Report Agilent Experiment 2, 10-24-2002
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Report Agilent Experiment 4, 11-12-2002 (version 1/8/2003) Outline of experiment In this experiment the hybridization patterns of 16S PCR fragments of six different species were studied. The 6 species show different levels of relatedness. They are: 1. Bacillus subtilis 2. Bacillus sphaericus 3. Bacillus thuringiensis 4. Staphylococcus aureus 5. Enterococcus faecalis 6. Streptococcus oralis All 6 PCR fragments were amplified with primers 8F and 1391R, and RP labeled with Cy5. They were hybed against Cy3 RP-labeled CR pool version 2 (with added B. subtilis). CR = Common reference pool (equimolar mixture of 16S PCR fragments of app. 180 strains) RP=random prime (labeling with random hexamers) Cy3 is the green channel Cy5 is the red channel Both the single species and the CR pool were used in an endconcentration of 100 ng/ml. Agilent barcode number 16011437010003 16011437010002 16011437010001 Barcode side (1) B. subtilis B. thuringiensis E. faecalis Non-Barcode side (2) B. sphaericus S. aureus S. oralis Signal Average of signal values on Bacillus subtilis 16S tiled sequence probes (253 probes) 16S PCR fragment of Bacillus subtilis Bacillus sphaericus Bacillus thuringiensis Staphyloccus aureus Enterococcus faecalis Streptococcus oralis gBGSubSignal 1463 1953 1564 1704 1985 2773 rBGSubSignal 39739 30108 34076 29112 18306 18766 The average signal of the CR (green channel) is higher than in experiment 2. There is a trend: the less related to B. subtilis, the lower the average red signal, and the higher the green signal. Competition? Plot rBGSubSignal or NormSignal vs. position on Bacillus subtilis 16S gene: Hybridizations with all six species yields an up and down curve, so not all probes bind the labeled PCR product with the same efficiency. Two possibilities: 1. some probes have higher affinity to labeled PCR products than others (all have same length so there will be variations in GC content, different Tm, secundary structure) 2. conserved probes compete with Common Reference pool, specific probes do not In most cases the CR (green signal) goes up and down together with the red signals. This suggests that effect 1 is stronger than effect 2. Comparison of the six species: starting with BacSubt and moving “up the tree” more and more peaks disappear. E.g. peaks at position 180, 450, 1000, and 1250 seem BacSubt specific, not present in other species in this genus. BGSubSignals 70000 60000 50000 Bacillus subtilis Bacillus sphaericus Bacillus thuringiensis 40000 Staphylococcus aureus Enterococcus faecalis 30000 Streptococcus oralis CR in Bacillus subtilis hybe 20000 10000 10 0 15 0 20 0 25 0 30 0 35 0 40 0 45 0 50 0 59 0 64 0 73 0 78 0 87 0 92 0 97 0 10 20 10 70 11 20 11 70 12 20 12 70 13 20 13 70 0 50 0 Simplified version: BGSubSignals 70000 60000 50000 40000 Bacillus subtilis Bacillus sphaericus Streptococcus oralis CR in Bacillus subtilis hybe 30000 20000 10000 1370 1320 1270 1220 1170 1120 1070 970 1020 920 870 780 730 640 590 500 450 400 350 300 250 200 150 50 100 0 0 Normalized signals 300000 250000 200000 Bacillus subtilis Bacillus sphaericus Bacillus thuringiensis 150000 Staphylococcus aureus Enterococcus faecalis Streptococcus oralis CR in Bacillus subtilis hybe 100000 50000 1370 1320 1270 