Gleis Week 8

Vibrio cholerae Statistical Analysis

Uploaded Files

Media:Merrell_Compiled_Raw_Data_Vibrio_Gleis_forGenMAPP.txt

Media:Merrell_Compiled_Raw_Data_Vibrio_Gleis_forGenMAPP.xls

Sanity Check

P<0.05 - 137

p<0.01 - 26

p<0.001 - 2

p<0.0001 - 0

p<0.05 and fold change>0 - 43

p<0.05 and fold change<0 - 94

p<0.05 and fold change>0.25 - 41

p<0.05 and fold change<-.025 - 93

Lab Journal

1. Visited https://xmlpipedb.cs.lmu.edu/biodb/fall2013/index.php/Week_82 for procedural instructions

2. Opened this link: Sample Microarray Analysis for Vibrio cholerae page and downloaded data from Merrel et al. Host-induced epidemic spread of the cholera bacterium (2002)

3.Copied and selected all data in the file and pasted into a new Excel spreadsheet titled "Normalized".

4. Inserted two new rows just under the top title row. The first row was labeled "Average" and the second row was labeled "STDEV".

5. The average and standard deviations were calculated for each column

6. The data was then normalized by subtracting the average from a particular cell and dividing that sum by the standard deviation.

7. The data was copied and pasted-values only- into a new Excel sheet titled "statistics"

8. The averages of the technical replicates of each biological sample were calculated in the next three Columns

9. One column contained the average values of each gene for patient A, then Patient B, and finally patient C

10. In the next column the average values of A,B, and C were average to find a total average for each gene

11. Next a column was used to calculate if the average log ratio differed significantly from 0 using the equation

=AVERAGE(N2:P2)/(STDEV(N2:P2)/SQRT(number of replicates))

12. Next a p-value was calculated for each gene using the equation

=TDIST(ABS(R2),degrees of freedom,2)

13. The data were then formatted to be accepted by GenMAPP.

Lab Journal part 2

1.Launch GenMAPP

2.To select a gene database I selected Data > Choose Gene Database

3.I downloaded the 2008 version of the database at http://sourceforge.net/projects/xmlpipedb/files/V.%20cholerae%20Gene%20Database/V.%20cholerae%2020090622/Vc-Std_External_20090622.zip/download

4.The downloaded file was then extracted from the zip download format

5.Re-launch GenMAPP and loaded the newly downloaded database.

6.Selected Data >Expression Dataset Manager.

7.When the Dataset manger window opened I selected New Dataset.

8.I selected the tab-delimited file created using the procedure outlined in Week 8 Lab Journal Part 1

9.The expression data manager then converted the data in the txt file previously selected.

10.A (.gex) file was saved with the converted data

11.772 lines were determined to contain an error in the process of data conversion.

The genes with errors were:

