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
|
<!DOCTYPE html>
<html xmlns="http://www.w3.org/1999/xhtml" lang="" xml:lang="">
<head>
<meta charset="utf-8" />
<meta name="generator" content="pandoc" />
<meta name="viewport" content="width=device-width, initial-scale=1.0, user-scalable=yes" />
<title>Restriction</title>
<style>
html {
line-height: 1.5;
font-family: Georgia, serif;
font-size: 20px;
color: #1a1a1a;
background-color: #fdfdfd;
}
body {
margin: 0 auto;
max-width: 36em;
padding-left: 50px;
padding-right: 50px;
padding-top: 50px;
padding-bottom: 50px;
hyphens: auto;
overflow-wrap: break-word;
text-rendering: optimizeLegibility;
font-kerning: normal;
}
@media (max-width: 600px) {
body {
font-size: 0.9em;
padding: 1em;
}
}
@media print {
body {
background-color: transparent;
color: black;
font-size: 12pt;
}
p, h2, h3 {
orphans: 3;
widows: 3;
}
h2, h3, h4 {
page-break-after: avoid;
}
}
p {
margin: 1em 0;
}
a {
color: #1a1a1a;
}
a:visited {
color: #1a1a1a;
}
img {
max-width: 100%;
}
h1, h2, h3, h4, h5, h6 {
margin-top: 1.4em;
}
h5, h6 {
font-size: 1em;
font-style: italic;
}
h6 {
font-weight: normal;
}
ol, ul {
padding-left: 1.7em;
margin-top: 1em;
}
li > ol, li > ul {
margin-top: 0;
}
blockquote {
margin: 1em 0 1em 1.7em;
padding-left: 1em;
border-left: 2px solid #e6e6e6;
color: #606060;
}
code {
font-family: Menlo, Monaco, 'Lucida Console', Consolas, monospace;
font-size: 85%;
margin: 0;
}
pre {
margin: 1em 0;
overflow: auto;
}
pre code {
padding: 0;
overflow: visible;
overflow-wrap: normal;
}
.sourceCode {
background-color: transparent;
overflow: visible;
}
hr {
background-color: #1a1a1a;
border: none;
height: 1px;
margin: 1em 0;
}
table {
margin: 1em 0;
border-collapse: collapse;
width: 100%;
overflow-x: auto;
display: block;
font-variant-numeric: lining-nums tabular-nums;
}
table caption {
margin-bottom: 0.75em;
}
tbody {
margin-top: 0.5em;
border-top: 1px solid #1a1a1a;
border-bottom: 1px solid #1a1a1a;
}
th {
border-top: 1px solid #1a1a1a;
padding: 0.25em 0.5em 0.25em 0.5em;
}
td {
padding: 0.125em 0.5em 0.25em 0.5em;
}
header {
margin-bottom: 4em;
text-align: center;
}
#TOC li {
list-style: none;
}
#TOC a:not(:hover) {
text-decoration: none;
}
code{white-space: pre-wrap;}
span.smallcaps{font-variant: small-caps;}
span.underline{text-decoration: underline;}
div.column{display: inline-block; vertical-align: top; width: 50%;}
div.hanging-indent{margin-left: 1.5em; text-indent: -1.5em;}
ul.task-list{list-style: none;}
pre > code.sourceCode { white-space: pre; position: relative; }
pre > code.sourceCode > span { display: inline-block; line-height: 1.25; }
pre > code.sourceCode > span:empty { height: 1.2em; }
.sourceCode { overflow: visible; }
code.sourceCode > span { color: inherit; text-decoration: inherit; }
div.sourceCode { margin: 1em 0; }
pre.sourceCode { margin: 0; }
@media screen {
div.sourceCode { overflow: auto; }
}
@media print {
pre > code.sourceCode { white-space: pre-wrap; }
pre > code.sourceCode > span { text-indent: -5em; padding-left: 5em; }
}
pre.numberSource code
{ counter-reset: source-line 0; }
pre.numberSource code > span
{ position: relative; left: -4em; counter-increment: source-line; }
pre.numberSource code > span > a:first-child::before
{ content: counter(source-line);
position: relative; left: -1em; text-align: right; vertical-align: baseline;
border: none; display: inline-block;
-webkit-touch-callout: none; -webkit-user-select: none;
-khtml-user-select: none; -moz-user-select: none;
-ms-user-select: none; user-select: none;
padding: 0 4px; width: 4em;
color: #aaaaaa;
}
pre.numberSource { margin-left: 3em; border-left: 1px solid #aaaaaa; padding-left: 4px; }
div.sourceCode
{ }
@media screen {
pre > code.sourceCode > span > a:first-child::before { text-decoration: underline; }
}
code span.al { color: #ff0000; font-weight: bold; } /* Alert */
code span.an { color: #60a0b0; font-weight: bold; font-style: italic; } /* Annotation */
code span.at { color: #7d9029; } /* Attribute */
code span.bn { color: #40a070; } /* BaseN */
code span.bu { } /* BuiltIn */
code span.cf { color: #007020; font-weight: bold; } /* ControlFlow */
code span.ch { color: #4070a0; } /* Char */
code span.cn { color: #880000; } /* Constant */
code span.co { color: #60a0b0; font-style: italic; } /* Comment */
code span.cv { color: #60a0b0; font-weight: bold; font-style: italic; } /* CommentVar */
code span.do { color: #ba2121; font-style: italic; } /* Documentation */
code span.dt { color: #902000; } /* DataType */
code span.dv { color: #40a070; } /* DecVal */
code span.er { color: #ff0000; font-weight: bold; } /* Error */
code span.ex { } /* Extension */
code span.fl { color: #40a070; } /* Float */
code span.fu { color: #06287e; } /* Function */
code span.im { } /* Import */
code span.in { color: #60a0b0; font-weight: bold; font-style: italic; } /* Information */
code span.kw { color: #007020; font-weight: bold; } /* Keyword */
code span.op { color: #666666; } /* Operator */
code span.ot { color: #007020; } /* Other */
code span.pp { color: #bc7a00; } /* Preprocessor */
code span.sc { color: #4070a0; } /* SpecialChar */
code span.ss { color: #bb6688; } /* SpecialString */
code span.st { color: #4070a0; } /* String */
code span.va { color: #19177c; } /* Variable */
code span.vs { color: #4070a0; } /* VerbatimString */
code span.wa { color: #60a0b0; font-weight: bold; font-style: italic; } /* Warning */
.display.math{display: block; text-align: center; margin: 0.5rem auto;}
</style>
</head>
<body>
<h1 id="working-with-restriction-enzymes">Working with restriction enzymes</h1>
<h2 id="table-of-contents">Table of contents</h2>
<ol class="incremental" type="1">
<li><a href="#1">The restriction enzymes classes</a>
<ol class="incremental" type="1">
<li><a href="#1.1">Importing the enzymes</a></li>
<li><a href="#1.2">Naming convention</a></li>
<li><a href="#1.3">Searching for restriction sites</a></li>
<li><a href="#1.4">Retrieving the sequences produced by a digestion</a></li>
<li><a href="#1.5">Analysing circular sequences</a></li>
<li><a href="#1.6">Comparing enzymes with each others</a></li>
<li><a href="#1.7">Other facilities provided by the enzyme classes</a></li>
</ol></li>
<li><a href="#2">The RestrictionBatch class: a class to deal with several enzymes</a>
<ol class="incremental" type="1">
<li><a href="#2.1">Creating a RestrictionBatch</a></li>
<li><a href="#2.2">Restricting a RestrictionBatch to a particular supplier</a></li>
<li><a href="#2.3">Adding enzymes to a RestrictionBatch</a></li>
<li><a href="#2.4">Removing enzymes from a RestrictionBatch</a></li>
<li><a href="#2.5">Manipulating RestrictionBatch</a></li>
<li><a href="#2.6">Analysing sequences with a RestrictionBatch</a></li>
<li><a href="#2.7">Other RestrictionBatch methods</a></li>
</ol></li>
<li><a href="#3">AllEnzymes and CommOnly: two preconfigured RestrictionBatches</a></li>
<li><a href="#4">The Analysis class: even simpler restriction analysis</a>
<ol class="incremental" type="1">
<li><a href="#4.1">Setting up an Analysis</a></li>
<li><a href="#4.2">Full restriction analysis</a></li>
<li><a href="#4.3">Changing the title</a></li>
<li><a href="#4.4">Customising the output</a></li>
<li><a href="#4.5">Fancier restriction analysis</a></li>
<li><a href="#4.6">More complex analysis</a></li>
</ol></li>
<li><a href="#5">Advanced features: the FormattedSeq class</a>
<ol class="incremental" type="1">
<li><a href="#5.1">Creating a FormattedSeq</a></li>
<li><a href="#5.2">Unlike Bio.Seq, FormattedSeq retains information about their shape</a></li>
<li><a href="#5.3">Changing the shape of a FormattedSeq</a></li>
<li><a href="#5.4">Using / and // operators with FormattedSeq</a></li>
</ol></li>
<li><a href="#6">More advanced features</a>
<ol class="incremental" type="1">
<li><a href="#6.1">Updating the enzymes from Rebase</a>
<ol class="incremental" type="1">
<li><a href="#6.1.1">Fetching the recent enzyme files manually from Rebase</a></li>
<li><a href="#6.1.2">Fetching the recent enzyme files with rebase_update.py</a></li>
<li><a href="#6.1.3">Compiling a new dictionary with ranacompiler.py</a></li>
</ol></li>
<li><a href="#6.2">Subclassing the class Analysis</a></li>
</ol></li>
<li><a href="#7">Limitation and caveat</a>
<ol class="incremental" type="1">
<li><a href="#7.1">All DNA are non methylated</a></li>
<li><a href="#7.2">No support for star activity</a></li>
<li><a href="#7.3">Safe to use with degenerated DNA</a></li>
<li><a href="#7.4">Non standard bases in DNA are not allowed</a></li>
<li><a href="#7.5">Sites found at the edge of linear DNA might not be accessible in a real digestion</a></li>
<li><a href="#7.6">Restriction reports cutting sites not enzyme recognition sites</a></li>
</ol></li>
<li><a href="#8">Annexe: modifying dir() to use with from Bio.Restriction import *</a></li>
</ol>
<h3 id="the-restriction-enzymes-classes"><a name="1"></a>1. The restriction enzymes classes</h3>
