Report Agilent Experiment 2, 10-24-2002
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Report Agilent Experiment 2, 10-24-2002 Outline of experiment In this experiment we wanted to investigate the difference in PCR labeling vs. RP labeling. In Experiment 1, the plot of the red channel vs. the position on the E. coli 16S gene in Expt. 1 showed a steep upwards slope. This could be explained by the low dNTP concentration since most of the labeled PCR fragments generated in these suboptimal conditions will not be full-length but only represent the 5’ and the 3’ ends of the molecule. Because only one of the two strands (the 3’ strand) is hybridized during the hybridization process, you see a a higher signal at the 3’ end of the molecule. In this second experiment the PCR labeling of the E. coli PCR fragment will be repeated, this time using the correct amount of dNTPs. Also, the PCR fragment will be labeled using an alternative method, random priming. CR = Common reference pool (equimolar mixture of 16S PCR fragments of 180 strains) RP=random prime (labeling with random hexamers) Cy3 is the green channel Cy5 is the red channel Slide position 1 = barcode side, 2 = non-barcode side Slide 16011437010008 1. E. coli 16S 8Fx1391R PCR fragment, Cy5 PCR-labeled (500 ng/ml) vs. Cy-3 PCR-labeled CR1 (500 ng/ml) 2. E. coli 16S 8Fx1391R PCR fragment, Cy5 RP-labeled (500 ng/ml) vs. Cy-3 PCR-labeled CR1 (500 ng/ml) Note: The CR pool used is the old one, the same as in Experiment 1, the one that was accidentically labeled using a too low amount of dNTPs. The RP and PCR labeled E. coli PCR fragments are new labeling reactions, using the correct amount of dNTPs. Signal Average of signal values: E. coli PCR product PCR labeled RP labeled gBGSubSignal (all features) 532 584 gBGSubSignal (only E.coli) 887 956 rBGSubSignal (all features) 2822 3102 rBGSubSignal (only E.coli) 14510 24803 In the experiment where the E. coli PCR fragment was RP labeled, the average signal is higher than with the PCR labeled fragment. The signal of the green channel is now much lower than in Experiment 1, although the same amount and same batch of common reference pool was used. This suggests competition between the PCR fragments in the pool, and the specific E. coli PCR fragments (used in much higher concentration than in expt. 1). Plot rBGSubSignal vs. position on E. coli 16S gene: The PCR labeled E. coli gives a up-and-down curve with a slope up towards position 1350, then drops (end of labeled product). The slope is less steep than the slope in Expt. 1, where a suboptimal labelling was performed (too low amount of dNTPs), but the slope effect is not completely removed by the higher dNTP concentration. The RP labeled E. coli gives an up-and-down curve with a slight dome effect, and sharply drops after position 1350 (end of the PCR fragment). Overall the signal in this hybe is higher than the PCR labeled hybe. In both plots, the PCR labeled CR pool (green signal) is very low compared to the red channel, and it gives a sharp rise towards the end of the molecule. This might be caused by the suboptimal labeling (see above). When comparing the PCR labeling vs. the RP labeling, the highs and lows are not always on the same position on the E. coli 16S gene. Overall the peaks are not as clear as in exp. 1. There seems to be less competition between the CR and the specific fragment than in expt. 1, probably because a much higher concentration of the specific fragments were used. 1 BGSubSignal E. coli hybridization 45000 40000 35000 30000 RP labeled E. coli 25000 PCR labeled E. coli PCR labeled CR in RP hybe 20000 PCR labeled CR in PCR hybe 15000 10000 5000 1440 1500 1450 1500 1380 1320 1200 1260 1140 1200 1080 1150 960 1020 900 840 780 720 660 600 540 480 420 360 300 240 180 120 0 60 0 Log Ratio E. coli hybridization 1.5 1 0.5 1400 1350 1300 1250 1100 1050 1000 950 900 850 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 0 -0.5 50 0 RP labeled E. coli PCR labeled E. coli -1 -1.5 -2 -2.5 2 Normalized signal in E. coli hybridization 4000000 3500000 3000000 2500000 RP labeled E. coli PCR labeled E. coli 2000000 PCR labeled CR in RP hybe PCR labeled CR in PCR hybe 1500000 1000000 500000 1500 1440 1380 1320 1260 1200 1140 1080 960 1020 900 840 780 720 660 600 540 480 420 360 300 240 180 120 0 60 0 Ranking The rBGSubSignal for each experiment was sorted in descending order (the highest signal on top). Then a ranking number was assigned (the top signal is given rank number 1, the lowest signal 8455). In the RP labeled experiment, 82 features in this top-100 are E. coli tiled sequences,18 features are 16SAVE sequences (all of them representing node 13, a broad range probe), and 1 is a B. subtilis tiled sequence, position 910 (one of the conserved positions on the 16S gene). In the PCR labeled experiment, 53 features in the top-100 are E. coli tiled sequences, 43 features are 16AVE sequences (all of them representing node 13),, and 4 are B. subtilis tiled sequences. So the RP labeling performs better in the ranking. 3 Top 100 PCR labeled E. coli: (ranked on rBGSubSignal) FeatureNumRow Col Start ProbeUID ProbeName GeneNa gBGSubS rBGSubSi Rank 834 6838 839 5306 7514 1395 42 8264 4843 8086 7645 1836 464 5135 6122 4233 273 7423 3324 1378 6913 934 5449 5857 5190 2753 5411 6169 4977 6825 7622 1363 3342 3628 830 4558 5105 7783 2307 5271 5733 2716 4910 2100 5816 2023 4290 2945 2021 4878 5481 7725 2473 5026 7827 6310 7855 1458 1150 5417 4867 512 1005 5928 3425 847 3230 7070 7626 2433 2292 5425 4595 1932 8026 6398 1771 2913 6811 5121 2540 1894 7862 4680 4687 5333 2773 7621 1578 6371 3646 3338 7492 6101 3016 6627 6450 4122 4367 6514 33 74 38 55 38 60 42 76 37 76 80 56 19 62 70 50 6 36 31 43 60 44 20 72 28 83 71 28 82 61 57 28 49 68 29 19 32 40 82 20 37 46 15 53 31 65 18 8 63 72 52 71 70 42 84 80 23 34 82 77 61 67 26 54 43 46 26 39 61 30 67 85 56 63 16 79 80 65 47 48 48 25 30 52 59 82 14 56 65 52 86 45 16 49 79 41 42 28 6 17 400080 401275 401265 401270 400085 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