you entered elsewhere in the submission, for example, any title
that was attached to the nucleotide sequence. For a description of
definition lines, see
<A HREF="#NucleotideDefinitionLine(Title)">
Nucleotide Definition Line (Title)
</A>
, above.
#Sort Unique Count by Group opens a new window which displays your record(s)
the number of times an individual line appears in the flatfile(s). This is
particularly useful when checking that all records in a large set contain the
required source or feature information.
>Options Menu
#This menu is only available when using Sequin in its network-aware mode.
*Font
#Use this item to change the display font. From the pop-up menus,
choose the style and size of type. For additional changes, mark the
Bold, Italic, or Underline check boxes. The default font is 10-point
Courier.
>Sequence Editor
#This editor allows you to modify the nucleotide or amino acid sequences
and corresponding annotation in your entry. Although the Sequence Editor
does allow you to undo changes you make to the sequence, we strongly
suggest that you save a copy of the entry before launching the Sequence
Editor so that you can revert to it if necessary.
*Starting the Sequence Editor
#The sequence that appears in the editor is dependent on the sequence(s)
selected in the Target Sequence pull-down list. There are two ways to
launch the sequence editor for nucleotide sequences. First, you can
double click within sequence in any display format of the record viewer.
A window containing the DNA sequence will appear. Second, in the record
viewer, select the sequence that you would like to edit in the Target
Sequence pop-up menu. Click on Edit Sequence under the Edit menu. You
can launch the editor for protein sequences by selecting the protein
sequence in the Target Sequence pop-up menu and double clicking within
the protein sequence. A window containing the protein sequence will
appear.
*Moving around the Sequence Editor
#The cursor can be moved with the mouse or the arrow keys. The display
window will change to show the position of the cursor. The sequence
location of the first residue on each line is indicated on the left side
of the window. The cursor location, or the range of sequences selected
by the mouse, is shown in the upper left corner of the window. If you
want to move the cursor to a specific location, type the number in the
box on the top left of the sequence editor window, and hit the Go to
button. If you want to look at a specific sequence, but not move the
cursor to it, type the number in the upper right box of the window and
hit the Look at button.
*Editing Sequence and Existing Annotation
#Select a piece of sequence by highlighting it with the mouse. To
select the entire sequence, click on a sequence location number on the
left side of the window. Any sequence that is highlighted in the
Sequence Editor will show up as a box on the sequence when it is viewed
in the Graphic Display Format.
#One way to insert and delete residues is with the mouse. Move the
cursor to the appropriate location and type. Text will be inserted to
the left of the cursor. Delete sequence with the backspace or delete
key. Text will be deleted to the left of the cursor. To delete a block
of sequence, highlight it with the mouse and use the delete or backspace
key.
#Another way to insert and delete residues is with options under the Edit
menu of the Sequence Editor. Use Cut to remove, or Copy to copy,
highlighted residues. Copied residues can then be pasted elsewhere
within the sequence by using the Paste option.
#Features annotated via the record viewer will be displayed in a
graphical format within the sequence editor. CDS features will be be
displayed as a blue line across the appropriate nucleotide location. All
other features will be displayed as a black line. To the left of the
line, the name of the feature is displayed. In the case of CDS or mRNA
features, the product name is shown. For gene features, the gene locus
is shown.
#Double-clicking on the feature will launch the feature editor just as in
the record viewer. However, you can also change the nucleotide location
of any feature within the graphical view. To move the entire feature,
select the feature and drag it to the appropriate location while holding
down the mouse button. To alter the 5' or 3' end of a feature, click on
the feature's end and drag to the new location while holding down the
mouse button.
#Before moving the nucleotide locations of a CDS feature, it may be
useful to view the codons in the current translation. You can do this by
clicking on the feature line and releasing the mouse button. A grid will
be displayed that shows the triplet location for the current annotation.
Once you have changed the nucleotide location of a CDS feature in the
graphical view, you can see the new translation by using the Translate
CDS button at the bottom of the window.
#To save changes you have made to the sequence, press the Accept button
at the bottom of the Sequence Editor display window. If you do not want
to save the changes, press the Cancel button at the bottom of the
Sequence Editor display window. Selecting either Accept or Cancel will
quit the Sequence Editor and return you to the record viewer. Any
changes you make will not become a permanent part of the Sequin record
until you Save the record in the record viewer.
