-
Notifications
You must be signed in to change notification settings - Fork 3
/
Copy pathprotocol_455.ann
535 lines (535 loc) · 15.5 KB
/
protocol_455.ann
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
T1 Action 347 353 washed
E1 Action:T1
T2 Action 432 439 blasted
E2 Action:T2
T3 Action 663 668 added
E3 Action:T3
T4 Action 686 694 agitated
E4 Action:T4
T5 Action 752 763 homogenized
E5 Action:T5
T6 Action 838 849 centrifuged
E6 Action:T6
T7 Action 892 903 transferred
E7 Action:T7
T8 Action 937 941 spun
E8 Action:T8
T9 Action 968 974 pellet
E9 Action:T9
T10 Action 1102 1110 layering
E10 Action:T10
T11 Action 1288 1299 centrifuged
E11 Action:T11
T12 Action 1405 1416 homogenized
E12 Action:T12
T13 Action 1477 1481 spun
E13 Action:T13
T14 Action 1527 1536 aliquoted
E14 Action:T14
T15 Action 1717 1726 disrupted
E15 Action:T15
T16 Action 1761 1772 centrifuged
E16 Action:T16
T18 Action 2160 2169 enumerate
E18 Action:T18
T19 Action 2079 2088 subjected
E19 Action:T19
T20 Action 2224 2231 treated
E20 Action:T20
T21 Action 2304 2313 extracted
E21 Action:T21
T22 Action 2324 2331 treated
E22 Action:T22
T23 Action 2424 2433 extracted
E23 Action:T23
T24 Action 2618 2624 reduce
E24 Action:T24
T25 Action 2896 2905 underwent
E25 Action:T25
T26 Action 2964 2970 mixing
E26 Action:T26
T27 Action 3141 3150 incubated
E27 Action:T27
T28 Action 3192 3198 placed
E28 Action:T28
T29 Action 3332 3341 incubated
E29 Action:T29
T30 Action 3374 3383 incubated
E30 Action:T30
T31 Action 3462 3468 pooled
E31 Action:T31
T32 Action 3473 3480 cleaned
E32 Action:T32
T33 Action 3520 3526 eluted
E33 Action:T33
T34 Action 3603 3612 amplified
E34 Action:T34
T35 Action 3717 3720 run
E35 Action:T35
T36 Action 3799 3805 pooled
E36 Action:T36
T37 Action 3810 3817 cleaned
E37 Action:T37
T38 Action 3847 3853 eluted
E38 Action:T38
T39 Action 3937 3947 sequencing
E39 Action:T39
T40 Action 4071 4078 convert
E40 Action:T40
T41 Action 4285 4290 added
E41 Action:T41
T42 Action 4341 4350 incubated
E42 Action:T42
T43 Action 4392 4398 placed
E43 Action:T43
T44 Action 4502 4507 added
E44 Action:T44
T45 Action 4512 4521 incubated
E45 Action:T45
T46 Action 4649 4655 adding
E46 Action:T46
T47 Action 4718 4727 underwent
E47 Action:T47
T48 Action 4781 4784 put
E48 Action:T48
T50 Action 4906 4915 incubated
E50 Action:T50
T51 Action 4958 4968 terminated
E51 Action:T51
T52 Action 5251 5257 heated
E52 Action:T52
T53 Action 5299 5305 cooled
E53 Action:T53
T54 Action 5401 5406 added
E54 Action:T54
T55 Action 5567 5578 synthesized
E55 Action:T55
T56 Action 5649 5658 incubated
E56 Action:T56
T57 Action 5865 5874 increased
E57 Action:T57
T58 Action 5935 5941 heated
E58 Action:T58
T59 Action 5989 5995 cooled
E59 Action:T59
T60 Action 6051 6062 synthesized
E60 Action:T60
T61 Action 6169 6179 terminated
E61 Action:T61
T62 Action 6361 6370 incubated
E62 Action:T62
T63 Action 6409 6418 underwent
E63 Action:T63
T64 Action 6472 6478 loaded
E64 Action:T64
T66 Action 6911 6920 incubated
E66 Action:T66
T67 Action 6959 6968 underwent
E67 Action:T67
T68 Action 7013 7020 cleaned
E68 Action:T68
T69 Action 7032 7040 assessed
E69 Action:T69
T70 Action 7076 7083 cleaned
E70 Action:T70
T71 Action 7197 7204 checked