VC2758 VC2758 VC2759 VC0015 VC0020 VC0021 VC0275 VC0276 VC0298 VC0328 VC0328 VC0328 VC0329 VC0329 VC0329 VC0556 VC0557 VC0602 VC1110 VC1115 VC1128 VC1133 VC1159 VC1166 VC1388 VC1692 VC1692 VC1922 VC1942 VC2209 VC2209 VC2214 VC2226 VC2245 VC2245 VC2245 VC2247 VC2248 VC2458 VC2480 VC2492 VC2501 VC2508 VC2514 VCA1031 VCA0744 VCA0745 VCA0748 VCA0751 VCA1045 VCA1046 VCA0161 VCA0180 VCA0183 VCA0207 VC0056 VC0093 VC0093 VC0095 VC0120 VC0339 VC0384 VC0385 VC0386 VC0626 VC0639 VC0956 VC1182 VC1202 VC1237 VC1476 VC1483 VC1704 VC1998 VC2019 VC2036 VC2262 VC2268 VC2277 VC2316 VC2571 VCA0522 VCA0534 VCA0805 VCA0805 VCA0824 VCA0825 VCA1079 VCA0016 VCA0235 VCA0276 VCA0276 VCA0278 VC2758 VC2759 VC0006 VC0020 VC0020 VC0276 VC0297 VC0298 VC0328 VC0328 VC0329 VC0329 VC0329 VC0556 VC0602 VC0889 VC1110 VC1128 VC1132 VC1133 VC1159 VC1166 VC1167 VC1388 VC1660 VC1692 VC1911 VC2201 VC2209 VC2209 VC2214 VC2225 VC2245 VC2245 VC2246 VC2247 VC2457 VC2461 VC2493 VC2501 VC2508 VC2510 VC2514 VCA0449 VCA0711 VCA0745 VCA0748 VCA0751 VCA1025 VCA1045 VCA1046 VCA1060 VCA0183 VCA0221 VC0055 VC0093 VC0100 VC0125 VC0384 VC0385 VC0390 VC0626 VC0639 VC0675 VC0902 VC0956 VC1202 VC1203 VC1219 VC1237 VC1242 VC1457 VC1759 VC2016 VC2018 VC2019 VC2036 VC2262 VC2262 VC2549 VCA0522 VCA0522 VCA0534 VCA0805 VCA0823 VCA0824 VCA0827 VCA0016 VCA0016 VCA0235 VCA0276 VCA0276 VCA0278 VCA0280 VC0162 VC0432 VC0444 VC0445 VC0465 VC0685 VC0704 VC0722 VC0723 VC0741 VC0745 VC0971 VC0971 VC0986 VC0999 VC1018 VC1257 VC1307 VC1513 VC1782 VC1784 VC2049 VC2049 VC2085 VC2107 VC2338 VC2338 VC2348 VC2349 VC2350 VC2616 VC2618 VC2629 VC2645 VC2646 VC2651 VC2670 VCA0572 VCA0596 VCA0606 VCA0614 VCA0616 VCA0875 VC0207 VC0222 VC0241 VC0242 VC0243 VC0487 VC0500 VC0518 VC0521 VC0526 VC0541 VC0759 VC0760 VC0773 VC0792 VC0793 VC0794 VC1038 VC1337 VC1585 VC1620 VC1620 VC1620 VC1625 VC1625 VC1875 VC2116 VC2174 VC2175 VC2180 VC2400 VC2401 VC2412 VC2427 VC2446 VC2447 VC2701 VC2709 VC2718 VC2738 VCA0699 VCA0925 VCA0946 VCA0984 VCA0128 VCA0150 VC0158 VC0162 VC0445 VC0465 VC0468 VC0692 VC0704 VC0722 VC0723 VC0723 VC0741 VC0748 VC0971 VC0987 VC0994 VC1018 VC1307 VC1513 VC1513 VC1532 VC1781 VC1784 VC1784 VC1807 VC2209 VC2049 VC2057 VC2085 VC2107 VC2338 VC2338 VC2347 VC2348 VC2349 VC2616 VC2618 VC2646 VC2646 VC2655 VCA0572 VCA0596 VCA0614 VCA0615 VCA0875 VCA0358 VC0207 VC0210 VC0240 VC0241 VC0242 VC0243 VC0487 VC0499 VC0501 VC0518 VC0521 VC0541 VC0545 VC0759 VC0760 VC0773 VC0774 VC0793 VC0793 VC0818 VC1098 VC1327 VC1337 VC1355 VC1585 VC1620 VC1620 VC1620 VC1625 VC1625 VC1877 VC2118 VC2157 VC2174 VC2180 VC2182 VC2400 VC2401 VC2412 VC2446 VC2447 VC2689 VC2701 VC2708 VC2738 VCA0687 VCA0699 VCA0925 VCA0946 VCA0984 VCA0133 VC2762 VC2763 VC2764 VC2766 VC0013 VC0015 VC0028 VC0046 VC0051 VC0284 VC0284 VC0304 VC0318 