Report Agilent Experiment 1, data 09-10-2002
Version 03-01-2003
Brief description of array
Array consists of app. 8500 probes. Probes are 60 mers consisting of a 40 mer specific sequence on a 20mer tether. The tether sequence is 100% identical to a sequence in the human glyceraldehyde-3-phosphate
dehydrogenase gene. There are four types of probes:
Control probes
Negative and positive Agilent controls (marked – and + respectively under Control type).
Tiled strains
The complete small ribosomal subunit genes of 4 different organisms (Escherichia coli, Bacillus subtilis,
Methanococcus jannaschii and Saccharomyces cerevisiae) are present as tiled sequences on the array.
These 40 mers have an overlap of 5 nucleotides.
NODE probes
The probes starting with “NODE..” represent sequences specific for nodes on a phylogenetic tree,
created by clustering the ATCC strains in the Relman lab collection. Some probes are specific for 1
species, some represent a genus, family or order.
16S probes
Species specific probes, designed by two different methods and labeled “16STRICT” or “16AVE”.
Outline of experiment
First experiment to test Agilent arrays. Four different bacteria (two of the tiled strains and two non-tiled
strains) were amplified with broad range bacterial PCR primers 8F and 1391R. The strains were:
Escherichia coli (tiled strain)
Bacillus subtilis (tiled strain)
Campylobacter jejuni (non-tiled strain)
Clostridium cellulyticum (non-tiled strain)
The PCR fragments were labeled with Cy5 in a second PCR. By accident, the wrong concentration (50 times
too low) of dNTPs was used, as was discovered later. This resulted in a very low concentration of PCR
product. The PCR product was purified with Microcon columns.
Each of the strains was hybed in a different experiment. All four hybridizations were performed using the
same Cy3 PCR-labeled Common Reference (CR) pool version 1, consisting of pooled PCR fragments of
about 150 ATCC bacterial strains.
The single species were used in an endconcentration of 50 ng/ml.
The CR pool was used in an endconcentration of 500 ng/ml. (10x more than single species because this is
complex mixture).
Agilent barcode number
16011437010010
16011437010009
Barcode side (1)
Escherichia coli
Campylobacter jejuni
Non-Barcode side (2)
Bacillus subtilis
Clostridium cellulyticum
Scans
PMT value was set at 30% to decrease the number of saturated spots.
Signal
Average of signal values on Escherichia coli 16S tiled sequence probes (301 probes)
16S PCR fragment of
Escherichia coli (tiled)
Bacillus subtilis (tiled)
Campylobacter jejuni (non-tiled)
Clostridium cellulyticum (non-tiled)
gBGSubSignal
20404.4
17657.1
18368.0
18338.4
rBGSubSignal
14760.3
6377.9
7330.7
5795.3
The signal of the CR is stronger than that of the E. coli fragment.
1
Average of signal values on Bacillus subtilis 16S tiled sequence probes (253 probes)
16S PCR fragment of
Escherichia coli (tiled)
Bacillus subtilis (tiled)
Campylobacter jejuni (non-tiled)
Clostridium cellulyticum (non-tiled)
gBGSubSignal
18068.7
17414.9
16379.7
16338.6
rBGSubSignal
6934.0
13673.5
6619.6
5720.6
For both sets of tiled sequences, the gBGSubSignal (CR signal) is similar in all four experiments. As
expected, the rBGSubSignal of the E. coli tiled probes is the highest in the E. coli hybe. Similarly, for the B.
subtilis sequences, the signal is the highest in the Bacillus hybe.
Plot rBGSubSignal (or NormSignal) vs. position on Escherichia coli 16S gene:
The red Background subtracted signal of the 301 E. coli tiled sequences in each of the four hybes was
plotted against the position of the E. coli 16S rRNA gene. For comparison, the green CR signal from the E.
coli hybe is also plotted. The CR plots from the other three hybes are similar (not shown). The green CR
signal is usually higher than the red specific signal. This might be explained by the fact that only 50 ng/ul of
the specific PCR fragment was used in each hybe, against 500 ng/ul of the common reference pool.
Overall, the signals in the E.coli hybe were the strongest. All four hybes give a “up and down” plot with a
general slope upwards, and a sudden drop at position 1380. This drop is expected, because the
hybridizations are performed with a PCR fragment spanning position 8 to 1391 of the E. coli 16S ribosomal
gene, while the tiled sequences range from position 0 to 1500.
