Hi there,

I have two queries about my DESeq2 results. I am running some metagenomics analysis but I guess my question should be applicable to DESeq2 in general as the input is a count data table:

• It seems like there are limits on the log2FoldChange results of +30 and -30 in my results as there are a number of results recording exactly +30 and -30 and nothing outside of these limits (but plenty within these limits). I understand that these are very high log2fold change results.

• My results also lead to a significant gap or jump in both the positive and negative log2FoldChange results whereby there are lots of results recording log2FoldChanges of ~5-10 and 20-30 but nothing in between 10-20. This is obvious when plotted as geom_points (see here: https://github.com/joey711/phyloseq/issues/1306 ).

Could this just be the nature of my data? Or is it an issue with my code/DESeq2?

Any help would be much appreciated?! Code below.

Thanks!

dds = phyloseq_to_deseq2(stool_visit0_narm, ~ armcode)
gm_mean = function(x, na.rm=TRUE){
  exp(sum(log(x[x > 0]), na.rm=na.rm) / length(x))
}
geoMeans = apply(counts(dds), 1, gm_mean)
dds = estimateSizeFactors(dds, geoMeans = geoMeans)
dds = DESeq(dds, fitType="local")

res = results(dds)
res = res[order(res$padj, na.last=NA), ]
alpha = 0.01
sigtab = res[which(res$padj < alpha), ]
sigtab = cbind(as(sigtab, "data.frame"), as(tax_table(stool_visit0_narm)[rownames(sigtab), ], "matrix"))

sigtabpos <- filter(sigtab, log2FoldChange > 0)
sigtabneg <- filter(sigtab, log2FoldChange < 0)

sigtabpos[order(sigtabpos$log2FoldChange, na.last=NA), ]