VC0331 VC0332 VC0336 VC0563 VC0583 VC0592 VC0602 VC0607 VC0853 VC0869 VC0869 VC0869 VC0875 VC0889 VC0891 VC1134 VC1135 VC1169 VC1170 VC1171 VC1173 VC1174 VC1399 VC1401 VC1428 VC1650 VC1650 VC1660 VC1704 VC1916 VC2209 VC2209 VC2215 VC2215 VC2234 VC2236 VC2254 VC2469 VC2490 VC2491 VC2503 VC2503 VCA0454 VCA0724 VCA0736 VCA0736 VCA1031 VCA1031 VCA1036 VCA1073 VCA1073 VCA0197 VCA0232 VCA0232 VC0125 VC0346 VC0372 VC0374 VC0390 VC0390 VC0631 VC0663 VC0664 VC0682 VC0682 VC0683 VC0941 VC0944 VC0956 VC1188 VC1190 VC1203 VC1204 VC1205 VC1220 VC1220 VC1226 VC1231 VC1491 VC1726 VC1727 VC1730 VC1730 VC1732 VC1735 VC2209 VC2043 VC2273 VC2274 VC2287 VC2290 VC2307 VC2309 VC2545 VC2558 VC2559 VC2602 VCA0815 VCA0828 VCA1089 VCA1090 VCA1104 VCA0287 VC2762 VC2764 VC2765 VC2766 VC2768 VC0013 VC0015 VC0028 VC0045 VC0051 VC0052 VC0284 VC0284 VC0304 VC0318 VC0320 VC0332 VC0336 VC0583 VC0591 VC0607 VC0839 VC0869 VC0869 VC0875 VC1134 VC1135 VC1137 VC1139 VC1170 VC1171 VC1172 VC1174 VC1395 VC1401 VC1428 VC1650 VC1650 VC1660 VC1704 VC1972 VC2209 VC2209 VC2209 VC2215 VC2215 VC2244 VC2254 VC2256 VC2258 VC2469 VC2490 VC2491 VC2492 VC2503 VC2503 VCA0724 VCA0736 VCA0744 VCA1031 VCA1036 VCA1073 VCA1073 VCA0180 VCA0197 VCA0232 VC0092 VC0126 VC0347 VC0372 VC0374 VC0390 VC0390 VC0631 VC0664 VC0682 VC0682 VC0941 VC0943 VC1188 VC1190 VC1204 VC1205 VC1220 VC1245 VC1476 VC1491 VC1726 VC1727 VC1730 VC1732 VC1736 VC2021 VC2023 VC2043 VC2273 VC2274 VC2287 VC2290 VC2309 VC2316 VC2559 VC2560 VC2602 VCA0815 VCA1089 VCA1091 VCA1104 VCA0287 VC0151 VC0154 VC0453 VC0456 VC0472 VC0473 VC0476 VC0477 VC0726 VC0748 VC0755 VC0756 VC0994 VC0997 VC1036 VC1263 VC1297 VC1312 VC1552 VC1807 VC2062 VC2074 VC2108 VC2109 VC2360 VC2363 VC2389 VC2389 VC2390 VC2623 VC2626 VC2641 VC2642 VC2643 VC2644 VCA0596 VCA0604 VCA0624 VCA0885 VCA0887 VCA0053 VCA0057 VCA0098 VC2751 VC0211 VC0215 VC1911 VC0528 VC0531 VC0545 VC0550 VC0551 VC0766 VC0768 VC0771 VC0786 VC0800 VC0819 VC1046 VC1047 VC1047 VC1053 VC1098 VC1327 VC1347 VC1595 VC1611 VC1620 VC1620 VC1620 VC1848 VC2183 VC2403 VC2404 VC2406 VC2437 VC2438 VC2438 VC2448 VC2700 VC2700 VC2746 VCA0674 VCA0678 VCA0678 VCA0687 VCA0692 VCA0938 VC0151 VC0154 VC0441 VC0454 VC0472 VCA0624 VC0476 VC0477 VC0755 VC0997 VC1010 VC1018 VC1036 VC1036 VC1263 VC1297 VC1312 VC1552 VC2062 VC2074 VC2108 VC2109 VC2116 VC2389 VC2389 VC2390 VC2391 VC2623 VC2624 VC2626 VC2628 VC2641 VC2642 VC2644 VC2645 VCA0604 VCA0623 VCA0624 VCA0885 VCA0887 VCA0057 VCA0073 VCA0098 VCA0098 VC0215 VC0221 VC0254 VC0275 VC0518 VC0529 VC0532 VC0545 VC0549 VC0550 VC0551 VC0766 VC0768 VC0786 VC0792 VC0801 VC0819 VC1046 VC1047 VC1336 VC1595 VC1620 VC1620 VC1847 VC1848 VC2184 VC2403 VC2404 VC2406 VC2437 VC2438 VC2438 VC2448 VC2700 VC2700 VC2700 VC2746 VC2751 VCA0674 VCA0678 VCA0692 VCA0128 VCA0161