1220 1170 1120 1070 970 1020 920 870 780 730 640 590 500 450 400 350 300 250 200 150 100 0 50 0 Common reference plot: (BGSubSignal) The CR BGSubsSignal (see below) is more of less the same in all six hybes. Overall signal much lower than single species hybe signal. The CR consists of a pool of PCR fragments of 180 different bacteria so only a few species (the ones closely related to Bacillus subtilis) can bind specifically. The CR signal is the highest in the Streptococcus oralis hybe, the species the least related to the Bacillus subtilis probes, suggesting competition between the common reference and the single species. CR (green channel) 9000 8000 7000 6000 Bacillus subtilis Bacillus sphaericus 5000 Bacillus thuringiensis Staphyloccus aureus 4000 Enterococcus faecalis Streptococcus oralis 3000 2000 1000 1380 1335 1290 1245 1200 1155 1110 1065 975 930 885 800 755 710 625 580 495 450 405 360 315 270 225 180 135 90 1020 . 45 0 0 BGSubSignals 70000 60000 50000 Bacillus subtilis Bacillus sphaericus Bacillus thuringiensis 40000 Staphylococcus aureus Enterococcus faecalis 30000 Streptococcus oralis CR in Bacillus subtilis hybe 20000 10000 10 0 15 0 20 0 25 0 30 0 35 0 40 0 45 0 50 0 59 0 64 0 73 0 78 0 87 0 92 0 97 0 10 20 10 70 11 20 11 70 12 20 12 70 13 20 13 70 0 50 0 Plot Log Ratio’s (R/G) Unclear: Is this logratio of the normalized or the BGSubSignal?? The logratio’s of Bacillus subtilis hybes are almost all above 0, so much stronger red signal than red. Peaks around position 190, 450, 1000 and 1280 (same as in plot BGSubSignal).The ratio’s of the less related Streptococcus oralis are more around 0. LogRatios 2.5 2 1.5 1 Bacillus subtilis Bacillus sphaericus Bacillus thuringiencis 0.5 Staphylococcus aureus Enterococcus faecalis Streptococcus oralis 1380 1335 1290 1245 1200 1155 1110 1065 975 1020 930 885 800 755 710 625 580 495 450 405 360 315 270 225 180 90 135 0 45 0 -0.5 -1 -1.5 Simplified version: LogRatios 2.5 2 1.5 1 Bacillus subtilis Bacillus sphaericus Streptococcus oralis 0.5 -1 1380 1335 1290 1245 1200 1155 1110 1065 1020 975 930 885 800 755 710 625 580 495 450 405 360 315 270 225 180 90 135 0 -0.5 45 0 Ranking The rBGSubSignal for both the Bacillus subtilis and the Streptococcus oralis hybes (the 2 most distant species) were sorted in descending order (the highest signal on top). Then a ranking number was assigned (the top signal was 1, the lowest signal 8000-something). The top 100 of the Bacillus subtilis hybe is printed below, with the ranking number in the Streptococcus oralis hybe next to it. FeatureNum Start ProbeUID ProbeName GeneName Rank BaiSubt hybe Rank in StrepOralis Hybe 5898 2432 4246 2401 5616 4526 4878 3144 3278 4100 7580 4127 587 6607 3546 853 5280 5360 3485 1826 7096 1360 2564 1415 432 7219 957 6168 1477 1674 2801 516 2149 2356 7658 594 1328 6545 4739 3937 3997 5840 878 4467 3137 2483 6878 4380 7218 5142 3259 3852 5529 7010 2454 7855 2441 4532 4644 5749 5144 5112 1144 1097 4353 5813 3750 8279 5705 1872 6009 2634 2996 7512 6610 8305 3608 5969 5788 1291 4277 5663 3433 1668 1622 4964 2433 3973 1822 1302 5961 4599 4372 4408 5639 5132 6070 2637 6856 8109 5775 2321 4128 2292 5498 4408 4760 3026 3160 3982 7419 4009 518 6468 3428 778 5162 5242 3367 1729 6947 1273 2451 1328 367 7066 880 6039 1388 1581 2683 449 2044 2247 7495 525 1243 6408 4621 3819 3879 