gse28619

Maria Teresa Rubio Martinez-Abarca and Guillermo Ayala

5/11/23

Introducción

The dataset has been downloaded from GEO

The summary given there is > Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease and occurs in patients with excessive alcohol intake It is characterized by marked hepatocellular damage, steatosis and pericellular fibrosis. Patients with severe AH have a poor short-term prognosis. Unfortunately, current therapies (i.e. corticosteroids and pentoxyphylline) are not effective in many patients and novel targeted therapies are urgently needed. The development of such therapies is hampered by a poor knowledge of the underlying molecular mechanisms. Based on studies from animal models, TNF alfa was proposed to play a pivotal role in the mechanisms of AH. Consequently, drugs interfering TNF alfa were tested in these patients. The results were disappointing due to an increased incidence of severe infections. Unluckily, there are not experimental models that mimic the main findings of AH in humans. To overcome this limitation, translational studies with human samples are required. We previously analyzed samples from patients with biopsy-proven AH. In these previous studies, we identified CXC chemokines as a potential therapeutic target for these patients. We expanded these previous observations by performing a high-throughout transcriptome analysis.

Downloading and preprocessing the dataset

The vignette tamidata::gse28619 details how to download and preprocessing the dataset. The final result obtained using the code given there is an ExpressionSet used from now on.

Marginal differential expression

  • Loading basic packages needed later.
library(Biobase)
library(tami)
  • Loading the dataset tamidata::gse28619.
data(gse28619,package="tamidata")
  • The number of features (genes) and samples are
dim(gse28619)
Features  Samples 
   54675       22
  • Phenotipic variable?
data(gse28619,package="tamidata")
  • The number of features (genes) and samples are
dim(gse28619)
Features  Samples 
   54675       22
  • Phenotipic variable?
pData(gse28619)[,"type"]
 [1] control   control   control   control   control   control   control  
 [8] alcoholic alcoholic alcoholic alcoholic alcoholic alcoholic alcoholic
[15] alcoholic alcoholic alcoholic alcoholic alcoholic alcoholic alcoholic
[22] alcoholic
Levels: control alcoholic
  • Gene identifiers (not shown)
fData(gse28619)

Marginal differential expression

The test used is rowt corresponding to the t-test assuming a common variance.

x1 = dema(x=gse28619, y = "type", test = rowt, correction = "BH",
fdr = 0.01,foutput = "gse28619")

Note that only those genes with an adjusted p-value lesser than fdr are returned. If we want the analysis for all genes then we have to set fdr = 1.

A data.frame with the results can be obtained.

x1_df = tidy(x1)

These results can be examined in a html file.

browseURL(glimpse(x1))

How many genes have an adjusted p-value lesser than fdr = 0.01?

nrow(x1_df)
[1] 9198

Which is the highest adjusted p-value for the returned genes?

max(x1_df[,"adjp"])
[1] 0.009994994

We want to know all the information about the gene with ENTREZID identifier 6898. First we need to know the row within the data.frame x1_df.

grep("^6898",x1_df[,"ENTREZID"])
[1]  174 4243

The expression “^6898” looks for ENTREZID beginning with this code. Let us see these rows

x1_df[grep("^6898",x1_df[,"ENTREZID"]),]
            PROBEID ENTREZID         ENSEMBL         GO EVIDENCE ONTOLOGY
22260  1555189_a_at     6898 ENSG00000198650 GO:0004838      IDA       MF
402759    214413_at     6898 ENSG00000198650 GO:0004838      IDA       MF
       statistic         rawp         adjp         qval
22260   5.146400 4.913519e-05 0.0006416569 0.0003251238
402759  3.742766 1.282815e-03 0.0081103027 0.0041094428

Why we have two rows? Note that the PROBEID’s are different for each row. They are different probe sets but they corresponds to the same gene.

Note that the ENSEMBL and GO identifiers are ENSG00000198650 and GO:0004838 respectively.