E71 Action:T71
T72 Action 7352 7355 run
E72 Action:T72
T73 Action 7723 7732 processed
E73 Action:T73
T74 Action 7779 7787 imported
E74 Action:T74
T75 Action 7841 7848 trimmed
E75 Action:T75
T76 Action 7865 7872 checked
E76 Action:T76
T77 Action 7961 7967 merged
E77 Action:T77
T78 Action 8161 8169 uploaded
E78 Action:T78
T79 Modifier 357 367 autoclaved
T80 Size 369 376 0.02 μm
T81 Modifier 377 385 filtered
T82 Modifier 386 396 virus-free
T83 Reagent 397 405 seawater
T84 Reagent 332 341 Fragments
T85 Reagent 421 427 tissue
T86 Reagent 449 463 coral skeleton
T87 Device 474 481 air-gun
T88 Amount 488 493 15 mL
T89 Size 494 501 0.02 μm
T90 Modifier 502 510 filtered
T91 Device 512 527 Anotop, Whatman
T92 Reagent 529 538 SM buffer
T93 Concentration 540 546 100 mM
T94 Reagent 547 551 NaCl
T95 Concentration 553 557 8 mM
T96 Reagent 558 563 MgSO4
T97 Concentration 565 570 50 mM
T98 Reagent 571 575 Tris
T99 pH 576 582 pH 7.5
T100 Location 589 601 zip-lock bag
T101 Modifier 603 610 Briefly
T102 Amount 612 616 5 mL
T103 Reagent 620 630 chloroform
T104 Amount 635 640 40 mL
T105 Reagent 644 658 coral blastate
T106 Reagent 673 680 samples
T107 Modifier 695 701 gently
T108 Time 706 709 1 h
T109 Temperature 713 729 room temperature
T110 Speed 767 775 5000 rpm
T111 Time 780 785 1 min
T112 Reagent 731 746 Coral blastates
T113 Device 787 810 Heidolph SilentCrusher™
T114 Reagent 813 820 Samples
T115 Modifier 826 837 immediately
T116 Speed 853 859 1000 g
T117 Time 864 870 15 min
T118 Reagent 876 887 supernatant
T119 Modifier 907 914 sterile
T120 Location 915 932 glass corex tubes
T121 Speed 945 953 12,000 g
T122 Time 958 964 15 min
T123 Modifier 979 987 majority
T124 Reagent 991 1006 microbial cells
T125 Reagent 1043 1058 cesium chloride
T126 Reagent 1060 1064 CsCl
T127 Reagent 1066 1082 density gradient
T128 Action 1092 1098 formed
E79 Action:T128
T129 Amount 1111 1115 1 mL
T130 Numerical 1119 1122 1.7
T131 Numerical 1124 1127 1.5
T132 Generic-Measure 1133 1144 1.35 g mL−1
T133 Reagent 1145 1149 CsCl
T134 Amount 1155 1162 13.2 mL
T135 Location 1163 1196 UltraClear™ ultracentrifuge tubes
T136 Amount 1220 1224 9 mL
T137 Reagent 1225 1231 sample
T138 Action 1232 1239 layered
E80 Action:T138
T139 Location 1247 1250 top
T140 Reagent 1258 1266 gradient
T141 Reagent 1268 1277 Gradients
T142 Amount 1304 1307 2 h
T143 Speed 1311 1319 60,000 g
T144 Temperature 1323 1326 4°C
T145 Speed 1332 1353 swinging bucket rotor
T146 Method 1362 1373 MECH method
T147 Reagent 1379 1400 coral tissue blastate
T148 Speed 1420 1430 10,000 rpm
T149 Time 1435 1440 1 min
T150 Reagent 1446 1467 coral tissue blastate
T151 Speed 1485 1490 400 g
T152 Time 1495 1500 5 min
T153 Reagent 1506 1517 supernatant
T154 Amount 1542 1548 1.5 mL
T155 Reagent 1549 1557 aliquots
T156 Amount 1561 1565 2 mL
T157 Location 1566 1581 eppendorf tubes
T158 Amount 1593 1599 0.3 mL
T159 Modifier 1600 1611 acid-washed
T160 Reagent 1612 1623 glass beads
T161 Size 1625 1635 425–600 μm
T162 Measure-Type 1636 1644 diameter
T163 Location 1667 1672 tubes
T164 Action 1678 1684 placed
E81 Action:T164
T165 Device 1690 1701 bead beater
T166 Reagent 1706 1711 cells
T167 Speed 1730 1738 5000 rpm
T168 Time 1743 1748 5 min
