METAbolic pathway testing combining POsitive and NEgative mode data (metapone)

The metapone package conducts pathway tests for untargeted metabolomics data. It has three main characteristics: (1) expanded database combining SMPDB and Mummichog databases, with manual cleaning to remove redundancies; (2) A new weighted testing scheme to address the issue of metabolite-feature matching uncertainties; (3) Can consider positive mode and negative mode data in a single analysis.

Compared to existing methods, the weighted testing scheme allows the user to apply different level of penalty for multiple-mapped features, in order to reduce their undue impact on the results. In addition, considering positive mode and negative mode data simultaneously can improve the statistical power of the test.

library(metapone)
#> Loading required package: BiocParallel
#> Loading required package: fields
#> Loading required package: spam
#> Spam version 2.8-0 (2022-01-05) is loaded.
#> Type 'help( Spam)' or 'demo( spam)' for a short introduction 
#> and overview of this package.
#> Help for individual functions is also obtained by adding the
#> suffix '.spam' to the function name, e.g. 'help( chol.spam)'.
#> 
#> Attaching package: 'spam'
#> The following objects are masked from 'package:base':
#> 
#>     backsolve, forwardsolve
#> Loading required package: viridis
#> Loading required package: viridisLite
#> 
#> Try help(fields) to get started.
#> Loading required package: markdown
#> Loading required package: fdrtool
#> Loading required package: fgsea
#> Loading required package: ggplot2
#> Loading required package: ggrepel
#> 
#> Attaching package: 'metapone'
#> The following object is masked from 'package:stats':
#> 
#>     ftable

The input should contain at least three clumns - m/z, retention time, and feature p-value. Here to illustrate the usage of the method, we borrow the test results from our published study “Use of high-resolution metabolomics for the identification of metabolic T signals associated with traffic-related air pollution” in Environment International. 120: 145–154.

The positive mode results are in the object “pos”.

data(pos)
head(pos)
#>                  mz     time      p-value F-statistics
#> pos_70.005 70.00462 393.5786 0.5113989716    0.6809306
#> pos_70.068 70.06843 392.4810 0.0001519284   10.8083621
#> pos_70.291 70.29056 431.2095 0.0094772595    5.1889005
#> pos_70.512 70.51164 428.0062 0.4159535303    0.8949039
#> pos_70.569 70.56866 393.4188 0.0009075295    8.2483533
#> pos_70.953 70.95306 388.7599 0.3471977081    1.0837069

The negative mode results are in the object “neg”. If both positive mode and negative mode data are present, each is input into the algorithm as a separate matrix

data(neg)
head(neg)
#>                  mz     time   p-value F-statistics
#> neg_70.029 70.02878 371.1481 0.4228952    0.8776863
#> neg_70.079 70.07884 373.2138 0.2181511    1.5764905
#> neg_70.288 70.28823 426.4280 0.1292893    2.1438317
#> neg_70.31  70.31043 281.8414 0.1485619    1.9918230
#> neg_70.532 70.53234 375.6303 0.1089732    2.3321718
#> neg_70.563 70.56271 363.9597 0.4777650    0.7511758

It is not required that both positive and negative mode results are present. Having a single ion mode is also OK. The test is based on HMDB identification. The common adduct ions are pre-processed and stored in:

data(hmdbCompMZ)
head(hmdbCompMZ)
#>       HMDB_ID      m.z ion.type
#> 1 HMDB0059597 1.343218     M+3H
#> 2 HMDB0001362 1.679159     M+3H
#> 3 HMDB0037238 2.341477     M+3H
#> 4 HMDB0005949 3.345944     M+3H
#> 5 HMDB0002387 4.011337     M+3H
#> 6 HMDB0002386 4.677044     M+3H

Pathway information that was summarized from Mummichog and smpdb is built-in:

data(pa)
head(pa)
#>        database                    pathway.name     HMDB.ID KEGG.ID category
#> 5092 Metapone 1 2-oxocarboxylic acid metabolism HMDB0000243  C00022 combined
#> 5157 Metapone 1 2-oxocarboxylic acid metabolism HMDB0000148  C00025 combined
#> 5200 Metapone 1 2-oxocarboxylic acid metabolism HMDB0000208  C00026 combined
#> 5288 Metapone 1 2-oxocarboxylic acid metabolism HMDB0000223  C00036 combined
#> 5377 Metapone 1 2-oxocarboxylic acid metabolism HMDB0000182  C00047 combined
#> 5407 Metapone 1 2-oxocarboxylic acid metabolism HMDB0000191  C00049 combined