Gene set collection

load("hsa_go.rda")
hsa_go
  • How many gene sets?
length(hsa_go)
[1] 22934

The first gene set is

hsa_go[1]
$`GO:0000002`
 [1] "5428"   "6742"   "11232"  "55186"  "56652"  "84275"  "92667"  "201973"
 [9] "1763"   "7157"   "9093"   "10891"  "80119"  "83667"  "201163" "142"   
[17] "1890"   "2021"   "3980"   "4358"   "4976"   "6240"   "7156"   "10000" 
[25] "50484"  "64863"  "219736" "4205"   "9361"   "291"

We have the name and the (ENTREZID) identifiers of the genes belonging to this gene set. The elements of the set can be accessed with

hsa_go[[1]]
 [1] "5428"   "6742"   "11232"  "55186"  "56652"  "84275"  "92667"  "201973"
 [9] "1763"   "7157"   "9093"   "10891"  "80119"  "83667"  "201163" "142"   
[17] "1890"   "2021"   "3980"   "4358"   "4976"   "6240"   "7156"   "10000" 
[25] "50484"  "64863"  "219736" "4205"   "9361"   "291"

We can know the number of elements in this groups with

length(hsa_go[[1]])
[1] 30

It is not necessary to evaluate the length (cardinal) of each group. It can be known the cardinality of each set simultaneously using lapply.

ngs = lapply(hsa_go,length)

What kind of object has been returned?

class(ngs)
[1] "list"

The (previously calculated) length of the first gene set is

ngs[1]
$`GO:0000002`
[1] 30

or

ngs[[1]]
[1] 30

The lengths of the first gene sets can be obtained with

ngs[1:10]
$`GO:0000002`
[1] 30

$`GO:0000003`
[1] 1506

$`GO:0000009`
[1] 2

$`GO:0000010`
[1] 2

$`GO:0000012`
[1] 12

$`GO:0000014`
[1] 10

$`GO:0000015`
[1] 4

$`GO:0000016`
[1] 1

$`GO:0000017`
[1] 2

$`GO:0000018`
[1] 134

The largest gene set is

which.max(ngs)
GO:0005575 
      2643

How many gene sets have exactly 10 genes?

table(ngs == 10)

FALSE  TRUE 
22506   428

Other possibility is

sum(ngs == 10)
[1] 428

Note that the lengths calculated ngs is a list. Sometimes it is useful to have a vector.

ngs0 = unlist(ngs)
class(ngs0)
[1] "integer"