<p>The restriction enzyme package is situated in <code>Bio.Restriction</code>. This package will allow you to work with restriction enzymes and realise restriction analysis on your sequence. Restriction make use of the facilities offered by <strong>REBASE</strong> and contains classes for more than 800 restriction enzymes. This chapter will lead you through a quick overview of the facilities offered by the <code>Restriction</code> package of Biopython. The chapter is constructed as an interactive Python session and the best way to read it is with a Python shell open alongside you.</p>
<h4 id="importing-the-enzymes"><a name="1.1"></a> 1.1 Importing the enzymes</h4>
<p>To import the enzymes, open a Python shell and type:</p>
<pre class="pycon"><code>>>> from Bio import Restriction
>>> dir()
['Restriction', '__annotations__', '__builtins__', '__doc__', '__loader__', '__name__', '__package__', '__spec__']
>>> Restriction.EcoRI
EcoRI
>>> Restriction.EcoRI.site
'GAATTC'
>>></code></pre>
<p>You will certainly notice that the package is quite slow to load. This is normal as each enzyme possess its own class and there is a lot of them. This will not affect the speed of Python after the initial import.</p>
<p>I don’t know for you but I find it quite cumbersome to have to prefix each operation with <code>Restriction.</code>, so here is another way to import the package.</p>
<pre class="pycon"><code>>>> from Bio.Restriction import *
>>> EcoRI
EcoRI
>>> EcoRI.site
'GAATTC'
>>></code></pre>
<p>However, this method has one big disadvantage: It is almost impossible to use the command <code>dir()</code> anymore as there is so much enzymes the results is hardly readable. A workaround is provided at the end of this tutorial. I let you decide which method you prefer. But in this tutorial I will use the second. If you prefer the first method you will need to prefix each call to a restriction enzyme with <code>Restriction.</code> in the remaining of the tutorial.</p>
<h4 id="naming-convention"><a name="1.2"></a>1.2 Naming convention</h4>
<p>To access an enzyme simply enter its name. You must respect the usual naming convention with the upper case letters and Latin numbering (in upper case as well):</p>
<pre class="pycon"><code>>>> EcoRI
EcoRI
>>> ecori
Traceback (most recent call last):
File "<pyshell#25>", line 1, in -toplevel-
ecori
NameError: name 'ecori' is not defined
>>> EcoR1
Traceback (most recent call last):
File "<pyshell#26>", line 1, in -toplevel-
EcoR1
NameError: name 'EcoR1' is not defined
>>> KpnI
KpnI
>>></code></pre>
<p><code>ecori</code> or <code>EcoR1</code> are not enzymes, <code>EcoRI</code> and <code>KpnI</code> are.</p>
<h4 id="searching-for-restriction-sites"><a name="1.3"></a>1.3 Searching for restriction sites</h4>
<p>So what can we do with these restriction enzymes? To see that we will need a DNA sequence. Restriction enzymes support both <code>Bio.Seq.MutableSeq</code>and <code>Bio.Seq.Seq</code> objects. Your sequence must comply with the IUPAC alphabet. That means using A, C, G and T or U, plus N for any base, and various other standard codes like S for C or G, and V for A, C or G.</p>
<pre class="pycon"><code>>>> from Bio.Seq import Seq
>>> my_seq = Seq('AAAAAAAAAAAAAA')</code></pre>
<p>Searching a sequence for the presence of restriction site for your preferred enzyme is as simple as:</p>
<pre class="pycon"><code>>>> EcoRI.search(my_seq)
[]</code></pre>
<p>The results is a list. Here the list is empty since there is obviously no EcoRI site in <em>my_seq</em>. Let’s try to get a sequence with an EcoRI site.</p>
<pre class="pycon"><code>>>> ecoseq = my_seq + Seq(EcoRI.site) + my_seq
>>> ecoseq
Seq('AAAAAAAAAAAAAAGAATTCAAAAAAAAAAAAAA')
>>> EcoRI.search(ecoseq)
[16]</code></pre>
<p>We therefore have a site at position 16 of the sequence <em>ecoseq</em>. The position returned by the method search is the first base of the downstream segment produced by a restriction (i.e. the first base after the position where the enzyme will cut). The <code>Restriction</code> package follows biological convention (the first base of a sequence is base 1). No need to make difficult conversions between your recorded biological data and the results produced by the enzymes in this package.</p>
<h4 id="retrieving-the-sequences-produced-by-a-digestion"><a name="1.4"></a>1.4 Retrieving the sequences produced by a digestion</h4>
<p><code>Seq</code> objects as all Python sequences, have different conventions and the first base of a sequence is base 0. Therefore to get the sequences produced by an EcoRI digestion of <em>ecoseq</em>, one should do the following:</p>
<pre class="pycon"><code>>>> ecoseq[:15], ecoseq[15:]
(Seq('AAAAAAAAAAAAAAG'), Seq('AATTCAAAAAAAAAAAAAA'))</code></pre>
<p>I hear you thinking “this is a cumbersome and error prone method to get these sequences”. To simplify your life, <code>Restriction</code> provides another method to get these sequences without hassle: <code>catalyse</code>. This method will return a tuple containing all the fragments produced by a complete digestion of the sequence. Using it is as simple as before:</p>
<pre class="pycon"><code>>>> EcoRI.catalyse(ecoseq)
(Seq('AAAAAAAAAAAAAAG'), Seq('AATTCAAAAAAAAAAAAAA'))</code></pre>
<p>BTW, you can also use spell it the American way <code>catalyze</code>:</p>
<pre class="pycon"><code>>>> EcoRI.catalyze(ecoseq)
(Seq('AAAAAAAAAAAAAAG'), Seq('AATTCAAAAAAAAAAAAAA'))</code></pre>
<h4 id="analysing-circular-sequences"><a name="1.5"></a>1.5 Analysing circular sequences</h4>
<p>Now, if you have entered the previous command in your shell you may have noticed that both <code>search</code> and <code>catalyse</code> can take a second argument <code>linear</code> which defaults to <code>True</code>. Using this will allow you to simulate circular sequences such as plasmids. Setting <code>linear</code> to <code>False</code> informs the enzyme to make the search over a circular sequence and to search for potential sites spanning over the boundaries of the sequence.</p>
<pre class="pycon"><code>>>> EcoRI.search(ecoseq, linear=False)
[16]
>>> EcoRI.catalyse(ecoseq, linear=False)
(Seq('AATTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAG'),)
>>> ecoseq # for memory
Seq('AAAAAAAAAAAAAAGAATTCAAAAAAAAAAAAAA')</code></pre>
<p>OK, this is quite a difference, we only get one fragment, which correspond to the linearised sequence. The beginning sequence has been shifted to take this fact into account. Moreover we can see another difference:</p>
<pre class="pycon"><code>>>> new_seq = Seq('TTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAA')
>>> EcoRI.search(new_seq)
[]
>>> EcoRI.search(new_seq, linear=False)
[33]</code></pre>
<p>As you can see using <code>linear=False</code>, make a site appearing in the sequence <em>new_seq</em>. This site does not exist in a linear sequence as the EcoRI site is split into two halves at the start and the end of the sequence. In a circular sequence however, the site is effectively present when the beginning and end of the sequence are joined.</p>
<h4 id="comparing-enzymes-with-each-others"><a name="1.6"></a>1.6 Comparing enzymes with each others</h4>
<p><code>Restriction</code> enzymes define 4 comparative operators <code>==</code>, <code>!=</code>, <code>>></code> and <code>%</code>. All these operator compares two enzymes together and either return <code>True</code> or <code>False</code>.</p>
<dl class="incremental">
<dt><code>==</code> (test identity)</dt>
<dd>It will return <code>True</code> if the two sides of the operator are the same. *Same” is defined as: same name, same site, same overhang (i.e. the only thing which is equal to <code>EcoRI</code> is <code>EcoRI</code>).
</dd>
<dt><code>!=</code> (test for different site or cutting)</dt>
<dd>It will return <code>True</code> if the two sides of the operator are different. Two enzymes are not different if the result produced by one enzyme will always be the same as the result produced by the other (i.e. true isoschizomers will not being the same enzymes, are not different since they are interchangeable).
</dd>
<dt><code>>></code> (test for neoschizomer)</dt>
<dd><code>True</code> if the enzymes recognise the same site, but cut it in a different way (i.e. the enzymes are neoschizomers).
</dd>
<dt><code>%</code> (test compatibilty)</dt>
<dd>Test the compatibility of the ending produced by the enzymes (will be <code>True</code> if the fragments produced with one of the enzyme can directly be ligated to fragments produced by the other).
</dd>
</dl>
<p>Let’s use <code>Acc65I</code> and its isoschizomers as example:</p>
<pre class="pycon"><code>>>> Acc65I.isoschizomers()
[Asp718I, KpnI]
>>> Acc65I.elucidate()
'G^GTAC_C'
>>> Asp718I.elucidate()
'G^GTAC_C'
>>> KpnI.elucidate()
'G_GTAC^C'
>>> # Asp718I and Acc65I are true isoschizomers,
>>> # they recognise the same site and cut it the
>>> # same way.
>>> # KpnI is a neoschizomers of the 2 others.