Some parts of the 16S gene (position 100, 345, 535, 765, 940, 1365) give a high signal in all four
hybridizations, and are also high in the green channel (CR). This might be caused by conserved regions or
by regions of higher affinity/accessibility.
Other parts (position 185, 440, 825, 1000, 1275) give a high signal in the E. coli hybe, while all other three
PCR products show a relatively low signal, suggesting regions of low conservation. In most of these peaks,
the green channel also shows a high signal, but the common reference pool also contains E. coli.
Fuchs et al. (1998, Appl. Environm. Microb. 64:4973): highest accessibility (as determined by flow cytometry
of tiled, labeled probes to E. coli cells) in regions 38 to 108, 181 to 215, 316 to 359, 871 to 933, 1383 to
1427, and 1473 to 1517. The 345 peak might be explained by good accessibility, the others not really. I am
not sure if accessibility plays a role in PCR products, as compaired to hybridization in cells, to RNA.
According to the same article, the least conserved (so most species specific) regions are found around
positions 80, 200, 570, 630, 840, 1000, 1130, 1260 and 1550. This corresponds to most of the E. coli specific
peaks. Conserved regions are found at positions 340, 510-560, 690, 800, 900-950, 1050-1100, 1200, 1340,
1390 and 1500.
If you compare Fuchs’ conservation plot to our results, regions with low conservation correspond to regions
with high E. coli specific signals.
The upwards slope effect might be explained by the very low concentration of dNTPs used (by accident).
This probably resulted in high amounts of unfinished, not full-length PCR fragments. Since the
oligonucleotides used on the array represent the 5’ strand, which hybridize the opposite strand, the probes
situated on the end of the 16S molecule might have more PCR fragment to hybridize to.
Plot rBGSubSignal or NormSignal vs. position on Bacillus subtilis 16S gene:
A similar plot was made comparing the hybridization signals of the tiled Bacillus subtilis sequences. Now the
Bacillus hybe shows the highest overall signals. As with the E. coli features, an “upwards slope” is observed.
Probes with a high signal in all four hybridizations are found around positions 550, 760, 930. This is roughly
at the same positions as with the E. coli probes, although the peaks are not as clear.
Note: Bacillus probes from position 510 to 545 were not in the feature data list. Are they
not included on the array???
Around positions 200, 430, 620, 815, 990, 1120, 1220 and 1265, Bacillus subtilis specific hybridization
signals are found (high Bacillus signals, low signals in the other three hybes). Again, this seem to correspond
to the known species-specific regions in the 16S gene, and to the Fuchs plot.
2
rBGSubSignals on tiled E coli features
70000
60000
rBGSubSignal
50000
Escherichia coli
Bacillus subtilis
Campylobacter jejuni
Clostridium cellulyticum
green signal CR (in E. coli hybe)
40000
30000
20000
10000
Position
1500
1440
1380
1320
1260
1200
1140
1080
1020
960
900
840
780
720
660
600
540
480
420
360
300
240
180
120
0
60
0
From Fuchs et al. 1998.
AEM 64:4973.
Avarage conservation
of E. coli probes in
arbitrary units, where
low values indicate low
conservation.
rBGSubSignals on tiled Bacillus subtilis
features
60000
50000
Escherichia coli
Bacillus subtilis
30000
Campylobacter jejuni
Clostridium cellulyticum
20000
10000
1370
1320
1270
1220
1170
1120
1070
1020
970
920
870
780
730
640
590
500
450
400
350
300
250
200
150
100
0
0
50
rBGSubSignal
40000
Position
3
Ranking
For each of the four hybes, all features were ranked according to the value of the red background subtracted
signal, such that the highest signal was given rank # 1 and the lowest rank # 8455. In the table on the
following page the top 100 features of the 4 different hybes are given.
rBGSubSignal
Escherichia coli
Bacillus subtilis
Campylobacter jejuni
Clostridium cellulyticum
Ranking # 1
56590
46576
56816
51119
Ranking # 100
26309
22301
32459
27257
As expected, in the Escherichia coli hybe, most features in the top 100 are E. coli features (the tiled
sequences labeled ESCCOLI400000 - 1380). Unexpectedly, on rank 10 is 16AVE04616, which is an
archaeal probe. Most other 16AVE probes in the top 100 are based on NODE13, which is a broad range
probe.
Similarly, in the Bacillus subtilis hybe, most top 100 scores are Bacillus tiled probes. The best score is
16AVE03907, designed in NODE5764 (Bacillus subtilis). 16AVE03906 designed on the same node is found
on rank # 34. Most other probes are based on NODE13. ESCCOLI tiled probes based on positions at the 3’
end of the 16S gene also end high.