We can evaluate a table of absolute frequencies with

table(ngs0)
ngs0
    1     2     3     4     5     6     7     8     9    10    11    12    13 
 4466  2747  1783  1292   989   789   665   590   476   428   400   368   287 
   14    15    16    17    18    19    20    21    22    23    24    25    26 
  256   268   213   216   206   177   147   155   145   125   124   123   121 
   27    28    29    30    31    32    33    34    35    36    37    38    39 
  119   108    89   106    90    92    68    82    66    73    78    66    63 
   40    41    42    43    44    45    46    47    48    49    50    51    52 
   64    52    52    61    61    50    52    55    58    45    42    48    46 
   53    54    55    56    57    58    59    60    61    62    63    64    65 
   37    45    36    40    46    33    26    30    34    30    29    35    28 
   66    67    68    69    70    71    72    73    74    75    76    77    78 
   21    29    27    21    29    20    33    29    25    24    23    21    25 
   79    80    81    82    83    84    85    86    87    88    89    90    91 
   18    32    14    21    21    10    18    24    23    20    16    17    21 
   92    93    94    95    96    97    98    99   100   101   102   103   104 
   26    21    26    15    17    23    14    21    13    10    21    18    21 
  105   106   107   108   109   110   111   112   113   114   115   116   117 
   14     9    19    11    24    12    10    14     8    13    15    15    13 
  118   119   120   121   122   123   124   125   126   127   128   129   130 
   14    10    12    11    16    17    11    10    17    14     6    17    13 
  131   132   133   134   135   136   137   138   139   140   141   142   143 
   19     9     8     9     7    10    11    10    11    17    12    12    13 
  144   145   146   147   148   149   150   151   152   153   154   155   156 
    6     6    14    11    10     5     9     7    13    10     8    13    15 
  157   158   159   160   161   162   163   164   165   166   167   168   169 
   11     3     6     9    10     5     8     7     7     7     6     6     5 
  170   171   172   173   174   175   176   177   178   179   180   181   182 
    8     5     8     7     2     8     7    13     5     7     7     5     8 
  183   184   185   186   187   188   189   190   191   192   193   194   195 
    6     9    13     5    12     4     6     8     5     6     5     7     7 
  196   197   198   199   200   201   202   203   204   205   206   207   208 
    5    13     4     2     4     5     7     4     3     3     3     7     6 
  209   210   211   212   213   214   215   216   217   218   219   220   221 
    5     4     4     7     5     8     5     2     4     2     3     4     3 
  222   223   224   225   226   227   228   229   230   231   232   233   234 
    3     2     4     7     4     5     3     3     6     5     3     6     8 
  235   236   237   238   239   240   241   242   243   244   245   246   247 
    5     3     3     3     4     4     5     3     6     3     5     2     4 
  248   249   250   251   252   253   254   255   256   257   258   259   260 
    4     6     9     3     4     3     3     6     2     2     2     5     1 
  261   262   263   264   265   266   267   268   269   270   271   272   273 
    2     3     3     1     3     3     1     4     3     3     4     2     4 
  274   275   276   277   278   279   280   281   282   283   284   285   286 
    2     3     4     2     3     1     3     2     1     3     2     2     2 
  288   289   290   292   293   294   295   296   297   298   299   300   301 
    1     3     3     1     2     2     2     3     3     2     2     5     5 
  302   303   304   305   306   307   308   309   310   311   312   313   314 
    3     6     5     2     3     1     6     2     1     2     4     3     2 
  315   317   319   320   321   322   323   324   325   327   328   329   330 
    3     5     7     6     1     3     3     4     4     9     5     3     2 
  331   332   333   334   336   337   338   339   341   342   343   344   345 
    2     3     4     1     1     2     2     5     3     2     1     1     4 
  346   347   348   349   351   352   353   354   355   357   359   360   361 
    2     2     3     5     2     2     3     2     1     1     2     1     3 
  362   363   364   365   366   367   368   369   370   371   372   373   374 
    1     1     2     2     3     2     1     2     1     2     1     3     4 
  375   376   377   378   379   381   382   383   384   385   387   388   389 
    4     2     2     2     1     3     3     2     1     2     1     1     1 
  390   392   393   394   395   396   397   398   400   401   402   404   408 
    4     1     1     2     1     2     1     2     1     2     1     3     1 
  409   410   413   414   415   417   418   419   420   421   422   423   425 
    1     2     2     1     6     1     2     1     1     3     4     3     2 
  426   427   428   429   430   431   432   433   434   435   438   439   440 
    3     3     2     1     1     1     4     2     2     2     2     3     2 
  441   442   444   445   446   447   450   452   453   455   457   460   461 
    2     1     1     2     2     2     3     3     2     1     2     1     3 
  463   464   465   466   467   468   472   473   474   475   477   478   479 
    2     1     1     2     3     2     1     1     1     3     1     3     3 
  482   483   484   486   487   488   489   490   491   492   495   496   497 
    1     3     1     1     2     4     2     1     1     2     5     1     4 
  500   503   504   505   506   507   508   510   513   514   515   516   517 
    2     4     1     2     1     1     3     2     2     1     1     1     1 
  518   519   524   530   533   534   535   536   537   542   543   546   547 
    2     1     1     1     1     1     2     2     2     1     1     2     1 
  549   551   553   554   555   556   557   560   561   562   565   566   567 
    1     2     1     2     2     1     1     1     1     1     5     1     1 
  568   569   571   572   573   575   576   577   580   581   583   587   588 
    1     2     2     1     1     1     1     3     1     1     2     1     1 
  589   591   593   594   595   596   598   599   601   602   605   607   608 