T169 Location 1750 1755 Tubes
T170 Speed 1776 1785 top speed
T171 Device 1791 1821 bench-top Eppendorf centrifuge
T172 Time 1826 1831 1 min
T173 Reagent 1837 1848 supernatant
T174 Action 1853 1862 collected
E82 Action:T174
T175 Method 1867 1886 viral fractionation
T176 Reagent 1893 1920 step CsCl density gradients
T17 Action 1925 1932 confirm
E17 Action:T17
T177 Method 1942 1953 MECH method
T178 Action 1958 1972 not disrupting
E83 Action:T178
T179 Reagent 1973 1988 virus particles
T180 Numerical 1990 1993 two
T181 Reagent 1994 2007 dsDNA viruses
T182 Reagent 2009 2014 OtV-2
T183 Reagent 2043 2049 EhV-86
T184 Modifier 2096 2100 same
T185 Method 2101 2131 mechanical disruption protocol
T186 Device 2133 2147 Flow cytometry
T187 Reagent 2170 2177 viruses
T188 Method 2178 2205 before and after disruption
T189 Modifier 2207 2210 All
T190 Reagent 2211 2218 samples
T191 Reagent 2237 2242 DNase
T192 Reagent 2247 2252 RNase
T194 Method 2271 2294 nucleic acid extraction
T193 Reagent 2296 2299 DNA
T195 Reagent 2318 2323 RNase
T196 Reagent 2340 2354 MasterPure kit
T197 Method 2387 2414 manufacturer's instructions
T198 Reagent 2416 2419 RNA
T199 Reagent 2442 2469 Qiagen QIAamp viral RNA kit
T200 Method 2489 2516 manufacturer's instructions
T201 Modifier 2532 2537 final
T202 Method 2538 2548 DNase step
T203 Modifier 2625 2629 some
T204 Method 2656 2688 multi-displacement amplification
T205 Action 2763 2772 converted
E84 Action:T205
T206 Reagent 2742 2757 DNA extractions
T207 Reagent 2776 2781 dsDNA
T208 Method 2791 2804 amplification
T209 Numerical 2806 2816 Triplicate
T210 Amount 2817 2823 10 μ L
T211 Reagent 2824 2832 aliquots
T212 Reagent 2840 2855 DNA extractions
T213 Reagent 2868 2870 ds
T214 Reagent 2875 2894 ssDNA viral genomes
T215 Method 2924 2939 Klenow reaction
T216 Amount 2971 2978 1.5 μ L
T217 Concentration 2982 2985 10×
T218 Reagent 2986 3001 reaction buffer
T219 Amount 3034 3041 1.5 μ L
T220 Reagent 3045 3050 dNTPs
T221 Concentration 3052 3058 2.5 mM
T222 Reagent 3059 3064 stock
T223 Amount 3067 3072 1 μ L
T224 Reagent 3076 3098 random hexamer primers
T225 Concentration 3100 3109 50 ng/μ L
T226 Reagent 3111 3121 Invitrogen
T227 Temperature 3154 3158 94°C
T228 Time 3163 3168 3 min
T229 Location 3202 3205 ice
T230 Time 3210 3215 3 min
T231 Method 3229 3245 primer annealing
T232 Amount 3247 3252 1 μ L
T233 Reagent 3256 3262 Klenow
T234 Action 3290 3299 incubated
E85 Action:T234
T235 Action 3280 3285 added
E86 Action:T235
T236 Temperature 3303 3307 25°C
T237 Time 3312 3318 10 min
T238 Temperature 3345 3349 37°C
T239 Time 3354 3360 60 min
T240 Method 3391 3407 termination step
T241 Temperature 3411 3415 75°C
T242 Time 3420 3426 20 min
T243 Reagent 3489 3515 Qiagen QIAamp DNA mini kit
T244 Reagent 3447 3456 reactions
T245 Amount 3530 3535 50 μL
T246 Reagent 3539 3548 Buffer AE
T247 Action 3550 3559 Replicate
E87 Action:T247
T248 Reagent 3560 3573 MDA reactions
T249 Amount 3619 3626 2.5 μ L
T250 Reagent 3627 3641 dsDNA template
T251 Reagent 3650 3668 Qiagen RepliG® kit
T252 Method 3679 3696 standard protocol
T253 Modifier 3698 3701 All
T254 Method 3702 3711 reactions
T255 Concentration 3726 3730 0.8%
T256 Reagent 3731 3742 agarose gel