The user can specify which adduct ions are allowed by setting the allowed adducts. For example:

pos.adductlist = c("M+H", "M+NH4", "M+Na", "M+ACN+H", "M+ACN+Na", "M+2ACN+H", "2M+H", "2M+Na", "2M+ACN+H")
neg.adductlist = c("M-H", "M-2H", "M-2H+Na", "M-2H+K", "M-2H+NH4", "M-H2O-H", "M-H+Cl", "M+Cl", "M+2Cl")

It is common for a feature to be matched to multiple metabolites. Assume a feature is matched to m metabolites, metapone weighs the feature by (1/m)^p, where p is a power term to tune the penalty. m can also be capped at a certain level such that the penalty is limited. These are controlled by parameters:

Setting p: fractional.count.power = 0.5 Setting the cap of n: max.match.count = 10

It is easy to see that when p=0, no penalty is assigned for multiple-matching. The higher p is, the larger penalty for features that are multiple matched.

Other parameters include p.threshold, which controls which metabolic feature is considered significant. If use.fgsea = FALSE, then testing is done by permutation. Otherwise, a GSEA type testing is conducted and the parameter use.meta controls using metabolite-based or feature-based testing. Overall, the analysis is conducted this way:

dat <- list(pos, neg)
type <- list("pos", "neg")
# permutation test
r<-metapone(dat, type, pa, hmdbCompMZ=hmdbCompMZ, pos.adductlist=pos.adductlist, neg.adductlist=neg.adductlist, p.threshold=0.05,n.permu=200,fractional.count.power=0.5, max.match.count=10, use.fgsea = FALSE)

# GSEA type testing based on metabolites
#r<-metapone(dat, type, pa, hmdbCompMZ=hmdbCompMZ, pos.adductlist=pos.adductlist, neg.adductlist=neg.adductlist, p.threshold=0.05,n.permu=100,fractional.count.power=0.5, max.match.count=10, use.fgsea = TRUE, use.meta = TRUE)

# GSEA type testing based on features
#r<-metapone(dat, type, pa, hmdbCompMZ=hmdbCompMZ, pos.adductlist=pos.adductlist, neg.adductlist=neg.adductlist, p.threshold=0.05,n.permu=100,fractional.count.power=0.5, max.match.count=10, use.fgsea = TRUE, use.meta = FALSE)

hist(ptable(r)[,1])

We can subset the pathways that are significant:

selection<-which(ptable(r)[,1]<0.025)
ptable(r)[selection,]
#>                                                 p_value
#> folate metabolism                                 0.020
#> secondary bile acid biosynthesis                  0.000
#> sphingolipid metabolism                           0.005
#> c21-steroid hormone biosynthesis and metabolism   0.010
#>                                                 n_significant_metabolites
#> folate metabolism                                                2.643594
#> secondary bile acid biosynthesis                                 2.136364
#> sphingolipid metabolism                                          7.680441
#> c21-steroid hormone biosynthesis and metabolism                  5.897251
#>                                                 n_mapped_metabolites
#> folate metabolism                                           4.850674
#> secondary bile acid biosynthesis                            3.314658
#> sphingolipid metabolism                                    19.223232
#> c21-steroid hormone biosynthesis and metabolism            16.779095
#>                                                 n_metabolites
#> folate metabolism                                          29
#> secondary bile acid biosynthesis                           18
#> sphingolipid metabolism                                    48
#> c21-steroid hormone biosynthesis and metabolism            77
#>                                                                                                                                                                                                 significant metabolites
#> folate metabolism                                                                                                                                                                   HMDB0000217,HMDB0000121,HMDB0001396
#> secondary bile acid biosynthesis                                        HMDB0000505,HMDB0000138,HMDB0000518,HMDB0000626,HMDB0000946,HMDB0000811,HMDB0000917,HMDB0000411,HMDB0000361,HMDB0000686,HMDB0000415,HMDB0000364
#> sphingolipid metabolism                         HMDB0001480,HMDB0000217,HMDB0001383,HMDB0004950,HMDB0000252,HMDB0001348,HMDB0010708,HMDB0000140,HMDB0000122,HMDB0004866,HMDB0000024,HMDB0000143,HMDB0004947,HMDB0004833
#> c21-steroid hormone biosynthesis and metabolism             HMDB0000653,HMDB0004026,HMDB0006760,HMDB0011653,HMDB0003259,HMDB0006754,HMDB0000935,HMDB0006753,HMDB0001231,HMDB0006893,HMDB0000774,HMDB0003193,HMDB0000363
#>                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          mapped_metabolites
#> folate metabolism                                                                                                                                                                                                                                                                                                                                                                                                                                                       HMDB0000217,HMDB0000121,HMDB0000538,HMDB0003470,HMDB0001533,HMDB0001396,HMDB0006825
#> secondary bile acid biosynthesis                                                                                                                                                                                                                                                                                                                                                    HMDB0000505,HMDB0000138,HMDB0000518,HMDB0000626,HMDB0000036,HMDB0000951,HMDB0000946,HMDB0000811,HMDB0000917,HMDB0000411,HMDB0000361,HMDB0000686,HMDB0000415,HMDB0000364
#> sphingolipid metabolism                                                                                                                                                                                                                         HMDB0001480,HMDB0000269,HMDB0000217,HMDB0000538,HMDB0001383,HMDB0001551,HMDB0004950,HMDB0000252,HMDB0010699,HMDB0001348,HMDB0006790,HMDB0001565,HMDB0010708,HMDB0000140,HMDB0000122,HMDB0000286,HMDB0000295,HMDB0004866,HMDB0000024,HMDB0000143,HMDB0004947,HMDB0012096,HMDB0000648,HMDB0004833,HMDB0004610
#> c21-steroid hormone biosynthesis and metabolism HMDB0000653,HMDB0000077,HMDB0004026,HMDB0006760,HMDB0000295,HMDB0011653,HMDB0000016,HMDB0004029,HMDB0002961,HMDB0000153,HMDB0000145,HMDB0003259,HMDB0006754,HMDB0000949,HMDB0000268,HMDB0001425,HMDB0003769,HMDB0000935,HMDB0006281,HMDB0006753,HMDB0002829,HMDB0012455,HMDB0001231,HMDB0006203,HMDB0006893,HMDB0006280,HMDB0001457,HMDB0000774,HMDB0000374,HMDB0000067,HMDB0006763,HMDB0006224,HMDB0004484,HMDB0005896,HMDB0000253,HMDB0003193,HMDB0000490,HMDB0000363,HMDB0003069,HMDB0000031,HMDB0006773
#>                                                 lfdr  adjust.p
#> folate metabolism                                  1 0.8290909
#> secondary bile acid biosynthesis                   1 0.0000000
#> sphingolipid metabolism                            1 0.5700000
#> c21-steroid hormone biosynthesis and metabolism    1 0.7600000

We note that applying the multiple-matching penalty using parameter fractional.count.power will effectively making fractional counts out of the multiple-matched features. Thus the mapped feature tables, you will see fractional counts, rather than integer counts.