#New features can be added using the Features menu.
*Sequence Editor Window Buttons
**Go to
#Moves the cursor to the indicated location.
**Look at
#Moves the window to the indicated location without moving the cursor.
**Merge Feature Mode/Split Feature Mode
#In merge mode, any new sequence that is entered into a region spanned
by an existing feature becomes part of that feature. For example, if
you enter new sequence in the middle of a CDS, that sequence will be
translated as part of the CDS. In split mode, the new sequence
interrupts the feature. For example, if you enter new sequence in the
middle of a CDS, the CDS will be interrupted by that sequence (see the
location of the CDS in the record viewer).
**Numbering
#Allows the sequence location numbering to be hidden, displayed on the
side, or displayed on the top of the sequence.
**Grid
#Allows the display to show a grid separating each feature and sequence
for easier viewing.
**Show/Hide Features
#This box toggles between hiding and showing the features on a sequence.
**Accept
#Closes the Sequence Editor after saving all of the changes made to
sequences and features.
**Cancel
#Closes the Sequence Editor without saving any changes made to sequences or
features.
**Translate CDS
#Allows translation of coding region features after the location has been
changed within the graphical view.
*Sequence Editor File Menu
**Export
#Allows the export of a range of sequence as a FASTA file or text file.
Using the text option will also export overlapping features if they are
displayed. If the features are first hidden, only the sequence will be
exported. All protein translations displayed at the time of export, will
be exported as well.
**Accept
#Closes the Sequence Editor after saving all of the changes made to
sequence and features.
**Cancel
#Closes the Sequence Editor without saving any changes made to sequences
of features.
*Sequence Editor Edit Menu
**Undo
#Undoes all actions performed in the Sequence Editor since the last save.
**Redo
#Restores changes removed with Undo option
**Cut
#Removes the highlighted sequence. This sequence can be pasted elsewhere.
**Paste
#Pastes a cut or copied sequence to the right of the cursor.
**Copy
#Copies the highlighted sequence. This sequence can be pasted elsewhere.
**Find
#Allows you to find DNA or amino acid sequence patterns in your sequence.
The search is case insensitive. To find an exact match to a DNA
sequence pattern, type the pattern in the box. The number of items found
will be displayed and you can toggle through each instance with the Find
Next button. To find the reverse complement of the pattern, click on
the reverse complement box at the bottom of the pop-up box.
#To find an exact match to an amino acid seqeunce pattern, type that
sequence in the box, and click on "translate sequence". Sequin will look
for all occurrences of that pattern in all six open reading frames. The
DNA sequence encoding that protein sequence in any of the six reading
frames will be hightlighted.
**Translate CDS
#Allows translation of coding region features after the location has been
changed within the graphical view.
**Complement
#Shows the complement of the submitted strand underneath the original.
**Reading Frames
#Shows the indicated phase translation of the selected coding sequence.
You can select any or all of the six reading frames, all reading frames
or all positive or negative frames.
**Protein Mismatches
#Indicates amino acid which does not match conceptual translation
following a nucleotide sequence change. The original amino acid sequence
will be displayed until the Translate CDS function is used. Differences
will be indicated by a red box around the amino acid abbreviation.
**On-the-fly Protein Translations
#Creates a second amino acid sequence in the display which retranslates
as the nucleotide sequence is changed to allow side-by-side comparison to
the original amino acid sequence.
*Sequence Editor Features Menu
#The menu contains a long list of all features that can be annotated on a
sequence. These features are the same as those that are accessible
through the main Sequin Annotate menu.
#You can annotate features either in the Annotate menu or in the Sequence
Editor. If you annotate them in the Annotate menu, you must type in the
nucleotide sequence location of the feature. However, if you add
features from the Sequence Editor, you can highlight the sequence that
the feature covers, and the location of the sequence will be
automatically entered in the feature location box. Additional
explanations of how to annotate features are provided in the section on
<A HREF="#Features">
Features.
</A>
>Working with Sets of Aligned Sequences
#Sequin allows you to work with aligned sets of closely related
nucleotide sequences that are part of a population, phylogenetic, or
mutation study. If the sequences are imported in a pre-aligned format,
such as PHYLIP, Sequin uses this alignment. If the sequences are
imported individually in FASTA format, Sequin can generate its own
alignment.