None of the probes listed in the top 100 of the Campylobacter jejuni hybe are species specific. On the array,
four Campylobacter jejuni specific probes are present, labeled 16AVE/STRICT00057 – 58. In the C. jejuni
hybe, the species-specific 16AVE00057 and -58 probes end on ranking position 314 and 1074 respectively,
while 16STRICT00057 and 58 end on position 427 and 1077 respectively. NODE 2484 also represents
Campylobacter jejuni, it is present on the array as probe numbers 16STRICT00495-501 and 16AVE014651475. Of these species-specific node-probes none ends up in the top 100. Nine probes in the top 100 are
designed to be specific for nodes 2451/2455/2458 (Node-children Campylobacter rectus, C. mucosalis, C.
hyointestinalis, C. jejuni and Arcobacter cryaerophilus, and Wolinella succinogenes). Apparently, this more
broad range probe has a higher affinity for the 16S PCR product than the C. jejuni specific probes.
The other probes in the top 100 of this hybe are mostly based on NODE13.
Specific Clostridium cellulyticum oligonucleotides on the array are 16AVE/STRICT00075 – 87. The top 2
scores in the top 100 of the C. cellulyticum hybe are species specific features, 16AVE00085 and
16STRICT00085. Most of the other top scores are probes representing NODE13, which is a broad range
probe. 16AVE00084 and 16STRICT00084 end up on position 57 and 67 respectively. No other CloCell
specific sequences ended up in the top 100.
Probe NODE1122 ends very high in the top 100 of all four hybridizations. It is a broad range probe designed
on NODE13. Similarly, probes BAISUBT301360 and -65 and ESCCOLI401365-75, and SCER01625 show
up in the top 100 of all four hybes, suggesting these are broad range primers based on conserved regions.
Four other Saccharomyces cerevisiae probes (SCER01605, 1610, 1615 and 1620) also end high in all four
hybes (all four in top 200).
These results suggests that for every species the best probe (out of a set of computer-designed
oligonucleotides) has to be determined by hybridization.
Some broad range primers, designed on conserved sequences (like NODE13 probes), always perform very
well, while others do not make it to the top 100.
4
Ranking top 100 per hybe (1 is highest rBGSubSignal, 8455 is lowest)
Rank
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
Escherichia coli
ProbeName FeatureNum
6838
ESCCOLI401275
5306
ESCCOLI401270
839
ESCCOLI401265
1395
ESCCOLI401280
273
ESCCOLI401285
7645
ESCCOLI401260
7423
ESCCOLI401290
6169
ESCCOLI401255
4558
ESCCOLI401000
2728
16AVE04616
5857
ESCCOLI401230
6398
ESCCOLI401250
6514
ESCCOLI401365
1161
ESCCOLI400995
5105
ESCCOLI401245
1771
16AVE00618
8026
16AVE00815
8041
ESCCOLI401375
1836
ESCCOLI401330
7619
16AVE01122
7862
ESCCOLI401370
6811
BAISUBT301360
8166
16AVE00932
8291
ESCCOLI401335
7986
16AVE01096
775
16AVE00781
5321
16AVE00992
3983
16AVE01033
7622
ESCCOLI401220
4233
ESCCOLI401225
3164
16AVE00713
7917
BAISUBT301365
5542
16AVE00541
3192
16AVE00823
7260
ESCCOLI401240
6654
ESCCOLI400440
4451
16AVE01016
830
ESCCOLI401215
5518
ESCCOLI401010
749
16AVE00756
3991