ftable(r)[which(ptable(r)[,1]<0.025 & ptable(r)[,2]>=2)]
#> $`folate metabolism`
#>      mz       time     p-value     F-statistics HMDB_ID       m.z     
#> [1,] 1511.155 335.4843 0.001683378 7.410698     "HMDB0000217" 1511.156
#> [2,] 924.3137 522.3555 0.02859562  3.857759     "HMDB0000121" 924.3132
#> [3,] 440.1676 73.31135 0.001323347 7.734161     "HMDB0001396" 440.1682
#>      ion.type   counts   
#> [1,] "2M+Na"    1        
#> [2,] "2M+ACN+H" 0.6435943
#> [3,] "M-H2O-H"  1        
#> 
#> $`secondary bile acid biosynthesis`
#>       mz       time     p-value    F-statistics HMDB_ID       m.z     
#>  [1,] 456.308  96.02325 0.03401312 3.654646     "HMDB0000361" 456.3084
#>  [2,] 456.308  96.02325 0.03401312 3.654646     "HMDB0000411" 456.3084
#>  [3,] 456.308  96.02325 0.03401312 3.654646     "HMDB0000518" 456.3084
#>  [4,] 456.308  96.02325 0.03401312 3.654646     "HMDB0000626" 456.3084
#>  [5,] 456.308  96.02325 0.03401312 3.654646     "HMDB0000686" 456.3084
#>  [6,] 456.308  96.02325 0.03401312 3.654646     "HMDB0000811" 456.3084
#>  [7,] 456.308  96.02325 0.03401312 3.654646     "HMDB0000946" 456.3084
#>  [8,] 826.6231 299.4422 0.03344936 3.674143     "HMDB0000361" 826.6191
#>  [9,] 826.6231 299.4422 0.03344936 3.674143     "HMDB0000411" 826.6191
#> [10,] 826.6231 299.4422 0.03344936 3.674143     "HMDB0000518" 826.6191
#> [11,] 826.6231 299.4422 0.03344936 3.674143     "HMDB0000626" 826.6191
#> [12,] 826.6231 299.4422 0.03344936 3.674143     "HMDB0000686" 826.6191
#> [13,] 826.6231 299.4422 0.03344936 3.674143     "HMDB0000811" 826.6191
#> [14,] 826.6231 299.4422 0.03344936 3.674143     "HMDB0000946" 826.6191
#> [15,] 858.608  295.2427 0.04060484 3.448908     "HMDB0000364" 858.609 
#> [16,] 858.608  295.2427 0.04060484 3.448908     "HMDB0000415" 858.609 
#> [17,] 858.608  295.2427 0.04060484 3.448908     "HMDB0000505" 858.609 
#> [18,] 858.608  295.2427 0.04060484 3.448908     "HMDB0000917" 858.609 
#> [19,] 972.6485 410.6006 0.00694524 5.57578      "HMDB0000138" 972.6519
#> [20,] 500.2789 73.83075 0.01139168 4.962465     "HMDB0000138" 500.2784
#>       ion.type   counts    
#>  [1,] "M+ACN+Na" 0.09090909
#>  [2,] "M+ACN+Na" 0.09090909
#>  [3,] "M+ACN+Na" 0.09090909
#>  [4,] "M+ACN+Na" 0.09090909
#>  [5,] "M+ACN+Na" 0.09090909
#>  [6,] "M+ACN+Na" 0.09090909
#>  [7,] "M+ACN+Na" 0.09090909
#>  [8,] "2M+ACN+H" 0.1111111 
#>  [9,] "2M+ACN+H" 0.1111111 
#> [10,] "2M+ACN+H" 0.1111111 
#> [11,] "2M+ACN+H" 0.1111111 
#> [12,] "2M+ACN+H" 0.1111111 
#> [13,] "2M+ACN+H" 0.1111111 
#> [14,] "2M+ACN+H" 0.1111111 
#> [15,] "2M+ACN+H" 0.0625    
#> [16,] "2M+ACN+H" 0.0625    
#> [17,] "2M+ACN+H" 0.0625    
#> [18,] "2M+ACN+H" 0.0625    
#> [19,] "2M+ACN+H" 0.2222222 
#> [20,] "M+Cl"     0.25      
#> 
#> $`sphingolipid metabolism`
#>       mz       time     p-value      F-statistics HMDB_ID       m.z     
#>  [1,] 566.5487 339.8191 0.01077019   5.031309     "HMDB0004950" 566.5507
#>  [2,] 717.5996 228.5805 0.023961     4.066429     "HMDB0010708" 717.5987
#>  [3,] 834.6823 410.2771 3.144121e-06 17.13239     "HMDB0000140" 834.6793
#>  [4,] 523.4812 295.2402 0.01671986   4.496276     "HMDB0004947" 523.4833
#>  [5,] 607.5751 370.3215 0.001152875  7.921133     "HMDB0004950" 607.5772
#>  [6,] 773.6387 303.5713 0.01702076   4.474803     "HMDB0001348" 773.6405
#>  [7,] 1612.113 310.8567 0.0299504    3.80341      "HMDB0004866" 1612.118
#>  [8,] 785.5168 286.4947 0.005572923  5.853094     "HMDB0001383" 785.518 
#>  [9,] 1511.155 335.4843 0.001683378  7.410698     "HMDB0000217" 1511.156