         baseMean log2FoldChange     lfcSE      stat       pvalue         padj   Kingdom         Phylum
43 6.156538e+03       2.944630 0.9053495  3.252478 1.144033e-03 3.278879e-03  Bacteria     Firmicutes
44 7.107569e+03       4.313601 1.3340480  3.233468 1.222971e-03 3.474103e-03  Bacteria     Firmicutes
41 2.582860e+04       4.352137 1.2554974  3.466465 5.273512e-04 1.596979e-03  Bacteria Proteobacteria
40 1.322164e+04       4.599755 1.1889702  3.868688 1.094224e-04 3.547939e-04  Bacteria     Firmicutes
37 5.119151e+03       5.545840 0.9656401  5.743175 9.291786e-09 3.468213e-08  Bacteria     Firmicutes
5  4.266353e+06       5.654552 0.5018312 11.267836 1.891616e-29 3.571817e-28  Bacteria Proteobacteria
46 2.521036e+03       6.124430 2.0562832  2.978398 2.897595e-03 7.949811e-03  Bacteria Proteobacteria
45 3.516809e+02       6.287914 1.9644933  3.200781 1.370554e-03 3.859193e-03  Bacteria     Firmicutes
38 4.333094e+03       6.730340 1.6122561  4.174486 2.986602e-05 1.030859e-04  Bacteria     Firmicutes
13 6.468068e+05       7.925411 0.8146315  9.728829 2.271929e-22 2.209968e-21  Bacteria Proteobacteria
39 1.742195e+03       8.077941 2.0264392  3.986273 6.711916e-05 2.221160e-04  Bacteria Proteobacteria
42 2.339207e+01       8.317912 2.4628372  3.377370 7.318258e-04 2.195477e-03  Bacteria     Firmicutes
12 9.380184e+05       8.858038 0.8941817  9.906306 3.908291e-23 3.920504e-22  Bacteria Proteobacteria
2  5.918264e+06       9.721039 0.7246594 13.414631 4.963976e-41 1.963594e-39  Bacteria Proteobacteria
3  1.028417e+06      10.746428 0.8624877 12.459804 1.236743e-35 3.053803e-34  Bacteria     Firmicutes
36 6.557735e+03      11.834363 1.6998467  6.962018 3.354330e-12 1.297277e-11  Bacteria     Firmicutes
35 7.272282e+00      22.507519 2.9196912  7.708870 1.269370e-14 4.969121e-14  Bacteria  Bacteroidetes
34 1.701994e+00      22.525718 2.9215741  7.710131 1.256891e-14 4.969121e-14  Bacteria Proteobacteria
33 2.707836e+00      23.167322 2.9208111  7.931811 2.159730e-15 8.888121e-15  Bacteria Proteobacteria
32 3.551821e+00      23.540703 2.9204526  8.060635 7.589899e-16 3.205734e-15  Bacteria     Firmicutes
31 4.185228e+00      23.768954 2.9202282  8.139416 3.971877e-16 1.699963e-15  Bacteria     Firmicutes
30 4.644595e+00      23.912441 2.9201036  8.188902 2.636209e-16 1.143545e-15  Bacteria  Bacteroidetes
29 5.103611e+00      24.025557 2.9200016  8.227926 1.904826e-16 8.376017e-16  Bacteria     Firmicutes
28 5.460097e+00      24.140696 2.9199334  8.267550 1.367394e-16 6.096296e-16  Bacteria Proteobacteria
27 5.652638e+00      24.188227 2.9199003  8.283922 1.191834e-16 5.388434e-16  Bacteria Actinobacteria
25 5.750061e+00      24.208280 2.9198848  8.290834 1.124573e-16 5.156970e-16  Bacteria     Firmicutes
26 5.749768e+00      24.209970 2.9198844  8.291414 1.119102e-16 5.156970e-16  Bacteria     Firmicutes
24 6.297300e+00      24.335834 2.9198043  8.334748 7.766322e-17 3.720880e-16 Eukaryota    Apicomplexa
23 6.473562e+00      24.375923 2.9197813  8.348544 6.910944e-17 3.412943e-16  Bacteria Proteobacteria
22 7.535019e+00      24.582965 2.9196655  8.419788 3.771660e-17 1.921751e-16  Bacteria Actinobacteria
21 7.611680e+00      24.594755 2.9196582  8.423847 3.643192e-17 1.886233e-16  Bacteria Actinobacteria
20 1.577834e+01      25.062006 2.9189132  8.586074 8.999136e-18 4.896140e-17  Bacteria     Firmicutes
19 1.093404e+01      25.107061 2.9193662  8.600175 7.959446e-18 4.405142e-17  Bacteria     Firmicutes
17 1.154469e+01      25.183238 2.8654306  8.788640 1.513811e-18 9.718668e-18  Bacteria     Firmicutes
18 1.874348e+01      25.432221 2.9187492  8.713397 2.949009e-18 1.820446e-17  Bacteria     Firmicutes
16 5.012748e+01      26.238523 2.9182019  8.991332 2.442516e-19 1.704451e-18  Bacteria Proteobacteria
15 2.658075e+01      26.343816 2.9184924  9.026515 1.772255e-19 1.264208e-18  Bacteria     Firmicutes
14 7.143871e+01      26.417134 2.9180924  9.052878 1.392494e-19 1.015887e-18  Bacteria Proteobacteria
4  4.776805e+01      27.165839 2.1860854 12.426705 1.871921e-35 4.005912e-34  Bacteria Actinobacteria
11 6.249270e+01      27.544150 2.7389145 10.056594 8.591998e-24 9.510452e-23  Bacteria     Firmicutes
6  8.685003e+01      27.981831 2.5665704 10.902421 1.122311e-27 2.001454e-26  Bacteria Proteobacteria
10 2.633885e+02      29.560437 2.9177364 10.131291 4.013144e-24 4.771182e-23  Bacteria     Firmicutes
9  2.862216e+02      29.678511 2.9177258 10.171796 2.649773e-24 3.402308e-23  Bacteria Proteobacteria
8  3.348759e+02      29.897896 2.9177079 10.247049 1.220120e-24 1.631910e-23  Bacteria     Firmicutes
1  1.755509e+03      30.000000 1.8538740 16.182330 6.720291e-59 1.078607e-56  Bacteria     Firmicutes
7  1.316807e+03      30.000000 2.9176296 10.282320 8.466491e-25 1.235338e-23 Eukaryota     Ascomycota


sigtabneg[order(sigtabneg$log2FoldChange, na.last=NA), ]