>>> # Here are the results of the 4 operators
>>> # for each pair of enzymes:
>>>
>>> ############# x == y (x is y)
>>> Acc65I == Acc65I # same enzyme => True
True
>>> Acc65I == KpnI # all other cases => False
False
>>> Acc65I == Asp718I
False
>>> Acc65I == EcoRI
False
>>> ############ x != y (x and y are not true isoschizomers)
>>> Acc65I != Acc65I # same enzyme => False
False
>>> Acc65I != Asp718I # different enzymes, but cut same manner => False
False
>>> Acc65I != KpnI # all other cases => True
True
>>> Acc65I != EcoRI
True
>>> ########### x >> y (x is neoschizomer of y)
>>> Acc65I >> Acc65I # same enzyme => False
False
>>> Acc65I >> Asp718I # same site, same cut => False
False
>>> Acc65I >> EcoRI # different site => False
False
>>> Acc65I >> KpnI # same site, different cut => True
True
>>> ########### x % y (fragments produced by x and fragments produced by y
>>> # can be directly ligated to each other)
>>> Acc65I % Asp718I
True
>>> Acc65I % Acc65I
True
>>> Acc65I % KpnI # KpnI -> '3 overhang, Acc65I-> 5' overhang => False
False
>>>
>>> SunI.elucidate()
'C^GTAC_G'
>>> SunI == Acc65I
False
>>> SunI != Acc65I
True
>>> SunI >> Acc65I
False
>>> SunI % Acc65I # different site, same overhang (5' GTAC) => True
True
>>> SmaI % EcoRV # 2 Blunt enzymes, all blunt enzymes are compatible => True
True</code></pre>
<h4 id="other-facilities-provided-by-the-enzyme-classes"><a name="1.7"></a>1.7 Other facilities provided by the enzyme classes</h4>
<p>The <code>Restriction</code> class provides quite a number of others methods. We will not go through all of them, but only have a quick look to the most useful ones.</p>
<p>Not all enzymes possess the same properties when it comes to the way they digest a DNA. If you want to know more about the way a particular enzyme cut you can use the three following methods. They are fairly straightforward to understand and refer to the ends that the enzyme produces: blunt, 5’ overhanging (also called 3’ recessed) sticky end and 3’ overhanging (or 5’ recessed) sticky end.</p>
<pre class="pycon"><code>>>> EcoRI.is_blunt()
False
>>> EcoRI.is_5overhang()
True
>>> EcoRI.is_3overhang()
False</code></pre>
<p>A more detailled view of the restriction site can be produced using the <code>elucidate()</code> method. The <code>^</code> refers to the position of the cut in the sense strand of the sequence, <code>_</code> to the cut on the antisense or complementary strand. <code>^_</code> means blunt.</p>
<pre class="pycon"><code>>>> EcoRI.elucidate()
'G^AATT_C'
>>> KpnI.elucidate()
'G_GTAC^C'
>>> EcoRV.elucidate()
'GAT^_ATC'</code></pre>
<p>The method <code>frequency()</code> will give you the statistical frequency of the enzyme site.</p>
<pre class="pycon"><code>>>> EcoRI.frequency()
4096
>>> XhoII.elucidate()
'R^GATC_Y'
>>> XhoII.frequency()
1024</code></pre>
<p>To get the length of a the recognition sequence of an enzyme use the built-in function <code>len()</code>:</p>
<pre class="pycon"><code>>>> len(EcoRI)
6
>>> BstXI.elucidate()
'CCAN_NNNN^NTGG'
>>> len(BstXI)
12
>>> FokI.site
'GGATG'
>>> FokI.elucidate() # FokI cut well outside its recognition site
'GGATGNNNNNNNNN^NNNN_N'
>>> len(FokI) # its length is the length of the recognition site
5</code></pre>
<p>Also interesting are the methods dealing with isoschizomers. For memory, two enzymes are <em>isoschizomers</em> if they share a same recognition site. A further division is made between isoschizomers (same name, recognise the same sequence and cut the same way) and <em>neoschizomers</em> which cut at different positions. <em>Equischizomer</em> is an arbitrary choice to design “isoschizomers_that_are_not_neoschizomers” as this last one was a bit long. Another set of method <code>one_enzyme.is_*schizomers(one_other_enzyme)</code>, allow to test 2 enzymes against each other.</p>
<pre class="pycon"><code>>>> Acc65I.isoschizomers()
[Asp718I, KpnI]
>>> Acc65I.neoschizomers()
[KpnI]
>>> Acc65I.equischizomers()
[Asp718I]
>>> KpnI.elucidate()
'G_GTAC^C'
>>> Acc65I.elucidate()
'G^GTAC_C'
>>> KpnI.is_neoschizomer(Acc65I)
True
>>> KpnI.is_neoschizomer(KpnI)
False
>>> KpnI.is_isoschizomer(Acc65I)
True
>>> KpnI.is_isoschizomer(KpnI)
True
>>> KpnI.is_equischizomer(Acc65I)
False
>>> KpnI.is_equischizomer(KpnI)
True</code></pre>
<p><code>suppliers()</code> will get you the list of all the suppliers of the enzyme. <code>all_suppliers()</code> will give you all the suppliers in the database.</p>
<h3 id="the-restrictionbatch-class-a-class-to-deal-with-several-enzymes"><a name="2"></a>2. The RestrictionBatch class: a class to deal with several enzymes</h3>
<p>If you want to make a restriction map of a sequence, using individual enzymes can become tedious and will endures a big overhead due to the repetitive conversion of the sequence to a <code>FormattedSeq</code> (see <a href="#5">Chapter 5</a>). <code>Restriction</code> provides a class to make easier the use of large number of enzymes in one go: <code>RestrictionBatch</code>. <code>RestrictionBatch</code> will help you to manipulate lots of enzymes with a single command. Moreover all the enzymes in the restriction batch will share the same converted sequence, reducing the overhead.</p>
<h4 id="creating-a-restrictionbatch"><a name="2.1"></a><span class="mozTocH4"></span>2.1 Creating a RestrictionBatch</h4>
<p>You can initiate a restriction batch by passing it a list of enzymes or enzyme names as argument.</p>
<pre class="pycon"><code>>>> rb = RestrictionBatch([EcoRI])
>>> rb
RestrictionBatch(['EcoRI'])
>>> rb2 = RestrictionBatch(['EcoRI'])
>>> rb2
RestrictionBatch(['EcoRI'])
>>> rb == rb2
True</code></pre>
<p>Adding a new enzyme to a restriction batch is easy:</p>
<pre class="pycon"><code>>>> rb.add(KpnI)
>>> rb
RestrictionBatch(['EcoRI', 'KpnI'])
>>> rb += EcoRV
>>> rb
RestrictionBatch(['EcoRI', 'EcoRV', 'KpnI'])])</code></pre>
<p>Another way to create a RestrictionBatch is by simply adding restriction enzymes together, this is particularly useful for small batches:</p>
<pre class="pycon"><code>>>> rb3 = EcoRI + KpnI + EcoRV
>>> rb3
RestrictionBatch(['EcoRI', 'EcoRV', 'KpnI'])</code></pre>
<h4 id="restricting-a-restrictionbatch-to-a-particular-supplier"><a name="2.2"></a>2.2 Restricting a RestrictionBatch to a particular supplier</h4>
<p>The Restriction package is based upon the <strong>REBASE</strong> database. This database gives a list of suppliers for each enzyme. It would be a shame not to make use of this facility. You can produce a <code>RestrictionBatch</code> containing only enzymes from one or a few supplier(s). Here is how to do it:</p>
<pre class="pycon"><code>>>> rb_supp = RestrictionBatch(first=[], suppliers=['C','B','E','I','K','J','M',
'O','N','Q','S','R','V','Y','X'])
>>> # This will create a RestrictionBatch with the all enzymes which possess a s
upplier.
>>> len(rb_supp) # May 2020
621</code></pre>
<p>The argument <code>suppliers</code> take a list of one or several single letter codes corresponding to the supplier(s). The codes are the same as defined in REBASE. As it would be a pain to have to remember each supplier code, <code>RestrictionBatch</code> provides a method which show the pair code <=> supplier:</p>
<pre class="pycon"><code>>>> RestrictionBatch.show_codes() # as of May 2016 REBASE release.
C = Minotech Biotechnology
B = Life Technologies
E = Agilent Technologies
I = SibEnzyme Ltd.
K = Takara Bio Inc.
J = Nippon Gene Co., Ltd.
M = Roche Applied Science
O = Toyobo Biochemicals
N = New England Biolabs
Q = Molecular Biology Resources - CHIMERx
S = Sigma Chemical Corporation
R = Promega Corporation
V = Vivantis Technologies
Y = SinaClon BioScience Co.
X = EURx Ltd.