    1     1     1     3     3     2     1     2     1     3     2     1     2 
  609   610   611   612   613   614   616   617   619   621   622   625   626 
    3     1     3     1     1     1     2     1     1     3     1     2     1 
  627   630   631   632   633   634   636   637   638   639   642   645   649 
    1     1     1     1     2     1     2     1     1     1     1     2     1 
  652   653   655   658   660   666   667   668   669   676   680   681   682 
    1     1     1     1     3     1     1     2     2     1     1     1     1 
  683   686   689   690   691   692   693   695   698   701   702   703   706 
    1     1     2     1     2     1     1     1     1     1     1     2     1 
  707   708   717   719   720   721   725   726   727   729   731   732   734 
    1     1     2     1     3     1     2     1     1     1     1     2     1 
  739   741   746   747   748   751   755   759   764   765   767   769   770 
    1     2     1     1     2     1     1     1     1     2     2     1     2 
  773   777   779   782   783   785   787   788   789   790   791   796   803 
    1     1     1     2     1     1     1     1     1     1     1     1     1 
  807   808   812   815   818   820   823   826   828   831   833   834   835 
    1     1     1     1     1     2     1     1     2     1     1     2     1 
  836   840   843   845   847   850   852   855   860   863   864   871   875 
    1     1     1     1     1     1     1     1     2     2     2     1     1 
  878   880   888   896   897   900   902   905   911   913   916   919   921 
    2     1     1     1     1     1     1     2     1     2     1     1     1 
  926   929   930   932   933   934   936   938   940   942   948   950   954 
    1     2     1     2     3     1     1     1     1     1     1     1     1 
  956   957   960   961   962   963   964   967   969   976   979   980   986 
    1     1     1     2     1     1     1     1     1     1     1     1     1 
  994  1001  1002  1011  1012  1017  1018  1019  1024  1025  1026  1029  1033 
    1     1     1     1     1     1     1     1     1     1     1     2     1 
 1036  1040  1043  1044  1045  1046  1051  1063  1066  1069  1074  1078  1080 
    1     1     1     1     1     1     1     1     2     1     1     1     2 
 1093  1094  1096  1099  1104  1107  1112  1122  1127  1137  1140  1143  1144 
    2     1     1     1     1     1     1     1     1     1     1     1     1 
 1149  1154  1165  1170  1175  1176  1188  1192  1193  1204  1208  1209  1212 
    1     2     2     1     1     1     1     1     1     1     1     1     1 
 1220  1221  1223  1224  1227  1240  1245  1246  1248  1250  1251  1256  1260 
    1     1     1     1     1     1     1     1     1     1     1     1     2 
 1277  1278  1284  1285  1288  1291  1292  1294  1306  1308  1312  1325  1327 
    1     1     1     1     1     1     1     1     1     1     1     1     1 
 1343  1351  1352  1357  1360  1370  1371  1384  1387  1389  1398  1407  1419 
    1     1     1     1     1     2     1     1     1     1     1     1     1 
 1429  1430  1443  1446  1455  1459  1461  1467  1468  1473  1477  1478  1488 
    1     1     1     1     1     1     1     1     1     1     1     1     2 
 1493  1498  1505  1506  1508  1509  1511  1512  1515  1518  1522  1524  1537 
    1     1     1     1     1     1     2     1     1     3     1     1     1 
 1546  1547  1548  1552  1558  1572  1573  1575  1579  1580  1588  1590  1611 
    1     1     2     1     1     1     1     1     1     2     1     1     1 
 1624  1626  1637  1640  1667  1669  1678  1685  1687  1689  1690  1706  1710 
    1     1     1     1     2     1     2     1     2     2     1     1     1 
 1713  1719  1741  1767  1769  1771  1772  1776  1778  1779  1790  1807  1813 
    1     1     1     1     1     1     1     1     1     1     1     1     1 
 1814  1815  1823  1824  1830  1850  1854  1876  1893  1903  1912  1928  1952 
    1     1     1     1     2     1     1     1     1     1     1     1     1 
 1975  1977  1979  1989  1995  2009  2014  2015  2031  2048  2053  2060  2063 
    1     1     1     1     1     1     1     1     1     1     1     1     2 
 2068  2134  2139  2140  2142  2143  2150  2164  2182  2211  2215  2242  2243 
    1     1     1     1     1     1     1     1     1     1     1     1     1 
 2245  2260  2290  2308  2309  2322  2343  2344  2356  2377  2379  2406  2414 
    1     1     1     1     2     1     1     1     1     1     2     1     1 
 2474  2476  2483  2484  2485  2487  2504  2539  2544  2605  2610  2646  2663 
    1     1     1     1     1     1     1     1     1     1     1     1     1 
 2702  2721  2736  2756  2782  2813  2815  2837  2851  2875  2961  3023  3087 
    1     1     1     2     1     1     1     1     1     1     1     1     1 
 3099  3101  3107  3157  3246  3248  3271  3273  3276  3323  3360  3362  3381 
    1     1     1     1     1     1     1     1     1     1     1     1     1 
 3397  3510  3518  3538  3547  3557  3562  3592  3593  3687  3822  3825  3837 
    1     1     1     1     1     1     1     1     1     1     1     1     1 
 3877  3897  3908  3911  3988  3999  4024  4060  4065  4097  4126  4155  4193 
    1     1     1     1     1     1     1     1     1     1     1     1     1 
 4209  4260  4316  4344  4346  4498  4517  4650  4676  4701  4707  4721  4862 
    1     1     1     1     1     1     1     1     1     1     1     1     1 
 4939  4944  5222  5306  5411  5430  5446  5516  5532  5589  5611  5668  5715 
    1     1     1     1     1     1     1     1     1     1     1     1     1 
 5724  5725  5731  5744  5790  5902  5917  5962  6024  6026  6066  6075  6133 
    1     1     1     1     1     1     1     1     1     1     3     1     1 
 6147  6185  6205  6217  6352  6374  6426  6460  6476  6517  6550  6566  6582 
    1     1     1     1     1     1     1     1     1     1     1     1     1 
 6628  6894  7070  7531  7803  7919  9141  9509  9930 10046 10156 10563 11047 
    1     1     1     1     1     1     1     1     1     1     1     1     1 
11721 12037 12044 12146 12378 12496 13520 13602 13998 14483 15196 16581 16932 
    1     1     1     1     1     1     1     1     1     1     1     1     1 
18369 18614 19103 19518 
    1     1     1     1