5719 803 4349 3019 2372 6733 4262 7065 5024 3141 3734 5411 6863 2343 7688 2330 4414 4526 5630 5026 4994 1063 1016 4235 5692 3632 8102 5586 1773 5884 2519 2878 7353 6471 8128 3490 5844 5667 1206 4159 5545 3315 1575 1529 4846 2322 3855 1725 1217 5838 4481 4254 4290 5521 5014 5943 2522 6711 7936 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 1547 6 1166 219 805 269 47 601 936 44 81 1021 148 1008 72 1141 270 75 4 1194 1506 1135 1105 1276 147 170 1801 966 2674 64 234 1292 130 750 10 94 1241 2311 361 37 1271 1510 720 1827 183 2258 398 1173 68 1124 967 116 1719 101 1091 55 287 2120 15 1283 1019 759 196 138 17 447 111 1047 35 127 1066 1048 107 879 118 2670 213 96 23 854 245 715 51 1259 950 333 184 137 233 1223 1058 172 452 189 206 265 685 279 1795 1001 300815 1109 300550 528 300265 301025 1145 300270 300085 300290 597 300695 300285 300065 1153 300800 300280 1101 301060 300070 300805 300705 301115 300555 301030 1022 300820 300080 300195 1039 761 300560 666 300690 580 738 300575 300200 800 1063 301010 300055 300275 300825 478 300205 300760 300075 1002 300260 300390 827 300595 987 300380 785 301185 300105 965 300215 300335 300685 1093 1056 400770 300890 634 300700 501 605 300370 300395 1110 300365 678 3904 647 693 1020 300505 810 997 897 300710 300360 949 915 933 625 300715 301015 300295 963 783 300955 1111 300795 558 300610 300375 BAISUBT300815 16AVE01109 BAISUBT300550 16AVE00528 BAISUBT300265 BAISUBT301025 16AVE01145 BAISUBT300270 BAISUBT300085 BAISUBT300290 16AVE00597 BAISUBT300695 BAISUBT300285 BAISUBT300065 16AVE01153 BAISUBT300800 BAISUBT300280 16AVE01101 BAISUBT301060 BAISUBT300070 BAISUBT300805 BAISUBT300705 BAISUBT301115 BAISUBT300555 BAISUBT301030 16AVE01022 BAISUBT300820 BAISUBT300080 BAISUBT300195 16AVE01039 16AVE00761 BAISUBT300560 16AVE00666 BAISUBT300690 16AVE00580 16AVE00738 BAISUBT300575 BAISUBT300200 16AVE00800 16AVE01063 BAISUBT301010 BAISUBT300055 BAISUBT300275 BAISUBT300825 16AVE00478 BAISUBT300205 BAISUBT300760 BAISUBT300075 16AVE01002 BAISUBT300260 BAISUBT300390 16AVE00827 BAISUBT300595 16AVE00987 BAISUBT300380 16AVE00785 BAISUBT301185 BAISUBT300105 16AVE00965 BAISUBT300215 BAISUBT300335 BAISUBT300685 16AVE01093 16AVE01056 ESCCOLI400770 BAISUBT300890 16AVE00634 BAISUBT300700 16AVE00501 16AVE00605 BAISUBT300370 BAISUBT300395 16AVE01110 BAISUBT300365 16AVE00678 16AVE03904 16AVE00647 16AVE00693 16AVE01020 BAISUBT300505 16AVE00810 16AVE00997 16AVE00897 BAISUBT300710 BAISUBT300360 16AVE00949 16AVE00915 16AVE00933 16AVE00625 BAISUBT300715 BAISUBT301015 BAISUBT300295 16AVE00963 16AVE00783 BAISUBT300955 16AVE01111 BAISUBT300795 16AVE00558 BAISUBT300610 BAISUBT300375 BAISUBT 16AVE BAISUBT 16AVE BAISUBT BAISUBT 16AVE BAISUBT BAISUBT BAISUBT 16AVE BAISUBT BAISUBT BAISUBT 16AVE BAISUBT BAISUBT 16AVE BAISUBT BAISUBT BAISUBT BAISUBT BAISUBT BAISUBT BAISUBT 16AVE BAISUBT BAISUBT BAISUBT 16AVE 16AVE BAISUBT 16AVE BAISUBT 16AVE 16AVE BAISUBT BAISUBT 16AVE 16AVE BAISUBT BAISUBT BAISUBT BAISUBT 16AVE BAISUBT BAISUBT BAISUBT 16AVE BAISUBT BAISUBT 16AVE BAISUBT 16AVE BAISUBT 16AVE BAISUBT BAISUBT 16AVE BAISUBT BAISUBT BAISUBT 16AVE 16AVE ESCCOLI BAISUBT 16AVE BAISUBT 16AVE 16AVE BAISUBT BAISUBT 16AVE BAISUBT 16AVE 16AVE 16AVE 16AVE 16AVE BAISUBT 16AVE 16AVE 16AVE BAISUBT BAISUBT 16AVE 16AVE 16AVE 16AVE BAISUBT BAISUBT BAISUBT 16AVE 16AVE BAISUBT 16AVE BAISUBT 16AVE BAISUBT BAISUBT