>>> # You can now choose a code and built your RestrictionBatch</code></pre>
<p>This way of producing a <code>RestrictionBatch</code> can drastically reduce the amount of useless output from a restriction analysis, limiting the search to enzymes that you can get hold of and limiting the risks of nervous breakdown. Nothing is more frustrating than to get the perfect enzyme for a sub-cloning only to find it’s not commercially available.</p>
<h4 id="adding-enzymes-to-a-restrictionbatch"><a name="2.3"></a>2.3 Adding enzymes to a RestrictionBatch</h4>
<p>Adding an enzyme to a batch if the enzyme is already present will not raise an exception, but will have no effects. Sometimes you want to get an enzyme from a <code>RestrictionBatch</code> or add it to the batch if it is not present. You will use the <code>get</code> method setting the second argument <code>add</code> to <code>True</code>.</p>
<pre class="pycon"><code>>>> rb3
RestrictionBatch(['EcoRI', 'EcoRV', 'KpnI'])
>>> rb3.add(EcoRI)
>>> rb3
RestrictionBatch(['EcoRI', 'EcoRV', 'KpnI'])
>>> rb3.get(EcoRI)
EcoRI
>>> rb3.get(SmaI)
Traceback (most recent call last):
File "<pyshell#4>", line 1, in -toplevel-
rb3.get(SmaI)
File "/usr/lib/Python2.3/site-packages/Bio/Restriction/Restriction.py", line 1800, in get
raise ValueError, 'enzyme %s is not in RestrictionBatch'%e.__name__
ValueError: enzyme SmaI is not in RestrictionBatch
>>> rb3.get(SmaI, add=True)
SmaI
>>> rb3
RestrictionBatch(['EcoRI', 'EcoRV', 'KpnI', 'SmaI'])</code></pre>
<h4 id="removing-enzymes-from-a-restrictionbatch"><a name="2.4"></a>2.4 Removing enzymes from a RestrictionBatch</h4>
<p>Removing enzymes from a batch is done using the <code>remove()</code> method. If the enzyme is not present in the batch this will raise a <code>KeyError</code>. If the value you want to remove is not an enzyme this will raise a <code>ValueError</code>.</p>
<pre class="pycon"><code>>>> rb3.remove(EcoRI)
>>> rb3
RestrictionBatch(['EcoRV', 'KpnI', 'SmaI'])
>>> rb3.remove(EcoRI)
Traceback (most recent call last):
File "<pyshell#14>", line 1, in -toplevel-
rb3.remove('EcoRI')
File "/usr/lib/Python2.3/site-packages/Bio/Restriction/Restriction.py", line 1839, in remove
return Set.remove(self, self.format(other))
File "/usr/lib/Python2.3/sets.py", line 534, in remove
del self._data[element]
KeyError: EcoRI
>>> rb3 += EcoRI
>>> rb3
RestrictionBatch(['EcoRI', 'EcoRV', 'KpnI', 'SmaI'])
>>> rb3.remove('EcoRI')
>>> rb3
RestrictionBatch(['EcoRV', 'KpnI', 'SmaI'])
>>> rb3.remove('spam')
Traceback (most recent call last):
File "<pyshell#18>", line 1, in -toplevel-
rb3.remove('spam')
File "/usr/lib/Python2.3/site-packages/Bio/Restriction/Restriction.py", line 1839, in remove
return Set.remove(self, self.format(other))
File "/usr/lib/Python2.3/site-packages/Bio/Restriction/Restriction.py", line 1871, in format
raise ValueError, '%s is not a RestrictionType'%y.__class__
ValueError: <type 'str'> is not a RestrictionType</code></pre>
<h4 id="manipulating-restrictionbatch"><a name="2.5"></a>2.5 Manipulating RestrictionBatch</h4>
<p>You can not, however, add batches together, as they are Python <code>sets</code>. You must use the pipe operator <code>|</code> instead. You can find the intersection between 2 batches using <code>&</code> (see the Python documentation about <code>sets</code> for more information.</p>
<pre class="pycon"><code>>>> rb3 = EcoRI + KpnI + EcoRV
>>> rb3
RestrictionBatch(['EcoRI', 'EcoRV', 'KpnI'])
>>> rb4 = SmaI + PstI
>>> rb4
RestrictionBatch(['PstI', 'SmaI'])
>>> rb3 + rb4
Traceback (most recent call last):
File "<pyshell#23>", line 1, in -toplevel-
rb3 + rb4
File "/usr/lib/Python2.3/site-packages/Bio/Restriction/Restriction.py", line 1829, in __add__
new.add(other)
File "/usr/lib/Python2.3/site-packages/Bio/Restriction/Restriction.py", line 1848, in add
return Set.add(self, self.format(other))
File "/usr/lib/Python2.3/site-packages/Bio/Restriction/Restriction.py", line 1871, in format
raise ValueError, '%s is not a RestrictionType'%y.__class__
ValueError: <class 'Bio.Restriction.Restriction.RestrictionBatch'> is not a RestrictionType
>>> rb3 | rb4
RestrictionBatch(['EcoRI', 'EcoRV', 'KpnI', 'PstI', 'SmaI'])
>>> rb3 & rb4
RestrictionBatch([])
>>> rb4 += EcoRI
>>> rb4
RestrictionBatch(['EcoRI', 'PstI', 'SmaI'])
>>> rb3 & rb4
RestrictionBatch(['EcoRI'])</code></pre>
<h4 id="analysing-sequences-with-a-restrictionbatch"><a name="2.6"></a>2.6 Analysing sequences with a RestrictionBatch</h4>
<p>To analyse a sequence for potential site, you can use the <code>search</code> method of the batch, the same way you did for restriction enzymes. The results is no longer a list however, but a dictionary. The keys of the dictionary are the names of the enzymes and the value a list of position site. <code>RestrictionBatch</code> does not implement a <code>catalyse</code> method, as it would not have a real meaning when used with large batch.</p>
<pre class="pycon"><code>>>> new_seq = Seq('TTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAA')
>>> rb.search(new_seq)
{'KpnI': [], 'EcoRV': [], 'EcoRI': []}
>>> rb.search(new_seq, linear=False)
{'KpnI': [], 'EcoRV': [], 'EcoRI': [33]}</code></pre>
<h4 id="other-restrictionbatch-methods"><a name="2.7"></a>2.7 Other RestrictionBatch methods</h4>
<p>Amongst the other methods provided by <code>RestrictionBatch</code>, <code>elements()</code> which return a list of all the element names alphabetically sorted, is certainly the most useful.</p>
<pre class="pycon"><code>>>> rb = EcoRI + KpnI + EcoRV
>>> rb.elements()
['EcoRI', 'EcoRV', 'KpnI']</code></pre>
<p>If you don’t care about the alphabetical order use the method <code>as_string()</code>, to get the same thing a bit faster. The list is not sorted. The order is random as Python sets are dictionary.</p>
<pre class="pycon"><code>>>> rb = EcoRI + KpnI + EcoRV
>>> rb.as_string()
['EcoRI', 'KpnI', 'EcoRV']</code></pre>
<p>Other <code>RestrictionBatch</code> methods are generally used for particular purposes and will not be discussed here. See the <a href="https://github.com/biopython/biopython/tree/master/Bio/Restriction">source</a> if you are interested.</p>
<h3 id="allenzymes-and-commonly-two-preconfigured-restrictionbatches"><a name="3"></a>3. AllEnzymes and CommOnly: two preconfigured RestrictionBatches</h3>
<p>While it is sometime practical to produce a <code>RestrictionBatch</code> of your own you will certainly more frequently use the two batches provided with the <code>Restriction</code> packages: <code>AllEnzymes</code> and <code>CommOnly</code>. These two batches contain respectively all the enzymes in the database and only the enzymes which have a commercial supplier. They are rather big, but that’s what make them useful. With these batch you can produce a full description of a sequence with a single command. You can use these two batch as any other batch.</p>
<pre class="pycon"><code>>>> len(AllEnzymes)
778
>>> len(CommOnly)
622
>>> AllEnzymes.search(new_seq) ...</code></pre>
<p>There is not a lot to say about them apart the fact that they are present. They are really normal batches, and you can use them as any other batch.</p>
<h3 id="the-analysis-class-even-simpler-restriction-analysis"><a name="4"></a>4. The Analysis class: even simpler restriction analysis</h3>
<p><code>RestrictionBatch</code> can give you a dictionary with the sites for all the enzymes in a batch. However, it is sometime nice to get something a bit easier to read than a Python dictionary. Complex restriction analysis are not easy with <code>RestrictionBatch</code>. Some refinements in the way to search a sequence for restriction sites will help. <code>Analysis</code> provides a serie of command to customise the results obtained from a pair restriction batch/sequence and some facilities to make the output sligthly more human readable.</p>
<h4 id="setting-up-an-analysis"><a name="4.1"></a>4.1 Setting up an Analysis</h4>
<p>To build a restriction analysis you will need a <code>RestrictionBatch</code> and a sequence and to tell it if the sequence is linear or circular. The first argument <code>Analysis</code> takes is the restriction batch, the second is the sequence. If the third argument is not provided, <code>Analysis</code> will assume the sequence is linear.</p>
<pre class="pycon"><code>>>> new_seq = Seq('TTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAA')
>>> rb = RestrictionBatch([EcoRI, KpnI, EcoRV])
>>> Ana = Analysis(rb, new_seq, linear=False)
>>> Ana
Analysis(RestrictionBatch(['EcoRI', 'EcoRV', 'KpnI']),Seq('TTCAAAAAAAAAAAAAAAAAA
AAAAAAAAAAGAA'),False)</code></pre>
<h4 id="full-restriction-analysis"><a name="4.2"></a>4.2 Full restriction analysis</h4>
<p>Once you have created your new <code>Analysis</code>, you can use it to get a restriction analysis of your sequence. The way to make a full restriction analysis of the sequence is:</p>
<pre class="pycon"><code>>>> Ana.full()
{'KpnI': [], 'EcoRV': [], 'EcoRI': [33]}</code></pre>
<p>This is much the same as the output of a <code>RestrictionBatch.search</code> method. You will get a more easy to read output with <code>print_that</code> used without argument:</p>
<pre class="pycon"><code>>>> # let's create a something a bit more complex to analyse.
>>>
>>> rb = RestrictionBatch([], ['C']) # we will explain the meaning of the
>>> # double list argument later.
>>>
>>> multi_site = Seq.Seq('AAA' + EcoRI.site + 'G' + KpnI.site + EcoRV.site +
'CT' + SmaI.site + 'GT' + FokI.site + 'GAAAGGGC' +
EcoRI.site + 'ACGT')
>>> Analong = Analysis(rb, multi_site)
>>> Analong.full()
{BglI: [], BstEII: [], AsuII: [], HinfI: [], SfiI: [], PspPI: [], BsiSI: [27], S
alI: [], SlaI: [], NcoI: [], NotI: [], PstI: [], StyI: [], BseBI: [], PvuII: [],
HindIII: [], BglII: [], ApaLI: [], TaqI: [], BssAI: [], AluI: [], SstI: [], Bse
CI: [], Sau3AI: [], HpaI: [], SnaBI: [], NheI: [], BclI: [], KpnI: [16], NruI: [
], MspCI: [], BshFI: [], CspAI: [], RsaI: [14], EcoRV: [20], SphI: [], BamHI: []
, MboI: [], SgrBI: [], SspI: [], ScaI: [], XbaI: [], SseBI: [], NaeI: [], EcoRI:
[5, 47], SmaI: [28], BseAI: []}
>>>
>>> # The results are here but it is difficult to read. let's try print_that
>>>
>>> Analong.print_that()
BsiSI : 27.
RsaI : 14.
EcoRI : 5, 47.
EcoRV : 20.
KpnI : 16.
SmaI : 28.
Enzymes which do not cut the sequence.
AluI BshFI MboI Sau3AI TaqI BseBI HinfI PspPI
ApaLI AsuII BamHI BclI BglII BseAI BseCI BssAI
CspAI HindIII HpaI MspCI NaeI NcoI NheI NruI
PstI PvuII SalI ScaI SgrBI SlaI SnaBI SphI
SseBI SspI SstI StyI XbaI BstEII NotI BglI
SfiI</code></pre>
<p>Much clearer, is’nt ? The output is optimised for a shell 80 columns wide. If the output seems odd, check that the width of your shell is at least 80 columns.</p>
<h4 id="changing-the-title"><a name="4.3"></a>4.3 Changing the title</h4>
<p>You can provide a title to the analysis and modify the sentence ‘Enzymes which do not cut the sequence’, by setting the two optional arguments of <code>print_that</code>, <code>title</code> and <code>s1</code>. No formatting will be done on these strings so if you have to include the newline (<code>\n</code>) as you see fit:</p>
<pre class="pycon"><code>>>> Analong.print_that(None, title='sequence = multi_site\n\n')
sequence = multi_site
BsiSI : 27.
RsaI : 14.
EcoRI : 5, 47.
EcoRV : 20.
KpnI : 16.
SmaI : 28.
Enzymes which do not cut the sequence.
AluI BshFI MboI Sau3AI TaqI BseBI HinfI PspPI
ApaLI AsuII BamHI BclI BglII BseAI BseCI BssAI
CspAI HindIII HpaI MspCI NaeI NcoI NheI NruI
PstI PvuII SalI ScaI SgrBI SlaI SnaBI SphI
SseBI SspI SstI StyI XbaI BstEII NotI BglI
SfiI
>>> Analong.print_that(None, title='sequence = multi_site\n\n',
s1='\n no site:\n\n')
sequence = multi_site
BsiSI : 27.