T257 Concentration 3746 3748 1×
T258 Reagent 3749 3752 TAE
T259 Generic-Measure 3756 3761 100 V
T260 Time 3766 3772 30 min
T261 Action 3776 3783 confirm
E88 Action:T261
T262 Method 3784 3797 amplification
T263 Reagent 3823 3842 QIAampl DNA minikit
T264 Amount 3857 3864 200 μ L
T265 Reagent 3868 3877 Buffer AE
T266 Action 3951 3958 confirm
E89 Action:T266
T267 Method 4012 4028 RepliG® protocol
T268 Reagent 4030 4045 Klenow Fragment
T269 Reagent 4083 4094 DNA genomes
T270 Reagent 4098 4103 dsDNA
T271 Reagent 4110 4126 RP-SISPA primers
T272 Reagent 4134 4160 3′ random hexamer sequence
T273 Method 4189 4206 PCR amplification
T274 Action 4211 4216 label
E90 Action:T274
T275 Modifier 4221 4226 first
T276 Reagent 4227 4233 strand
T277 Reagent 4243 4258 RP-SISPA primer
T278 Amount 4260 4264 5 μL
T279 Reagent 4268 4280 nucleic acid
T280 Amount 4294 4298 9 μL
T281 Reagent 4299 4311 reaction mix
T282 Reagent 4328 4336 reaction
T283 Temperature 4354 4358 94°C
T284 Time 4363 4368 3 min
T285 Reagent 4374 4382 reaction
T286 Location 4402 4405 ice
T287 Time 4410 4415 3 min
T288 Method 4429 4445 primer annealing
T289 Amount 4447 4451 1 μL
T290 Reagent 4455 4470 Klenow Fragment
T291 Temperature 4525 4529 37°C
T292 Time 4534 4540 60 min
T293 Reagent 4560 4584 Klenow Fragment reaction
T294 Reagent 4632 4644 SISPA primer
T295 Amount 4670 4674 1 μL
T296 Reagent 4678 4684 primer
T297 Reagent 4694 4698 dNTP
T298 Amount 4689 4693 1 μL
T299 Reagent 4704 4712 reaction
T300 Temperature 4730 4734 94°C
T301 Time 4739 4744 3 min
T302 Time 4796 4801 3 min
T303 Location 4788 4791 ice
T304 Reagent 4763 4771 reaction
T305 Reagent 4807 4815 reaction
T306 Amount 4851 4855 1 μL
T307 Reagent 4859 4874 Klenow Fragment
T308 Temperature 4919 4923 37°C
T309 Time 4928 4934 60 min
T310 Reagent 4893 4901 reaction
T311 Action 4839 4847 addition
E91 Action:T311
T49 Temperature 4972 4976 75°C
T312 Time 4981 4987 20 min
T313 Reagent 4940 4948 reaction
T314 Modifier 4989 4996 Briefly
T315 Action 5072 5077 mixed
E49 Action:T315
T316 Modifier 5040 5048 purified
T317 Reagent 5049 5067 RNA viral template
T318 Amount 5033 5039 10 μ L
T319 Amount 5083 5088 1 μ L
T320 Concentration 5092 5098 2.5 mM
T321 Reagent 5099 5104 dNTPs
T322 Amount 5109 5116 1.3 μ L
T323 Reagent 5176 5191 FR40RV-T primer
T324 Reagent 5120 5128 FR26RV-N
T325 Concentration 5158 5164 10 μ M
T326 Concentration 5220 5225 50 nM
T327 Reagent 5017 5031 cDNA synthesis
T328 Reagent 5238 5246 reaction
T329 Temperature 5261 5265 65°C
T330 Time 5270 5275 5 min
T331 Reagent 5281 5289 reaction
T332 Location 5309 5312 ice
T333 Time 5317 5322 3 min
T334 Reagent 5336 5343 primers
T335 Action 5347 5353 anneal
E92 Action:T335
T336 Location 5370 5373 ice
T337 Amount 5375 5379 1 μL
T338 Reagent 5380 5383 DTT
T339 Reagent 5414 5422 reaction
T340 Reagent 5429 5457 enzyme stabilization reagent
T341 Amount 5463 5467 1 μL
T342 Reagent 5468 5477 RNase OUT
T343 Action 5494 5501 protect
E93 Action:T343
T344 Reagent 5506 5512 sample
T345 Method 5518 5532 RNAse activity
T346 Modifier 5538 5551 complementary
T347 Reagent 5552 5562 DNA strand
T348 Generic-Measure 5584 5589 200 U
T349 Reagent 5593 5630 Superscript III reverse transcriptase