16AVE01045
2961
16AVE00565
7185
16AVE00856
3411
ESCCOLI401005
360
16AVE01007
2164
16AVE00871
2233
16AVE04620
8053
16AVE00893
6101
16AVE00493
3596
16AVE00887
512
ESCCOLI401325
2716
ESCCOLI401015
180
16AVE00962
5020
16AVE01006
7105
16AVE00468
1404
BAISUBT301375
7626
ESCCOLI401340
7773
16AVE00771
2540
16AVE00467
224
16AVE00972
941
16AVE00646
3425
ESCCOLI401235
4776
16AVE00995
4977
ESCCOLI401210
4910
ESCCOLI400825
6518
ESCCOLI400435
4744
16AVE00652
8230
ESCCOLI400445
7719
16AVE00848
2620
16AVE00500
3297
16AVE01071
8361
BAISUBT301380
6161
SCER01610
6412
16AVE00888
2621
16AVE01012
1431
SCER01625
3473
BAISUBT301355
7180
16AVE00975
1583
16AVE00512
5107
ESCCOLI401385
7745
ESCCOLI400820
4595
ESCCOLI401360
2641
16AVE01099
6827
16AVE00609
3342
ESCCOLI401355
2510
BAISUBT301370
5258
16AVE04617
5038
ESCCOLI401140
5077
ESCCOLI400990
2091
SCER01615
8153
16AVE01036
7739
16AVE00894
1898
ESCCOLI400450
7741
ESCCOLI401380
5411
ESCCOLI401150
2215
16AVE01128
457
16AVE01041
6371
16AVE00578
5164
SCER01620
7894
ESCCOLI400430
Bacillus subtilis
ProbeName FeatureNum
2489
16AVE03907
3178
BAISUBT301265
6773
BAISUBT301255
1829
BAISUBT301260
6947
BAISUBT301270
2959
BAISUBT301250
1719
BAISUBT301220
1255
BAISUBT301225
1367
BAISUBT301215
6904
BAISUBT301230
6502
BAISUBT301235
5121
BAISUBT301210
8344
BAISUBT301240
2777
BAISUBT301205
3636
BAISUBT300620
7619
16AVE01122
5542
16AVE00541
2961
16AVE00565
8041
ESCCOLI401375
858
BAISUBT301275
360
16AVE01007
7185
16AVE00856
8026
16AVE00815
7986
16AVE01096
7862
ESCCOLI401370
6734
BAISUBT300990
775
16AVE00781
3991
16AVE01045
6811
BAISUBT301360
3983
16AVE01033
8166
16AVE00932
2164
16AVE00871
1771
16AVE00618
6260
16AVE03906
5321
16AVE00992
7917
BAISUBT301365
180
16AVE00962
388
BAISUBT301245
6514
ESCCOLI401365
4451
16AVE01016
749
16AVE00756
3297
16AVE01071
5605
BAISUBT300995
3192
16AVE00823
7719
16AVE00848
8053
16AVE00893
3473
BAISUBT301355
6101
16AVE00493
3164
16AVE00713
1058
BAISUBT300625
4139
BAISUBT301195
1775
BAISUBT301200
3067
BAISUBT301320
3596
16AVE00887
2620
16AVE00500
7105
16AVE00468
5020
16AVE01006
7773
16AVE00771
6563
BAISUBT300615
941
16AVE00646
8198
16AVE01121
8077
BAISUBT301120
7180
16AVE00975
5898
BAISUBT300815
7676
16AVE00843
2540
16AVE00467
2564
BAISUBT301115
224
16AVE00972
1431
SCER01625
4744
16AVE00652
7855
16AVE00785
830
ESCCOLI401215
1500
BAISUBT301000
1303
BAISUBT300985
4776
16AVE00995
6412
16AVE00888
5107
ESCCOLI401385
1404
BAISUBT301375
4867
16AVE01095
5957
16AVE00780
8153
16AVE01036
6692
BAISUBT301280
2728
16AVE04616
4359
BAISUBT301325
2215
16AVE01128
425
BAISUBT300630
6827
16AVE00609
2510
BAISUBT301370
8361
BAISUBT301380
457
16AVE01041
1583
16AVE00512
6371
16AVE00578
7622
ESCCOLI401220
4940
BAISUBT301005
4367
16AVE00607
2352
BAISUBT301190
6239
16AVE00708
1674
16AVE01039
2621
16AVE01012
6026
16AVE00977
Campylobacter jejuni
ProbeName FeatureNum
3192
16AVE00823
7185
16AVE00856
5020
16AVE01006
7986
16AVE01096
3596
16AVE00887
1771
16AVE00618
360
16AVE01007
4776
16AVE00995
7917
BAISUBT301365
7619
16AVE01122
3983
16AVE01033
7180
16AVE00975
5321
16AVE00992
7719
16AVE00848
3297
16AVE01071
775