RsaI : 14.
EcoRI : 5, 47.
EcoRV : 20.
KpnI : 16.
SmaI : 28.
no site:
AluI BshFI MboI Sau3AI TaqI BseBI HinfI PspPI
ApaLI AsuII BamHI BclI BglII BseAI BseCI BssAI
CspAI HindIII HpaI MspCI NaeI NcoI NheI NruI
PstI PvuII SalI ScaI SgrBI SlaI SnaBI SphI
SseBI SspI SstI StyI XbaI BstEII NotI BglI
SfiI</code></pre>
<h4 id="customising-the-output"><a name="4.4"></a>4.4 Customising the output</h4>
<p>You can modify some aspects of the output interactively. There is three main type of output, two listing types (alphabetically sorted and sorted by number of site) and map-like type. To change the output, use the method <code>print_as()</code> of <code>Analysis</code>. The change of output is permanent for the instance of <code>Analysis</code> (that is until the next time you use <code>print_as()</code>). The argument of <code>print_as()</code> are strings: <code>'map'</code>, <code>'number'</code> or <code>'alpha'</code>. As you have seen previously the default behaviour is an alphabetical list (<code>'alpha'</code>).</p>
<pre class="pycon"><code>>>> Analong.print_as('map')
>>> Analong.print_that()
5 EcoRI
|
| 14 RsaI
| |
| | 16 KpnI
| | |
| | | 20 EcoRV
| | | |
| | | | 27 BsiSI
| | | | |
| | | | |28 SmaI
| | | | ||
| | | | || 47 EcoRI
| | | | || |
AAAGAATTCGGGTACCGATATCCTCCCGGGGTGGATGGAAAGGGCGAATTCACGT
|||||||||||||||||||||||||||||||||||||||||||||||||||||||
TTTCTTAAGCCCATGGCTATAGGAGGGCCCCACCTACCTTTCCCGCTTAAGTGCA
1 55
Enzymes which do not cut the sequence.
AluI BshFI MboI Sau3AI TaqI BseBI HinfI PspPI
ApaLI AsuII BamHI BclI BglII BseAI BseCI BssAI
CspAI HindIII HpaI MspCI NaeI NcoI NheI NruI
PstI PvuII SalI ScaI SgrBI SlaI SnaBI SphI
SseBI SspI SstI StyI XbaI BstEII NotI BglI
SfiI
>>> Analong.print_as('number')
>>> Analong.print_that()
enzymes which cut 1 times :
BsiSI : 27.
RsaI : 14.
EcoRV : 20.
KpnI : 16.
SmaI : 28.
enzymes which cut 2 times :
EcoRI : 5, 47.
Enzymes which do not cut the sequence.
AluI BshFI MboI Sau3AI TaqI BseBI HinfI PspPI
ApaLI AsuII BamHI BclI BglII BseAI BseCI BssAI
CspAI HindIII HpaI MspCI NaeI NcoI NheI NruI
PstI PvuII SalI ScaI SgrBI SlaI SnaBI SphI
SseBI SspI SstI StyI XbaI BstEII NotI BglI
SfiI
>>></code></pre>
<p>To come back to the previous behaviour:</p>
<pre class="pycon"><code>>>> Analong.print_as('alpha')
>>> Analong.print_that()
BsiSI : 27.
RsaI : 14.
EcoRI : 5, 47.
EcoRV : 20.
etc ...</code></pre>
<h4 id="fancier-restriction-analysis"><a name="4.5"></a>4.5 Fancier restriction analysis</h4>
<p>I will not go into the detail for each single method, here are all the functions that are available. Most are perfectly self explanatory and the others are fairly well documented (use <code>help('Analysis.command_name')</code>). The methods are:</p>
<div class="sourceCode" id="cb36"><pre class="sourceCode python"><code class="sourceCode python"><span id="cb36-1"><a href="#cb36-1" aria-hidden="true" tabindex="-1"></a>full(<span class="va">self</span>, linear<span class="op">=</span><span class="va">True</span>)</span>
<span id="cb36-2"><a href="#cb36-2" aria-hidden="true" tabindex="-1"></a>blunt(<span class="va">self</span>, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-3"><a href="#cb36-3" aria-hidden="true" tabindex="-1"></a>overhang5(<span class="va">self</span>, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-4"><a href="#cb36-4" aria-hidden="true" tabindex="-1"></a>overhang3(<span class="va">self</span>, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-5"><a href="#cb36-5" aria-hidden="true" tabindex="-1"></a>defined(<span class="va">self</span>, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-6"><a href="#cb36-6" aria-hidden="true" tabindex="-1"></a>with_sites(<span class="va">self</span>, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-7"><a href="#cb36-7" aria-hidden="true" tabindex="-1"></a>without_site(<span class="va">self</span>, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-8"><a href="#cb36-8" aria-hidden="true" tabindex="-1"></a>with_N_sites(<span class="va">self</span>, N, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-9"><a href="#cb36-9" aria-hidden="true" tabindex="-1"></a>with_number_list(<span class="va">self</span>, <span class="bu">list</span>, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-10"><a href="#cb36-10" aria-hidden="true" tabindex="-1"></a>with_name(<span class="va">self</span>, names, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-11"><a href="#cb36-11" aria-hidden="true" tabindex="-1"></a>with_site_size(<span class="va">self</span>, site_size, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-12"><a href="#cb36-12" aria-hidden="true" tabindex="-1"></a>only_between(<span class="va">self</span>, start, end, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-13"><a href="#cb36-13" aria-hidden="true" tabindex="-1"></a>between(<span class="va">self</span>, start, end, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-14"><a href="#cb36-14" aria-hidden="true" tabindex="-1"></a>show_only_between(<span class="va">self</span>, start, end, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-15"><a href="#cb36-15" aria-hidden="true" tabindex="-1"></a>only_outside(<span class="va">self</span>, start, end, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-16"><a href="#cb36-16" aria-hidden="true" tabindex="-1"></a>outside(<span class="va">self</span>, start, end, dct<span class="op">=</span><span class="va">None</span>)</span>
<span id="cb36-17"><a href="#cb36-17" aria-hidden="true" tabindex="-1"></a>do_not_cut(<span class="va">self</span>, start, end, dct<span class="op">=</span><span class="va">None</span>)</span></code></pre></div>
<p>Using these methods is simple:</p>
<pre class="pycon"><code>>>> new_seq = Seq('TTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAA')
>>> rb = RestrictionBatch([EcoRI, KpnI, EcoRV])
>>> Ana = Analysis(rb, new_seq, linear=False)
>>> Ana
Analysis(RestrictionBatch(['EcoRI', 'EcoRV', 'KpnI']),Seq('TTCAAAAAAAAAAAAAAAAAA
AAAAAAAAAAGAA'),False)
>>> Ana.blunt() # output only the result for enzymes which cut blunt
{'EcoRV': []}
>>> Ana.full() # all the enzymes in the RestrictionBatch
{'KpnI': [], 'EcoRV': [], 'EcoRI': [33]}
>>> Ana.with_sites() # output only the result for enzymes which have a site
{'EcoRI': [33]}
>>> Ana.without_site() # output only the enzymes which have no site
{'KpnI': [], 'EcoRV': []}
>>> Ana.only_between(1, 20) # the enzymes which cut between position 1 and 20
{}
>>> Ana.only_between(20, 34) # etc...
{'EcoRI': [33]}
>>> Ana.only_outside(20, 34)
{}
>>> Ana.with_name([EcoRI])
{'EcoRI': [33]}
>>></code></pre>
<p>To get a nice output, you still use <code>print_that</code> but this time with the command you want executed as argument.</p>
<pre class="pycon"><code>>>> Ana.print_that(Ana.blunt())
Enzymes which do not cut the sequence.
EcoRV
>>> pt = Ana.print_that
>>> pt(Ana.with_sites())
EcoRI : 33.
>>> pt(Ana.without_site())
Enzymes which do not cut the sequence.