How many gene sets have more than 5 genes?

sum(ngs0 > 5)
[1] 11657

Or greater or equal to 5?

sum(ngs0 >= 5)
[1] 12646

How many groups have a number of genes greater than 7 and less than 90?

sum(ngs0 > 7 & ngs0 < 90)
[1] 7604

How many groups have less than 34 or more than 67 genes?

sum(ngs0 < 34 | ngs0 > 67)
[1] 21321

Over representation analysis

We have loaded the gene set collection hsa_go previously downloaded.

x1_ora = overRepresentation(x1,minsize=5,maxsize = 100,
                            correction = "BH",
                            GeneSetList = hsa_go,
                            foutput="x1_ora")

A data.frame with the results.

x1_ora_df = tidy(x1_ora)

A html report

glimpse(x1_ora)
[1] "./reports/x1_ora.html"

that can be opened with

browseURL(glimpse(x1_ora))

Gene set analysis

x1_self_mean = GeneSetTest(x = gse28619 ,y="type",
         test = rowt,association="pvalue",correction="BH",
         GeneNullDistr = "randomization",minsize = 5,
         GeneSetNullDistr = "self-contained",
         alternative="less",nmax = 1000,
         id = "ENTREZID",gsc=hsa_go,descriptive=mean,
         foutput = "x1_self_mean")
x1_self_mean_df = tidy(x1_self_mean)
glimpse(x1_self_mean)
[1] "./reports/x1_self_mean.html"
browseURL(glimpse(x1_self_mean))