     baseMean log2FoldChange     lfcSE       stat       pvalue         padj  Kingdom         Phylum
1  1.467333e+03     -30.000000 1.7311502 -17.329519 2.816745e-67 9.041753e-65 Bacteria     Firmicutes
3  3.325310e+02     -30.000000 2.0197619 -14.853236 6.629002e-50 5.319774e-48 Bacteria     Firmicutes
5  5.016380e+02     -30.000000 2.1129425 -14.198209 9.398336e-46 5.028110e-44 Bacteria     Firmicutes
16 3.075333e+02     -30.000000 2.9206233 -10.271780 9.444577e-25 1.318134e-23 Bacteria     Firmicutes
8  2.498202e+02     -29.999817 2.2782830 -13.167731 1.345817e-39 4.320074e-38 Bacteria     Firmicutes
10 2.301883e+02     -29.999566 2.3718071 -12.648400 1.141759e-36 3.100233e-35 Bacteria     Firmicutes
15 9.918695e+01     -29.996101 2.8951526 -10.360801 3.738167e-25 5.714056e-24 Bacteria     Firmicutes
2  2.388144e+02     -29.683833 1.8747975 -15.833088 1.839673e-56 1.968451e-54 Bacteria     Firmicutes
17 2.071310e+02     -29.644658 2.9206278 -10.150098 3.310314e-24 4.086965e-23 Bacteria     Firmicutes
6  2.101074e+02     -29.634807 2.1829475 -13.575594 5.588915e-42 2.562917e-40 Bacteria     Firmicutes
18 3.243128e+02     -29.499090 2.9206228 -10.100274 5.508828e-24 6.315478e-23 Bacteria     Firmicutes
4  1.672339e+02     -29.328274 2.0039303 -14.635377 1.670481e-48 1.072449e-46 Bacteria     Firmicutes
20 1.264129e+02     -28.950765 2.9206366  -9.912485 3.673940e-23 3.804305e-22 Bacteria     Firmicutes
9  1.053924e+02     -28.655545 2.2657575 -12.647225 1.158965e-36 3.100233e-35 Bacteria     Firmicutes
11 1.359821e+02     -28.259942 2.2713082 -12.442143 1.543068e-35 3.538034e-34 Bacteria     Firmicutes
21 6.283718e+01     -27.990462 2.8964685  -9.663652 4.302483e-22 4.062050e-21 Bacteria  Bacteroidetes
22 6.223275e+01     -27.971905 2.9206598  -9.577256 9.965775e-22 9.140040e-21 Bacteria     Firmicutes
13 6.152894e+01     -27.960136 2.5970320 -10.766189 4.971582e-27 8.399357e-26 Bacteria Actinobacteria
12 5.981266e+01     -27.879234 2.4712448 -11.281454 1.620453e-29 3.251033e-28 Bacteria Proteobacteria
7  4.647158e+01     -27.550543 2.0549443 -13.406954 5.505405e-41 1.963594e-39 Bacteria     Firmicutes
24 4.186865e+01     -27.432888 2.9206820  -9.392631 5.851992e-21 5.076998e-20 Bacteria     Firmicutes
19 3.817422e+01     -27.306449 2.7284550 -10.008026 1.405285e-23 1.503655e-22 Bacteria     Firmicutes
25 3.268306e+01     -27.080533 2.9207011  -9.271929 1.828097e-20 1.544261e-19 Bacteria  Bacteroidetes
26 3.721897e+01     -26.850748 2.9206905  -9.193288 3.810145e-20 3.136042e-19 Bacteria     Firmicutes
27 2.618299e+01     -26.789877 2.9207228  -9.172345 4.628384e-20 3.714279e-19 Bacteria   Spirochaetes
28 5.604820e+01     -26.750101 2.9206648  -9.158908 5.242507e-20 4.104500e-19 Bacteria     Firmicutes
29 3.531454e+01     -26.605304 2.9206946  -9.109238 8.296223e-20 6.340685e-19 Bacteria     Firmicutes
14 2.397975e+01     -26.572117 2.5618285 -10.372325 3.313634e-25 5.318382e-24 Bacteria     Firmicutes
32 1.378633e+01     -25.908784 2.9208204  -8.870379 7.289744e-19 4.974519e-18 Bacteria     Firmicutes
31 1.372390e+01     -25.902220 2.9208214  -8.868129 7.438532e-19 4.974519e-18 Bacteria Actinobacteria
33 1.207378e+01     -25.729850 2.9208497  -8.809029 1.262344e-18 8.269642e-18 Bacteria Proteobacteria
34 1.148286e+01     -25.662807 2.9208618  -8.786039 1.549255e-18 9.751192e-18 Bacteria     Firmicutes
30 1.040547e+01     -25.518486 2.8114063  -9.076769 1.118453e-19 8.349384e-19 Bacteria Actinobacteria
23 2.974136e+01     -25.497256 2.7131160  -9.397776 5.572853e-21 4.969127e-20 Bacteria     Firmicutes
35 1.019120e+01     -25.401479 2.9208933  -8.696476 3.423486e-18 2.073470e-17 Bacteria     Firmicutes
36 8.952357e+00     -25.322187 2.9209318  -8.669215 4.351091e-18 2.586482e-17 Bacteria     Firmicutes