EcoRV KpnI
>>> # etc ...</code></pre>
<h4 id="more-complex-analysis"><a name="4.6"></a>4.6 More complex analysis</h4>
<p>All of these methods (except <code>full()</code> which, well … do a full restriction analysis) can be supplied with an additional dictionary. If no dictionary is supplied a full restriction analysis is used as starting point. Otherwise the dictionary provided by the argument <code>dct</code> is used. The dictionary must be formatted as the result of <code>RestrictionBatch.search</code>. Therefore of the form <code>{'enzyme_name': [position1, position2],...}</code>, where <em>position1</em> and <em>position2</em> are integers. All methods list previously output such dictionaries and can be used as starting point.</p>
<p>Using this method you can build really complex query by chaining several method one after the other. For example if you want all the enzymes which are 5’ overhang and cut the sequence only once, you have two ways to go:</p>
<p>The hard way consist to build a restriction batch containing only 5’ overhang enzymes and use this batch to create a new <code>Analysis</code> instance and then use the method <code>with_N_sites()</code> as follow:</p>
<pre class="pycon"><code>>>> rbov5 = RestrictionBatch([x for x in rb if x.is_5overhang()])
>>> Anaov5 = Analysis(rbov5, new_seq, linear=False)
>>> Anaov5.with_N_sites(1)
{'EcoRI' : [33]}</code></pre>
<p>The easy solution is to chain several <code>Analysis</code> methods. This is possible since each method return a dictionary as results and is able to take a dictionary as input:</p>
<pre class="pycon"><code>>>> Ana.with_N_sites(1, Ana.overhang5())
{'EcoRI': [33]}</code></pre>
<p>The dictionary is always the last argument whatever the command you use.</p>
<p>The way to prefer certainly depends of the conditions you will use your <code>Analysis</code> instance. If you are likely to frequently reuse the same batch with different sequences, using a dedicated <code>RestrictionBatch</code> might be faster as the batch is likely to be smaller. Chaining methods is generally quicker when working with an interactive shell. In a script, the extended syntax may be easier to understand in a few months.</p>
<h3 id="advanced-features-the-formattedseq-class"><a name="5"></a>5. Advanced features: the FormattedSeq class</h3>
<p>Restriction enzymes require a much more strict formatting of the DNA sequences than <code>Bio.Seq</code> object provides. For example, the restriction enzymes expect to find an ungapped (no space) upper-case sequence, while <code>Bio.Seq</code> object allow sequences to be in lower-case separated by spaces. Therefore when a restriction enzyme analyse a <code>Bio.Seq</code> object (be it a <code>Seq</code> or a <code>MutableSeq</code>), the object undergoes a conversion. The class <code>FormattedSeq</code> ensure the smooth conversion from a <code>Bio.Seq</code> object to something which can be safely be used by the enzyme.</p>
<p>While this conversion is done automatically by the enzymes if you provide them with a <code>Seq</code> or a <code>MutableSeq</code>, there is time where it will be more efficient to realise the conversion before hand. Each time a <code>Seq</code> object is passed to an enzyme for analysis you pay a overhead due to the conversion. When analysing the same sequence over and over, it will be faster to convert the sequence, store the conversion and then use only the converted sequence.</p>
<h4 id="creating-a-formattedseq"><a name="5.1"></a>5.1 Creating a FormattedSeq</h4>
<p>Creating a <code>FormattedSeq</code> from a <code>Bio.Seq</code> object is simple. The first argument of <code>FormattedSeq</code> is the sequence you wish to convert. You can specify a shape with the second argument <code>linear</code>, if you don’t the <code>FormattedSeq</code> will be linear:</p>
<pre class="pycon"><code>>>> from Bio.Restriction import *
>>> from Bio.Seq import Seq
>>> seq = Seq('TTCAAAAAAAAAAGAATTCAAAAGAA')
>>> linear_fseq = FormattedSeq(seq, linear=True)
>>> default_fseq = FormattedSeq(seq)
>>> circular_fseq = FormattedSeq(seq, linear=False)
>>> linear_fseq
FormattedSeq(Seq('TTCAAAAAAAAAAGAATTCAAAAGAA'), linear=True)
>>> linear_fseq.is_linear()
True
>>> default_fseq.is_linear()
True
>>> circular_fseq.is_linear()
False
>>> circular_fseq
FormattedSeq(Seq('TTCAAAAAAAAAAGAATTCAAAAGAA'), linear=False)</code></pre>
<h4 id="unlike-bio.seq-formattedseq-retains-information-about-their-shape"><a name="5.2"></a>5.2 Unlike Bio.Seq, FormattedSeq retains information about their shape</h4>
<p><code>FormattedSeq</code> retains information about the shape of the sequence. Therefore unlike with <code>Seq</code> and <code>MutableSeq</code> you don’t need to specify the shape of the sequence when using <code>search()</code> or <code>catalyse()</code>:</p>
<pre class="pycon"><code>>>> EcoRI.search(linear_fseq)
[15]
>>> EcoRI.search(circular_fseq) # no need to specify the shape
[15, 25]</code></pre>
<p>In fact, the shape of a FormattedSeq is not altered by the second argument of the commands <code>search()</code> and <code>catalyse()</code>:</p>
<pre class="pycon"><code>>>> # In fact the shape is blocked.
>>> # The 3 following commands give the same results
>>> # which correspond to a circular sequence
>>> EcoRI.search(circular_fseq)
[15, 25]
>>> EcoRI.search(circular_fseq, linear=True)
[15, 25]
>>> EcoRI.search(circular_fseq, linear=False)
[15, 25]</code></pre>
<h4 id="changing-the-shape-of-a-formattedseq"><a name="5.3"></a>5.3 Changing the shape of a FormattedSeq</h4>
<p>You can however change the shape of the <code>FormattedSeq</code>. The command to use are:</p>
<div class="sourceCode" id="cb44"><pre class="sourceCode python"><code class="sourceCode python"><span id="cb44-1"><a href="#cb44-1" aria-hidden="true" tabindex="-1"></a>FormattedSeq.to_circular() <span class="co"># new FormattedSeq, shape will be circular.</span></span>
<span id="cb44-2"><a href="#cb44-2" aria-hidden="true" tabindex="-1"></a>FormattedSeq.to_linear() <span class="co"># new FormattedSeq, shape will be linear</span></span>
<span id="cb44-3"><a href="#cb44-3" aria-hidden="true" tabindex="-1"></a>FormattedSeq.circularise() <span class="co"># change the shape of FormattedShape to circular</span></span>
<span id="cb44-4"><a href="#cb44-4" aria-hidden="true" tabindex="-1"></a>FormattedSeq.linearise() <span class="co"># change the shape of FormattedShape to linear</span></span></code></pre></div>
<pre class="pycon"><code>>>> circular_fseq
FormatedSeq(Seq('TTCAAAAAAAAAAGAATTCAAAAGAA'), linear=False)
>>> circular_fseq.is_linear()
False
>>> circular_fseq == linear_fseq
False
>>> newseq = circular_fseq.to_linear()
>>> circular_fseq
FormatedSeq(Seq('TTCAAAAAAAAAAGAATTCAAAAGAA'), linear=False)
>>> newseq
FormatedSeq(Seq('TTCAAAAAAAAAAGAATTCAAAAGAA'), linear=True)
>>> circular_fseq.linearise()
>>> circular_fseq
FormatedSeq(Seq('TTCAAAAAAAAAAGAATTCAAAAGAA'), linear=True)
>>> circular_fseq.is_linear()
True
>>> circular_fseq == linear_fseq
True
>>> EcoRI.search(circular_fseq) # which is now linear
[15]</code></pre>
<h4 id="using-and-operators-with-formattedseq"><a name="5.4"></a>5.4 Using / and // operators with FormattedSeq</h4>
<p>Not having to specify the shape of the sequence to analyse gives you the opportunity to use the shorthand ‘/’ and ‘//’ with restriction enzymes:</p>
<pre class="pycon"><code>>>> EcoRI/linear_fseq # <=> EcoRI.search(linear_fseq)
[15]
>>> linear_fseq/EcoRI # <=> EcoRI.search(linear_fseq)
[15]
>>> EcoRI//linear_fseq # <=> linear_fseq//EcoRI <=> EcoRI.catalyse(linear_fseq)
(Seq('TTCAAAAAAAAAAG'), Seq('AATTCAAAAGAA'))</code></pre>
<p>Another way to avoid the overhead due to a repetitive conversion from a <code>Seq</code> object to a <code>FormattedSeq</code> is to use a <a href="#2"><code>RestrictionBatch</code></a>.</p>
<p>To conclude, the performance gain achieved when using a <code>FormattedSeq</code> instead of a <code>Seq</code> is not huge. The analysis of a 10 kb sequence by all the enzymes in <code>AllEnzymes</code> (<code>for x in AllEnzymes: x.search(seq)</code>, 867 enzymes) is 7 % faster when using a <code>FormattedSeq</code> than a <code>Seq</code>. Using a <code>RestrictionBatch</code> (<code>AllEnzymes.search(seq)</code>) is about as fast as using a <code>FormattedSeq</code> the first time the search is run. This however is dramatically reduced in subsequent runs with the same sequence (<code>RestrictionBatch</code> keeps in memory the result of their last run while the sequence is not changed).</p>
<h3 id="more-advanced-features"><a name="6"></a>6. More advanced features</h3>
<p>This chapter addresses some more advanced features of the packages, most users can safely ignore it.</p>
<h4 id="updating-the-enzymes-from-rebase"><a name="6.1"></a>6.1 Updating the enzymes from REBASE</h4>
<p>Most people will certainly not need to update the enzymes. The restriction enzyme package will be updated in with each new release of Biopython. But if you wish to get an update in between Biopython-releases here is how to do it.</p>
<p>First, you have to download the two scripts <code>rebase_update.py</code> and <code>ranacompile.py</code>: Go to https://github.com/biopython/biopython/tree/master/Scripts/Restriction, click on the respective file and press the ‘<strong>Raw</strong>’ button on the top right of the code window. Then, with right-click, save the file. Both scripts must be in the same directory.</p>
<h5 id="fetching-the-recent-enzyme-files-manually-from-rebase"><a name="6.1.1"></a>6.1.1 Fetching the recent enzyme files manually from REBASE</h5>
<p>Each month, <a href="http://rebase.neb.com/rebase/rebase.html">REBASE</a> release a new compilation of data about restriction enzymes. While the enzymes do not change so frequently, you may wish to update the restriction enzymes classes. The first thing to do is to get the last rebase file. You can find the release of REBASE at http://rebase.neb.com/rebase/rebase.files.html. The file you are interested in are in the EMBOSS format. You can download the files directly from the REBASE ftp server using your browser. The file are situated at ftp://ftp.neb.com/pub/rebase. You will have to download 3 files: <code>emboss_e.###</code>, <code>emboss_r.###</code>, and <code>emboss_s.###</code>. The <code>###</code> is a 3 digit number corresponding to the year and month of the release. The first digit is the year, the two last are the month: so July 2015 will be: 507; October 2016: 610, etc… Download the three file corresponding to the current month and place them in the same folder as your <code>rebase_update.py</code> and <code>ranacompiler.py</code> scripts.</p>
<h5 id="fetching-the-recent-enzyme-files-with-rebase_update.py"><a name="6.1.2"></a>6.1.2 Fetching the recent enzyme files with rebase_update.py</h5>
<p>Another way to do the same thing is to use the <code>rebase_update.py</code> script. It will connect directly to the rebase ftp server and download the last batch of emboss files. From a DOS or Unix shell do the following:</p>
<div class="sourceCode" id="cb47"><pre class="sourceCode bash"><code class="sourceCode bash"><span id="cb47-1"><a href="#cb47-1" aria-hidden="true" tabindex="-1"></a><span class="ex">$</span> cd path_to_the_update_script</span>
<span id="cb47-2"><a href="#cb47-2" aria-hidden="true" tabindex="-1"></a><span class="ex">$</span> rebase_update.py <span class="at">-p</span> http://www.somewhere.com:8000</span>
<span id="cb47-3"><a href="#cb47-3" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb47-4"><a href="#cb47-4" aria-hidden="true" tabindex="-1"></a><span class="ex">Please</span> wait, trying to connect to Rebase</span>