12.An exception file was created with all data and the data with errors denoted.

Media:Gleis - Merrell Compiled Raw Data Vibrio.EX.txt
Errors- 772 errors vs. 121 for my partner Mitchell.
My older version of database is more susceptible to error.

13.The new expression dataset was customized

14.The customization was performed by opening the expression dataset manager window then selecting create a color set.

15.Two color sets were created, one titled "Increase" for the set of genes whose expression increased and one set titled "Decrease" for the set of genes whose expression decreased.

16.The Increase color set was set to pink and contains criteria [Avg_LogFC_all] > 0.25 AND [Pvalue] < 0.05

17.The Decrease color set was set to green and contains criteria [Avg_LogFC_all] < -0.25 AND [Pvalue] < 0.05

18.The color set was then saved by selecting save from the toolbar of the expression dataset manager menu

19.The expression dataset manager was closed by clicking the close button in the top right corner.

20.The file was saved as a (.gex) file

Media:Gleis - Merrell Compiled Raw Data Vibrio.gex

21.Launched the MAPPFinder program

22.The correct species was already loaded for MAPPFinder

23.Clicked calculate new results

24.Selected "Find FIle" and browsed for the .gex file saved previously

25.Selected the "Decreased" color set described previously

26.Checked boxes next to "Gene Ontology" and "p value"

27.Clicked the "Browse" button and saved the file as Media:Deacreased Expression Vibrio Gleis-Criterion1-GO.txt

28.Clicked "Run MAPPFinder"

29.A new window opens with a tree of results

30.Clicked on "Show Ranked List"

31.Top 10 Gene Ontology Terms 1.Localization 2.Transporter Activity 3.Cellular Biopolymer biosynthetic process 4.Biopolymer Synthetic Process 5.Cellular macromolecule biosynthetic process 6.macromolecule biosynthetic process 7.Macromolecule metabolic process 8.Transport 9.Establishment of Localization 10.cellular macromolecule metabolic process

The terms are not the same as my partner Mitchell's. This is likely because the newer dataset contained fewer errors and resulted in a greater number of genes processed by MAPPFinder.

32.Entered Gene IDs mentioned by Merrell et al. (2002) into Gene ID search.

33.The GO term results are below: parameters included all Go terms with 0 percent of genes changed and pvalue < 0.05

VC0028

branched chain family amino acid biosynthetic process
cellular amino acid biosynthetic process
metabolic process
metal ion binding
iron-sulfur cluster binding
4 iron, 5 sulfur cluster binding
catalytic activity
lyase activity
dihydroxy-acid dehydratase activity

VC0941

glycine metabolic process
L-serine metabolic process
one-carbon metabolic process
cytoplasm
pyridoxal phosphate binding
catalytic activity
transferase activity
glycine hydromethyltransferase activity

VC0869

glutamine metabolic process
purine nucleotide biosynthetic process
'de novo' IMP biosynthetic process
cytoplasm
nucleotide binding
ATP binding
catalytic activity
ligase activity
phosphoribosylformylglycinamidine synthase activity

VC0051

purine nucleotide biosynthetic process
'de novo' IMP biosynthetic process
nucleotide binding
ATP binding

8catalytic activity

lyase activity
carboxy-lyase activity
phosphoribosylaminoimidazole carboxylase activity

VC0647

mRNA catabolic process
RNA processing
cytoplasm
mitochondrion
RNA binding
3'-5'-exoribonuclease activity
transferase activity
polyribonucleotide nucleotidyltransferase activity

VC0468

glutathione biosynthetic process
metal ion binding
nucleotide binding
ATP binding
catalytic activity
ligase activity
glutathione synthase activity

VC2350

deoxyribonucleotide catabolic process
metabolic process
cytoplasm
catalytic activity
lyase activity
deoxyribose-phosphate aldolase activity

VCA0583

transport
outer membrane-bounded periplasmic space
transporter activity

34.My results corresponded with my partner Mitchell's results

35.Clicked on outer-membrane bounded periplasmic space under VCA0583

36.A results window opened

37.Searched uniport database with geneID of interest to determine expression from results of GO term search

38.VCA0583 is also known as Q9KM06_VIBCH and it showed little change in expression.

39.Clicked on the result box of Q9KM06_VIBCH

40.A new internet explorer window opened.

41.Clicked on the UniProt link and learned that the function of the protein is membrane transport

42.The MAPP file for this search is Media:Outer membrane-bounded periplasmic space.mapp

43.The results of the MAPPFinder were saved as: Media:Decreased Expression Vibrio Gleis-Criterion1-GO-Criterion0-GO.txt

44.The txt file was opened in Excel and compared with my partners data

45.Our data is different because a different number of genes were used for calculations due to differences in the databases.

46.Filtered data by: Z Score (in column N) greater than 2 PermuteP (in column O) less than 0.05 Number Changed (in column I) greater than or equal to 4 AND less than 100 Percent Changed (in column L) greater than or equal to 30%

47.Compared data in Excel to data in MAPPFinder

48.Comparisons were highlighted using a similar color scheme

Media:Decreased Expression Vibrio Gleis-Criterion1-GO-Criterion0-GO.xls

49.The expression of genes involved in catabolic processes decreased while expression of cell metabolic and cell cycle processes increased. This suggests that the patient-derived Vibrio is expending great amounts of energy and dividing rapidly while the lab-grown Vibrio is building itself up and reserving energy. From this information we can see that at a molecular level the patient-derived Vibrio appear to be much more aggressive in finding food and dividing while the lab-grown Vibrio seem to be suing food sources to build themselves up and divide at a slower rate

50.Media:Gleis - Merrell Compiled Raw Data Vibrio.gmf