37 8.727856e+00     -25.290041 2.9209401  -8.658185 4.793331e-18 2.797562e-17 Bacteria Proteobacteria
38 8.495663e+00     -25.246482 2.9209490  -8.643246 5.463793e-18 3.131924e-17 Bacteria  Bacteroidetes
39 7.852646e+00     -25.139972 2.9209763  -8.606702 7.519267e-18 4.234535e-17 Bacteria  Bacteroidetes
40 1.500412e+01     -24.990180 2.9208038  -8.555926 1.169268e-17 6.255582e-17 Bacteria     Firmicutes
41 5.513471e+00     -24.661788 2.9211300  -8.442551 3.104866e-17 1.633872e-16 Bacteria Actinobacteria
42 5.128694e+00     -24.539706 2.9211681  -8.400648 4.440204e-17 2.227040e-16 Bacteria Actinobacteria
43 4.675466e+00     -24.378818 2.9212222  -8.345417 7.096298e-17 3.451381e-16 Bacteria Proteobacteria
44 4.358225e+00     -24.230076 2.9212665  -8.294374 1.091588e-16 5.152937e-16 Bacteria  Bacteroidetes
45 6.451312e+00     -23.436860 2.9210548  -8.023424 1.028378e-15 4.287136e-15 Bacteria Actinobacteria
46 5.614217e+00     -23.007801 2.9211208  -7.876361 3.370537e-15 1.369547e-14 Bacteria Actinobacteria
47 4.672575e+00     -22.894407 2.9212227  -7.837269 4.604522e-15 1.847565e-14 Bacteria Actinobacteria
49 9.060837e+02     -11.788604 1.9693335  -5.986088 2.149479e-09 8.117443e-09 Bacteria     Firmicutes
48 1.517017e+03      -9.881046 1.5454835  -6.393498 1.621327e-10 6.195784e-10 Bacteria     Firmicutes
61 5.591574e+02      -6.908173 1.8927308  -3.649844 2.623994e-04 8.339625e-04 Bacteria  Bacteroidetes
52 7.128570e+02      -6.517375 1.3992597  -4.657731 3.197135e-06 1.153124e-05 Bacteria     Firmicutes
70 1.526088e+04      -6.355776 2.1247330  -2.991329 2.777659e-03 7.753292e-03 Bacteria  Bacteroidetes
72 7.109215e+02      -6.168665 2.1101567  -2.923321 3.463195e-03 9.421063e-03 Bacteria     Firmicutes
50 3.306764e+03      -5.963276 1.2609386  -4.729236 2.253664e-06 8.315242e-06 Bacteria     Firmicutes
60 5.579851e+03      -5.761839 1.4906810  -3.865240 1.109802e-04 3.562463e-04 Bacteria Actinobacteria
51 1.161831e+04      -5.651046 1.2030558  -4.697244 2.636957e-06 9.618898e-06 Bacteria  Bacteroidetes
62 1.617506e+03      -4.699028 1.3093260  -3.588891 3.320880e-04 1.045100e-03 Bacteria     Firmicutes
58 1.968689e+03      -4.352766 1.0674314  -4.077794 4.546494e-05 1.520234e-04 Bacteria     Firmicutes
63 2.400277e+03      -4.219640 1.1824143  -3.568665 3.588053e-04 1.113655e-03 Bacteria     Firmicutes
55 4.723151e+04      -4.119505 0.9499206  -4.336683 1.446490e-05 5.046991e-05 Bacteria     Firmicutes
68 7.488602e+03      -4.059534 1.2334207  -3.291281 9.973227e-04 2.910369e-03 Bacteria     Firmicutes
69 6.055383e+03      -3.784590 1.1522241  -3.284595 1.021290e-03 2.953459e-03 Bacteria Actinobacteria
56 1.060519e+05      -3.736996 0.8973273  -4.164586 3.119186e-05 1.065169e-04 Bacteria Actinobacteria
53 3.751369e+04      -3.308140 0.7184328  -4.604662 4.131370e-06 1.473522e-05 Bacteria     Firmicutes
64 3.243909e+03      -3.277236 0.9187128  -3.567204 3.608102e-04 1.113655e-03 Bacteria Actinobacteria
71 1.751304e+04      -3.219020 1.0791403  -2.982949 2.854857e-03 7.900078e-03 Bacteria     Firmicutes
65 1.077656e+04      -3.113812 0.8881506  -3.505951 4.549791e-04 1.390936e-03 Bacteria     Firmicutes
66 3.020734e+04      -2.953332 0.8817957  -3.349224 8.103816e-04 2.408634e-03 Bacteria     Firmicutes
59 1.228834e+05      -2.868606 0.7256320  -3.953251 7.709649e-05 2.525303e-04 Bacteria     Firmicutes
57 1.531834e+05      -2.547096 0.6221992  -4.093699 4.245457e-05 1.434517e-04 Bacteria Actinobacteria
54 2.003971e+05      -2.042014 0.4699114  -4.345530 1.389395e-05 4.901053e-05 Bacteria     Firmicutes
67 4.499925e+05      -1.914759 0.5725579  -3.344219 8.251457e-04 2.430016e-03 Bacteria  Bacteroidetes

These are from genes where you have {positive counts} vs {0's}. The MLE is not finite in this case.

You can obtain more reliable LFC estimates with lfcShrink if you like.