<span id="cb47-5"><a href="#cb47-5" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb47-6"><a href="#cb47-6" aria-hidden="true" tabindex="-1"></a><span class="ex">copying</span> ftp://ftp.neb.com/pub/rebase/emboss_e.407</span>
<span id="cb47-7"><a href="#cb47-7" aria-hidden="true" tabindex="-1"></a><span class="ex">to</span> /cvsroot/bioPython/Bio/Restriction/Scripts/emboss_e.407</span>
<span id="cb47-8"><a href="#cb47-8" aria-hidden="true" tabindex="-1"></a><span class="ex">copying</span> ftp://ftp.neb.com/pub/rebase/emboss_s.407</span>
<span id="cb47-9"><a href="#cb47-9" aria-hidden="true" tabindex="-1"></a><span class="ex">to</span> /cvsroot/bioPython/Bio/Restriction/Scripts/emboss_s.407</span>
<span id="cb47-10"><a href="#cb47-10" aria-hidden="true" tabindex="-1"></a><span class="ex">copying</span> ftp://ftp.neb.com/pub/rebase/emboss_r.407</span>
<span id="cb47-11"><a href="#cb47-11" aria-hidden="true" tabindex="-1"></a><span class="ex">to</span> /cvsroot/bioPython/Bio/Restriction/Scripts/emboss_r.407</span></code></pre></div>
<p>Some explanation are needed: <code>-p</code> is the switch to indicate to the script you are using a proxy. If you use a ftp proxy enter its address and the connection port after the ‘<code>:</code>’.</p>
<h5 id="compiling-a-new-dictionary-with-ranacompiler.py"><a name="6.1.3"></a>6.1.3 Compiling a new dictionary with ranacompiler.py</h5>
<p>Once you have got the recent emboss files you can compile a new module containing the data necessary to create restriction enzyme.</p>
<p>Note: if the emboss files are not present in the current directory or if they are not up to date, <code>ranacompiler.py</code> will invoke the script <a href="#6.1.2"><code>rebase_update.py</code></a>, which needs to be installed in the same folder. You will need to use the same options as before (ie <code>-m</code> and <code>-p</code>). See the previous paragraph on <a href="#6.1.2"><code>rebase_update.py</code></a> for more details.</p>
<p>For simplicity let’s assume we have put the emboss files in the same folder as the files which contains the script <code>ranacompiler.py</code>. You may have the change the mode of the file to make it executable:</p>
<div class="sourceCode" id="cb48"><pre class="sourceCode bash"><code class="sourceCode bash"><span id="cb48-1"><a href="#cb48-1" aria-hidden="true" tabindex="-1"></a><span class="ex">$</span> cd path_to_the_ranacompiler_script</span>
<span id="cb48-2"><a href="#cb48-2" aria-hidden="true" tabindex="-1"></a><span class="ex">$</span> chmod <span class="st">'+x'</span> ranacompiler.py</span></code></pre></div>
<p>Now execute the script:</p>
<div class="sourceCode" id="cb49"><pre class="sourceCode bash"><code class="sourceCode bash"><span id="cb49-1"><a href="#cb49-1" aria-hidden="true" tabindex="-1"></a><span class="ex">$</span> Python ranacompiler.py <span class="co"># or ./ranacompiler.py</span></span></code></pre></div>
<p>You get normally the following message:</p>
<div class="sourceCode" id="cb50"><pre class="sourceCode bash"><code class="sourceCode bash"><span id="cb50-1"><a href="#cb50-1" aria-hidden="true" tabindex="-1"></a><span class="ex">$</span> ./ranacompiler.py</span>
<span id="cb50-2"><a href="#cb50-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-3"><a href="#cb50-3" aria-hidden="true" tabindex="-1"></a> <span class="ex">Using</span> the files : emboss_e.407, emboss_r.407, emboss_s.407</span>
<span id="cb50-4"><a href="#cb50-4" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-5"><a href="#cb50-5" aria-hidden="true" tabindex="-1"></a><span class="ex">WARNING</span> : HaeIV cut twice with different overhang length each time.</span>
<span id="cb50-6"><a href="#cb50-6" aria-hidden="true" tabindex="-1"></a> <span class="ex">Unable</span> to deal with this behaviour.</span>
<span id="cb50-7"><a href="#cb50-7" aria-hidden="true" tabindex="-1"></a> <span class="ex">This</span> enzyme will not be included in the database. Sorry.</span>
<span id="cb50-8"><a href="#cb50-8" aria-hidden="true" tabindex="-1"></a> <span class="ex">Checking</span> :</span>
<span id="cb50-9"><a href="#cb50-9" aria-hidden="true" tabindex="-1"></a><span class="ex">Anyway,</span> HaeIV is not commercially available.</span>
<span id="cb50-10"><a href="#cb50-10" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-11"><a href="#cb50-11" aria-hidden="true" tabindex="-1"></a><span class="ex">WARNING</span> : HpyUM037X has two different sites.</span>
<span id="cb50-12"><a href="#cb50-12" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-13"><a href="#cb50-13" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-14"><a href="#cb50-14" aria-hidden="true" tabindex="-1"></a><span class="ex">The</span> new database contains 867 enzymes.</span>
<span id="cb50-15"><a href="#cb50-15" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-16"><a href="#cb50-16" aria-hidden="true" tabindex="-1"></a><span class="ex">Writing</span> the dictionary containing the new Restriction classes...</span>
<span id="cb50-17"><a href="#cb50-17" aria-hidden="true" tabindex="-1"></a><span class="ex">OK.</span></span>
<span id="cb50-18"><a href="#cb50-18" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-19"><a href="#cb50-19" aria-hidden="true" tabindex="-1"></a><span class="ex">Writing</span> the dictionary containing the suppliers datas...</span>
<span id="cb50-20"><a href="#cb50-20" aria-hidden="true" tabindex="-1"></a><span class="ex">OK.</span></span>
<span id="cb50-21"><a href="#cb50-21" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-22"><a href="#cb50-22" aria-hidden="true" tabindex="-1"></a><span class="ex">Writing</span> the dictionary containing the Restriction types....</span>
<span id="cb50-23"><a href="#cb50-23" aria-hidden="true" tabindex="-1"></a><span class="ex">OK.</span></span>
<span id="cb50-24"><a href="#cb50-24" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-25"><a href="#cb50-25" aria-hidden="true" tabindex="-1"></a> <span class="ex">******************************************************************************</span></span>
<span id="cb50-26"><a href="#cb50-26" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-27"><a href="#cb50-27" aria-hidden="true" tabindex="-1"></a> <span class="ex">Compilation</span> of the new dictionary : OK.</span>
<span id="cb50-28"><a href="#cb50-28" aria-hidden="true" tabindex="-1"></a> <span class="ex">Installation</span> : No.</span>
<span id="cb50-29"><a href="#cb50-29" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-30"><a href="#cb50-30" aria-hidden="true" tabindex="-1"></a> <span class="ex">You</span> will find the newly created <span class="st">'Restriction_Dictionary.py'</span> file</span>
<span id="cb50-31"><a href="#cb50-31" aria-hidden="true" tabindex="-1"></a> <span class="er">in</span> <span class="ex">the</span> :</span>
<span id="cb50-32"><a href="#cb50-32" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-33"><a href="#cb50-33" aria-hidden="true" tabindex="-1"></a> <span class="ex">/path/where/you/run/ranacompiler.py</span></span>
<span id="cb50-34"><a href="#cb50-34" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-35"><a href="#cb50-35" aria-hidden="true" tabindex="-1"></a> <span class="ex">Make</span> a copy of <span class="st">'Restriction_Dictionary.py'</span> and place it with</span>
<span id="cb50-36"><a href="#cb50-36" aria-hidden="true" tabindex="-1"></a> <span class="ex">the</span> other Restriction libraries.</span>
<span id="cb50-37"><a href="#cb50-37" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-38"><a href="#cb50-38" aria-hidden="true" tabindex="-1"></a> <span class="ex">note</span> :</span>
<span id="cb50-39"><a href="#cb50-39" aria-hidden="true" tabindex="-1"></a> <span class="ex">This</span> folder should be :</span>
<span id="cb50-40"><a href="#cb50-40" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-41"><a href="#cb50-41" aria-hidden="true" tabindex="-1"></a> <span class="ex">path_to_python/site-packages/Bio/Restriction</span></span>
<span id="cb50-42"><a href="#cb50-42" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb50-43"><a href="#cb50-43" aria-hidden="true" tabindex="-1"></a> <span class="ex">******************************************************************************</span></span></code></pre></div>
<p>The first line indicate which emboss files have been used for the present compilation. You can safely ignore the warnings as long as the <code>compilation of the new dictionary : OK.</code> is present in the last part of the output. They are here for debugging purpose. The number of enzymes in the new module is indicated as well as a list of the dictionary which have been compiled. The last part indicate that the module has been succesfully created but not installed. To finish the update you must copy the file <code>Restriction_Dictionary.py</code> into the folder <code>/your_python_path/site-packages/Bio/Restriction/</code> as indicated by the script. Looking into the present folder, you will see to new files: the newly created dictionary <code>Restriction_Dictionary.py</code> and <code>Restriction_Dictionary.old</code>. This last file containing the old dictionary to which you can revert in case anything the new file is corrupted (this should not happen since the script is happy enough the new dictionary is good, but if there is a problem it is always nice to know you can revert to the previous setting without having to reinstall the whole thing.</p>
<p>If you whish, the script may install the folder for you as well, but you will have to run it as root if your normal user has no write access to your Python installation (and it should’nt). Use the command <code>ranacompiler.py -i</code> or <code>ranacompiler.py --install</code> for this.</p>
<p>If anything goes wrong (you have no write access to the destination folder for example) the script will let you know it did not perform the installation. It will however still save the new module in the current directory.</p>
<p>As you can see the script is not very bright and will redo the compilation each time it is invoked, no matter if a previous version of the module is already present.</p>
<h4 id="subclassing-the-class-analysis"><a name="6.2"></a>6.2 Subclassing the class Analysis</h4>
<p>As seen previously, you can modify some aspects of the <code>Analysis</code> output interactively. However if you want to write your own <code>Analysis</code> class, you may wish to provide others output facilities than is given in this package. Depending on what you want to do you may get away with simply changing the <code>make_format</code> method of your derived class or you will need to provide new methods. Rather than get into a long explanation, here is the implementation of a rather useless <code>Analysis</code> class:</p>
<pre class="pycon"><code>>>> class UselessAnalysis(Analysis):
def __init__(self, rb=RestrictionBatch(), seq=Seq(''), lin=True):
"""UselessAnalysis -> A class that waste your time"""
#
# Unless you want to do something more fancy all
# you need to do here is instantiate Analysis.
# Don't forget the self in __init__
#
Analysis.__init__(self, rb, seq, lin)
def make_format(self, cut=[], t='', nc=[], s=''):
"""not funny"""
#
# Generally, you don't need to do anything else here
# This will tell to your new class to default to the
# _make_joke format.
#
return self._make_joke(cut, t, nc, s)
def print_as(self, what='joke'):
"""Never know somebody might want to change the behaviour of
this class."""