T350 Reagent 5636 5644 reaction
T351 Modifier 5659 5668 initially
T352 Temperature 5672 5676 25°C
T353 Time 5681 5687 10 min
T355 Reagent 5714 5723 hexamer 3
T354 Action 5697 5706 annealing
E94 Action:T354
T356 Reagent 5732 5747 primer FR26RV-N
T357 Reagent 5756 5772 poly(T)20 3′ end
T358 Reagent 5776 5791 primer FR40RV-T
T359 Action 5799 5807 template
E95 Action:T359
T360 Method 5814 5828 cDNA synthesis
T361 Measure-Type 5844 5855 temperature
T362 Temperature 5878 5882 50°C
T363 Reagent 5887 5893 60 min
T364 Modifier 5899 5904 first
T365 Reagent 5905 5930 strand synthesis reaction
T366 Modifier 5942 5953 immediately
T367 Temperature 5957 5961 94°C
T368 Time 5966 5971 3 min
T369 Modifier 5981 5988 rapidly
T370 Location 5999 6002 ice
T371 Modifier 6006 6019 complementary
T372 Reagent 6020 6033 second strand
T373 Modifier 6038 6050 subsequently
T374 Temperature 6066 6070 37°C
T375 Time 6075 6081 60 min
T376 Action 6091 6099 addition
E96 Action:T376
T377 Amount 6103 6107 1 μL
T378 Reagent 6108 6123 Klenow Fragment
T379 Reagent 6149 6164 Klenow reaction
T380 Action 6193 6203 incubation
E97 Action:T380
T381 Modifier 6187 6192 final
T382 Temperature 6207 6211 75°C
T383 Time 6216 6222 20 min
T384 Action 6224 6241 PCR amplification
E98 Action:T384
T385 Reagent 6249 6278 SISPA primer labeled template
T386 Numerical 6305 6315 triplicate
T387 Amount 6316 6321 25 μL
T388 Reagent 6322 6331 reactions
T389 Reagent 6348 6356 reaction
T390 Temperature 6374 6378 95°C
T391 Time 6383 6389 10 min
T392 Reagent 6395 6403 reaction
T393 Generic-Measure 6419 6428 30 cycles
T394 Method 6432 6444 denaturation
T395 Reagent 6453 6466 PCR reactions
T396 Concentration 6487 6491 0.8%
T397 Reagent 6492 6503 agarose gel
T398 Concentration 6507 6509 1×
T399 Reagent 6509 6512 TAE
T400 Generic-Measure 6516 6521 100 V
T401 Time 6526 6532 30 min
T65 Reagent 6898 6906 reaction
T402 Temperature 6924 6928 95°C
T403 Time 6933 6939 10 min
T404 Reagent 6945 6953 reaction
T405 Generic-Measure 6969 6977 5 cycles
T406 Method 6981 6993 denaturation
T407 Reagent 6998 7007 Reactions
T408 Measure-Type 7025 7027 QC
T409 Reagent 7063 7070 samples
T410 Reagent 7091 7111 QIAamp® DNA Mini kit
T411 Reagent 7141 7171 MinElute® PCR purification kit
T412 Reagent 7184 7191 Samples
T413 Measure-Type 7209 7223 quantification
T414 Reagent 7232 7255 Quant-iT PicoGreen® kit
T415 Device 7261 7292 NanoDrop 3300 fluorospecrometer
T416 Measure-Type 7298 7305 quality
T417 Generic-Measure 7307 7321 260:280 ratios
T418 Device 7328 7341 NanoDrop 2000
T419 Concentration 7361 7365 0.8%
T420 Reagent 7366 7377 agarose gel
T421 Concentration 7381 7383 1×
T422 Reagent 7384 7387 TAE
T423 Generic-Measure 7391 7396 100 V
T424 Time 7401 7407 30 min
T425 Action 7411 7418 confirm
E65 Action:T425
T426 Modifier 7432 7443 appropriate
T427 Action 7550 7559 sequenced
E99 Action:T427
T428 Reagent 7533 7544 metagenomes
T429 Modifier 7529 7532 All
T430 Device 7566 7582 Nextera XT MiSeq
T431 Generic-Measure 7583 7589 250 bp
T432 Method 7590 7611 paired-end sequencing
T433 Reagent 7699 7717 Raw sequence reads
T434 Device 7736 7762 CLC Genomics Workbench 5.5
T435 Reagent 7764 7773 Sequences
T436 Reagent 7791 7816 Illumina paired-end reads