#> [10,] 179.0553 36.83502 0.02430136   4.049723     "HMDB0000122" 179.0561
#> [11,] 179.0553 36.83502 0.02430136   4.049723     "HMDB0000143" 179.0561
#> [12,] 179.0553 432.7855 0.006187688  5.720927     "HMDB0000122" 179.0561
#> [13,] 179.0553 432.7855 0.006187688  5.720927     "HMDB0000143" 179.0561
#> [14,] 564.5361 379.4879 0.009946984  5.129183     "HMDB0004950" 564.5361
#> [15,] 890.6378 357.302  7.38564e-05  11.90184     "HMDB0000024" 890.6397
#> [16,] 280.2646 404.9412 2.737722e-08 26.54871     "HMDB0000252" 280.264 
#> [17,] 280.2646 404.9412 2.737722e-08 26.54871     "HMDB0001480" 280.264 
#> [18,] 842.6031 311.6396 0.0001615305 10.7171      "HMDB0004833" 842.5994
#> [19,] 846.6625 444.0693 0.00593383   5.773763     "HMDB0000140" 846.6595
#>       ion.type  counts    
#>  [1,] "M+H"     0.2637522 
#>  [2,] "M+NH4"   0.00407332
#>  [3,] "M+Na"    0.5946036 
#>  [4,] "M+ACN+H" 1         
#>  [5,] "M+ACN+H" 0.2637522 
#>  [6,] "M+ACN+H" 1         
#>  [7,] "2M+H"    1         
#>  [8,] "2M+Na"   0.1719822 
#>  [9,] "2M+Na"   1         
#> [10,] "M-H"     0.07142857
#> [11,] "M-H"     0.07142857
#> [12,] "M-H"     0.07142857
#> [13,] "M-H"     0.07142857
#> [14,] "M-H"     0.3296902 
#> [15,] "M-H"     0.09534626
#> [16,] "M-H2O-H" 0.5       
#> [17,] "M-H2O-H" 0.5       
#> [18,] "M-H2O-H" 0.07692308
#> [19,] "M+Cl"    0.5946036 
#> 
#> $`c21-steroid hormone biosynthesis and metabolism`
#>       mz       time     p-value      F-statistics HMDB_ID       m.z     
#>  [1,] 482.2738 126.6953 0.03922185   3.489025     "HMDB0003193" 482.2748
#>  [2,] 508.3468 127.6048 0.001470928  7.591605     "HMDB0000653" 508.3455
#>  [3,] 508.3465 694.251  0.01177125   4.922325     "HMDB0000653" 508.3455
#>  [4,] 530.33   664.3211 0.005241671  5.930785     "HMDB0000653" 530.3274
#>  [5,] 447.2836 458.7022 4.979651e-06 16.32296     "HMDB0003259" 447.2853
#>  [6,] 447.2836 458.7022 4.979651e-06 16.32296     "HMDB0006753" 447.2853
#>  [7,] 447.2836 458.7022 4.979651e-06 16.32296     "HMDB0006754" 447.2853
#>  [8,] 447.2836 458.7022 4.979651e-06 16.32296     "HMDB0006760" 447.2853
#>  [9,] 479.2574 130.7585 0.001269227  7.79065      "HMDB0000774" 479.2574
#> [10,] 549.3747 130.1223 0.005832964  5.795415     "HMDB0000653" 549.3721
#> [11,] 687.4566 451.9255 0.02257464   4.137142     "HMDB0000363" 687.4595
#> [12,] 687.4566 451.9255 0.02257464   4.137142     "HMDB0004026" 687.4595
#> [13,] 687.4566 451.9255 0.02257464   4.137142     "HMDB0011653" 687.4595
#> [14,] 895.7013 449.1108 0.0006073895 8.805532     "HMDB0001231" 895.6997
#> [15,] 1202.101 722.7504 3.475685e-06 16.95446     "HMDB0000935" 1202.102
#> [16,] 403.3571 272.3139 0.0004006864 9.393566     "HMDB0006893" 403.3582
#> [17,] 501.282  73.84088 0.003108978  6.601882     "HMDB0000653" 501.2811
#>       ion.type   counts   
#>  [1,] "M+NH4"    0.5      
#>  [2,] "M+ACN+H"  0.3535534
#>  [3,] "M+ACN+H"  0.3535534
#>  [4,] "M+ACN+Na" 0.3535534
#>  [5,] "M+2ACN+H" 0.2222222
#>  [6,] "M+2ACN+H" 0.2222222
#>  [7,] "M+2ACN+H" 0.2222222
#>  [8,] "M+2ACN+H" 0.2222222
#>  [9,] "M+2ACN+H" 0.7071068
#> [10,] "M+2ACN+H" 0.3535534
#> [11,] "2M+Na"    0.1818182
#> [12,] "2M+Na"    0.1818182
#> [13,] "2M+Na"    0.1818182
#> [14,] "2M+Na"    0.3333333
#> [15,] "2M+ACN+H" 0.5773503
#> [16,] "M-H"      0.5773503
#> [17,] "M+Cl"     0.3535534

We can also use bbplot1d() and bbplot2d to visualize the pathway testing result.

bbplot1d(r@test.result, 0.025)

bbplot2d(r@test.result, 0.025)