16AVE00781
8166
16AVE00932
7862
ESCCOLI401370
8041
ESCCOLI401375
8026
16AVE00815
6514
ESCCOLI401365
3991
16AVE01045
5542
16AVE00541
6811
BAISUBT301360
224
16AVE00972
4451
16AVE01016
2215
16AVE01128
8053
16AVE00893
2164
16AVE00871
2961
16AVE00565
749
16AVE00756
3164
16AVE00713
6101
16AVE00493
7112
16AVE01279
8153
16AVE01036
941
16AVE00646
2754
16AVE01334
7105
16AVE00468
7773
16AVE00771
2620
16AVE00500
7676
16AVE00843
2621
16AVE01012
8423
16AVE01271
180
16AVE00962
1404
BAISUBT301375
706
16AVE00598
7741
ESCCOLI401380
5107
ESCCOLI401385
1583
16AVE00512
2540
16AVE00467
8183
16AVE00855
4025
16AVE01391
7739
16AVE00894
4561
16AVE00767
4744
16AVE00652
6827
16AVE00609
6371
16AVE00578
6412
16AVE00888
6026
16AVE00977
6239
16AVE00708
3016
16AVE00966
4758
16AVE00469
3376
16AVE00836
6905
16AVE01325
5049
16AVE00720
3473
BAISUBT301355
4962
16AVE00635
2641
16AVE01099
1503
16AVE00805
3706
ESCCOLI401390
2510
BAISUBT301370
1265
16AVE01393
8361
BAISUBT301380
1462
16AVE00508
8300
16AVE01319
707
16AVE00520
8198
16AVE01121
6403
16AVE00643
2133
BAISUBT301350
1431
SCER01625
4595
ESCCOLI401360
5377
16AVE00991
4867
16AVE01095
2649
16AVE01080
6644
16AVE00870
3301
16AVE00786
8209
16AVE00814
237
16AVE01379
6217
16AVE00608
2091
SCER01615
6417
16AVE01392
7590
16AVE01015
1737
16AVE01050
5957
16AVE00780
7771
16STRICT00587
1270
16AVE00809
1299
16AVE01779
6161
SCER01610
3367
16AVE00764
2548
16AVE01602
Clostridium cellulyticum
ProbeName FeatureNum
2985
16AVE00085
260
16STRICT00085
5542
16AVE00541
2961
16AVE00565
4451
16AVE01016
8041
ESCCOLI401375
7619
16AVE01122
2164
16AVE00871
180
16AVE00962
7862
ESCCOLI401370
3983
16AVE01033
8026
16AVE00815
775
16AVE00781
8166
16AVE00932
7917
BAISUBT301365
8053
16AVE00893
6811
BAISUBT301360
7986
16AVE01096
360
16AVE01007
5321
16AVE00992
1771
16AVE00618
7185
16AVE00856
3991
16AVE01045
6101
16AVE00493
749
16AVE00756
6514
ESCCOLI401365
3192
16AVE00823
2540
16AVE00467
3164
16AVE00713
3297
16AVE01071
7105
16AVE00468
7773
16AVE00771
2620
16AVE00500
941
16AVE00646
3376
16AVE00836
3473
BAISUBT301355
3596
16AVE00887
4776
16AVE00995
7180
16AVE00975
4744
16AVE00652
224
16AVE00972
7676
16AVE00843
5377
16AVE00991
6412
16AVE00888
5020
16AVE01006
2621
16AVE01012
7719
16AVE00848
2215
16AVE01128
6239
16AVE00708
2510
BAISUBT301370
6371
16AVE00578
4758
16AVE00469
6827
16AVE00609
7739
16AVE00894
1583
16AVE00512
7741
ESCCOLI401380
1026
16AVE00084
8153
16AVE01036
8198
16AVE01121
4561
16AVE00767
6026
16AVE00977
1404
BAISUBT301375
1431
SCER01625
2641
16AVE01099
5049
16AVE00720
5107
ESCCOLI401385
239
16STRICT00084
707
16AVE00520
8183
16AVE00855
3016
16AVE00966
706
16AVE00598
8361
BAISUBT301380
4867
16AVE01095
6403
16AVE00643
6644
16AVE00870
2649
16AVE01080
5957
16AVE00780
1428
16AVE00707
1462
16AVE00508
8209
16AVE00814
2728
16AVE04616
6217
16AVE00608
2133
BAISUBT301350
5164
SCER01620
6161
SCER01610
3301
16AVE00786
7590
16AVE01015
1503
16AVE00805
1737
16AVE01050
1270
16AVE00809
4962
16AVE00635
2091
SCER01615
4595
ESCCOLI401360
3706
ESCCOLI401390
2233
16AVE04620
3367
16AVE00764
2503
16AVE00908
5281
SCER01605
5572
16AVE00905
4086
16STRICT00083
5