#
# add your new option to print_as
#
if what == 'joke':
self.make_format = self._make_joke
return
else:
#
# The other options will be treated as before
#
return Analysis.print_as(self, what)
def _make_joke(self, cut=[], title='', nc=[], s1=''):
"""UA._make_joke(cut, t, nc, s) -> new analysis output"""
#
# starting your new method with '_make_'
# will give a hint to what it is suppose to do
#
# We will not process the non-cutting enzymes
# Their names are in nc
# s1 is the string printed before them
#
if not title:
title = '\nYou have guessed right the following enzymes:\n\n'
for name, sites in cut:
#
# cut contains:
# - the name of the enzymes which cut the sequence (name)
# - a list of the site positions (sites)
#
guess = raw_input("next enzyme is %s, Guess how many sites ?\n>>> "%name)
try:
guess = int(guess)
except:
guess = None
if guess == len(sites):
print 'You did guess right. Good. Next.'
result = '%i site' % guess
if guess > 1:
result += 's'
#
# now we format the line. See the PrintFormat module
# for some examples
# PrintFormat.__section_list and _make_map are good start.
#
title=''.join((title, str(name).ljust(self.NameWidth),
' : ', result, '.\n'))
print '\nNo more enzyme.'
return title
#
# I you want to print the non cutting enzymes use
# the following return instead of the previous one:
#
#return title + t + self._make_nocut_only(nc,s1)
>>> # You initiate and use it as before
>>> rb = RestrictionBatch([], ['A'])
>>> multi_site = Seq('AAA' + EcoRI.site +'G' + KpnI.site + EcoRV.site + 'CT' +\
SmaI.site + 'GT' + FokI.site + 'GAAAGGGC' + EcoRI.site + 'ACGT')
>>>
>>> b = UselessAnalysis(rb, multi_site)
>>> b.print_that() # Well, I let you discover if you haven't already guessed</code></pre>
<p>Using this example, as a template you should now be able to subclass <code>Analysis</code> as you wish. You will found more implementation details in the module <code>Bio.Restriction.PrintFormat</code> which contains the class providing all the <code>_make_*</code> methods.</p>
<h3 id="limitation-and-caveat"><a name="7"></a>7. Limitation and caveat</h3>
<p>Particularly, the class <code>Analysis</code> is a quick and dirty implementation based on the facilities furnished by the package. Please check your results and report any fault.</p>
<p>On a more general basis, <code>Restriction</code> have some other limitations:</p>
<h4 id="all-dna-are-non-methylated"><a name="7.1"></a>7.1 All DNA are non methylated</h4>
<p>No facility to work with methylated DNA has been implemented yet. As far as the enzyme classes are concerned all DNA is non methylated DNA. Implementation of methylation sensibility will possibly occur in the future. But for now, if your sequence is methylated, you will have to check if the site is methylated using other means.</p>
<h4 id="no-support-for-star-activity"><a name="7.2"></a>7.2 No support for star activity</h4>
<p>As before no support has been yet implemented to find site mis-recognised by enzymes under high salt concentration conditions, the so-called star activity. This will be implemented as soon as I can get a good source of information for that.</p>
<h4 id="safe-to-use-with-degenerated-dna"><a name="7.3"></a>7.3 Safe to use with degenerated DNA</h4>
<p>It is safe to use degenerated DNA as input for the query. You will not be flooded with meaningless results. But this come at a price: GAA<strong><em>N</em></strong>TC will not be recognised as a potential EcoRI site for example, in fact it will not be recognised at all. Degenerated sequences will not be analysed. If your sequence is not fully sequenced, you will certainly miss restriction sites:</p>
<pre class="pycon"><code>>>> a = Seq('nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnGAATTCrrrrrrrrrrr')
>>> EcoRI.search(a)
[36]
>>> b = Seq('nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnGAAnTCrrrrrrrrrrr')
>>> EcoRI.search(b)
[]</code></pre>
<h4 id="non-standard-bases-in-dna-are-not-allowed"><a name="7.4"></a>7.4 Non standard bases in DNA are not allowed</h4>
<p>While you can use degenerated DNA, using non standard base alphabet will make the enzymes choke, even if <code>Bio.Seq.Seq</code> accepts them. However, space-like characters (’ ‘,’‘,’, …) and digit will be removed but will not stop the enzyme analysing the sequence. You can use them but the fragments produced by <code>catalyse</code> will have lost any formatting. <code>catalyse</code> tries to keep the original case of the sequence (i.e lower case sequences will generate lower case fragments, upper case sequences upper case fragments), but mixed case will return upper case fragments:</p>
<pre class="pycon"><code>>>> c = Seq('xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxGAANTCrrrrrrrrrrr')
>>> EcoRI.search(c)
Traceback (most recent call last):
File "<stdin>", line 1, in <module>
File "/usr/lib/python3.6/site-packages/Bio/Restriction/Restriction.py", line 553, in search
cls.dna = FormattedSeq(dna, linear)
File "/usr/lib/python3.6/site-packages/Bio/Restriction/Restriction.py", line 171, in __init__
self.data = _check_bases(stringy)
File "/usr/lib/python3.6/site-packages/Bio/Restriction/Restriction.py", line 122, in _check_bases
raise TypeError("Invalid character found in %s" % repr(seq_string))
TypeError: Invalid character found in 'XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXGAANTCRRRRRRRRRRR'
>>> d = Seq('1 nnnnn nnnnn nnnnn nnnnn nnnnn \n\
26 nnnnn nnnnG AATTC rrrrr rrrrr \n\
51 r')
>>> d
Seq('1 nnnnn nnnnn nnnnn nnnnn nnnnn \n26 nnnnn nnnnG AATTC rrrrr rrrrr \n51 r')
>>> EcoRI.search(d)
[36]
>>> EcoRI.catalyse(d)
(Seq('AATTCRRRRRRRRRRR'), Seq('NNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNG'))
>>> e = Seq('nnnnGAATTCrr')
>>> f = Seq('NNNNGAATTCRR')
>>> g = Seq('nnnngaattcrr')
>>> EcoRI.catalyse(e)
(Seq('NNNNG'), Seq('AATTCRR'))
>>> EcoRI.catalyse(f)
(Seq('NNNNG'), Seq('AATTCRR'))
>>> EcoRI.catalyse(g)
(Seq('nnnng'), Seq('aattcrr'))</code></pre>
<p>Not allowing other letters than IUPAC might seems drastic but this is really to limit errors. It is not totally fool proof but it does help.</p>
<h4 id="sites-found-at-the-edge-of-linear-dna-might-not-be-accessible-in-a-real-digestion"><a name="7.5"></a>7.5 Sites found at the edge of linear DNA might not be accessible in a real digestion</h4>
<p>While sites clearly outsides a sequence will not be reported, nothing has been done to try to determine if a restriction site at the end of a linear sequence is valid:</p>
<pre class="pycon"><code>>>> d = Seq('GAATTCAAAAAAAAAAAAAAAAAAAAAAAAAAGGATG')
>>> FokI.site # site present
'GGATG'
>>> FokI.elucidate() # but cut outside the sequence
'GGATGNNNNNNNNN^NNNN_N'
>>> FokI.search(d) # therefore no site found
[]
>>> EcoRI.search(d)
[2]</code></pre>
<p><code>EcoRI</code> finds a site at position 2 even if it is highly unlikely that EcoRI accepts to cut this site in a tube. It is generally considered that at about 5 nucleotides must separate the site from the edge of the sequence to be reasonably sure the enzyme will work correctly. This “security margin” is variable from one enzyme to the other. In doubt consult the documentation for the enzyme.</p>
<h4 id="restriction-reports-cutting-sites-not-enzyme-recognition-sites"><a name="7.6"></a>7.6 Restriction reports cutting sites not enzyme recognition sites</h4>
<p>Some enzymes will cut twice each time they encounter a restriction site. The enzymes in this package report both cut not the site. Other software may only reports restriction sites. Therefore the output given for some enzymes might seems to be the double when compared with the results of these software. It is not a bug.</p>
<pre class="pycon"><code>>>> AloI.cut_twice()
True
>>> AloI.fst5 # first cut
-7
>>> AloI.scd5 # second cut
25
>>> AloI.site
'GAACNNNNNNTCC'
>>> b = Seq('AAAAAAAAAAA'+ AloI.site + 'AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA')
>>> b
Seq('AAAAAAAAAAAGAACNNNNNNTCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA')
>>> AloI.search(b) # one site, two cuts -> two positions
[5, 37]</code></pre>
<h3 id="annex-modifying-dir-to-use-with-from-bio.restriction-import"><a name="8"></a>8. Annex: modifying dir() to use with from Bio.Restriction import *</h3>
<p>Having all the enzymes imported directly in the shell is useful when working in an interactive shell (even if it is not recommended by the purists). Here is a little hack to get some sanity back when using dir() in those conditions:</p>
<pre class="pycon"><code>>>> # we will change the builtin dir() function to get ride of the enzyme names.
>>> import sys
>>> def dir(object=None):
"""dir([object]) -> list of string.
over-ride the built-in function to get some clarity."""
if object:
# we only want to modify dir(),
# so here we return the result of the builtin function.
return __builtins__.dir(object)
else:
# now the part we want to modify.
# All the enzymes are in a RestrictionBatch (we will talk about
# that later, for the moment simply believe me).
# So if we remove from the results of dir() everything which is
# in AllEnzymes we will get a much shorter list when we do dir()
#
# the current level is __main__ ie dir() is equivalent to
# ask what's in __main__ at the moment.
# we can't access __main__ directly.
# so we will use sys.modules['__main__'] to reach it.
# the following list comprehension remove from the result of
# dir() everything which is also present in AllEnzymes.
#
return [x for x in __builtins__.dir(sys.modules['__main__'])
if not x in AllEnzymes]
>>> # now let's see if it works.
>>> dir()
['AllEnzymes', 'Analysis', 'CommOnly', 'NonComm', 'PrintFormat', 'RanaConfig',
'Restriction', 'RestrictionBatch', 'Restriction_Dictionary', '__builtins__',
'__doc__', '__name__', 'dir', 'sys']
>>> # ok that's much better.
>>> # The enzymes are still there
>>> EcoRI.site
'GAATTC'</code></pre>
</body>
</html>
|