#You can view the aligned sequences in the Sequence Alignment Editor. In
the record viewer, select All Sequences in the Target Sequences menu,
and select the Alignment Display Format.
#The Alignment Assistant is launched by selecting Alignment Assistant
from the Edit menu in the record viewer. It can be used to apply
features to the whole set of sequences using the alignment coordinates.
Rather than calculating the nucleotide coordinates for every feature on
every nucleotide sequence, you may select the feature's location using
its alignment coordinates and apply it to every member of the set
simultaneously. Sequin will calculate the nucleotide locations as they
apply to each member of the set.
*Alignment Assistant Window Buttons
**Go to
#The Go to alignment position and Go to sequence position buttons both
scroll the aligment assistant so that the requested position is
visible. If the requested position is already visible, nothing will
happen. Unlike the Sequence editor window, the 'go to' button does not
control the cursor position.
**Numbering
#Allows the sequence location numbering to be hidden, displayed on the
side, or displayed on the top of the sequence.
**Grid
#Allows the display to show a grid separating each feature and sequence for
easier viewing.
**Features Toggle
#It is possible to view annotated features in the aligment assistant.
The features are displayed as a bar underneath the coordinates for that
feature. The identity of the feature is displayed in the left-hand
column. The default selection is to have the features Hidden. You may
display the features associated only with the Target Sequence or
features annotated on All Sequences in the alignment.
*Alignment Assistant File Menu
**Export
#Allows you to export the alignment to a file in three different
formats. The contiguous and interleaved options export the alignment
accordingly in FASTA+GAP format. The text representation option saves
the alignment as it appears in the Alignment Assistant. Note that with
this option features are included if they are displayed at the time of
export.
**Close
#Closes the Alignment Assistant window and saves any changes made.
*Alignment Assistant Edit Menu
**Remove Sequences from Alignment
#Allows you to remove selected sequence(s) from the alignment. Select
the sequence by clicking on it. You can select multiple sequences by
holding down the control key. The sequence will then be highlighted in
grey. Note that this option will remove the sequence from the
alignment, but it is still present in your submission.
**Validate Alignment
#Checks for problems with the alignment. If errors are reported, please
review and attempt to fix your alignment before submission.
**Propagate Features
#This function is the same as that available under the Edit Menu in the
record viewer. A full description is available
<A HREF="#FeaturePropagate">
above
</A>
.
*Alignment Assistant View Menu
**Target
#Allows you to select a sequence within the alignment as the target
sequence. This can also be done by double-clicking on the sequence
within the alignment. The SeqID of the target sequence will be
displayed in red. Features can be displayed on the target sequence
only and it is the sequence used for comparison in the
<A HREF="#ShowSubstitutions">
Show Substitutions
</A>
view.
**Show Substitutions
#Changes the alignment view so that identities are replaced with a "."
and only substitutions are shown. The substitutions and identities are
relative to the selected target sequence.
*Alignment Assistant Features Menu
#Allows the annotation of features to a single sequence or all sequences
within the alignment. All features available in this menu are
discussed through the main Sequin Annotate menu.
#Select the feature location by clicking the start location on one of
the sequences, keeping the mouse button depressed, drag the cursor to
the end of the feature location. The selected area will now be
underlined and red and the alignment coordinates of this area will be
displayed in the upper left of the Alignment Assistant window.
**Apply to Target Sequence
#Allows you to choose a feature to be applied only to the target
sequence. The locations may be entered manually or can be determined
based on highlighting the sequence as described above.
**Apply to Alignnent
#Allows you to add the selected feature to all sequences within your
alignment based on the alignment coordinates you have selected. Note
that in the feature pop-up boxes in this menu, the Location will always
be entered as the location relative to the alignment coordinates.
<HR>
<CENTER>
<P> 
<P CLASS=medium1><B>Questions or Comments?</B>
<BR>Write to the <A HREF="mailto:info@ncbi.nlm.nih.gov">NCBI Service
Desk</A></P>
<P CLASS=medium1>Revised October 31, 2007
</CENTER>
<!-- end of content -->
</body>
</html>
|
