3  Explore composition

3.1 Pre-processing

The trans() function contains many normalization methods, suitable for pre-processing of different omics, some refer to vegan::decostand().

Table 3.1: Normalization methods used in omics
Method Description
cpm Counts per million
minmax linear transfer to (min, max)
acpm Counts per million, then asinh transfer
log1 log(n+1) transformat
total divide by total
max divide by maximum
frequency divide by total and multiply by the number of non-zero items, so that the average of non-zero entries is one.
normalize make margin sum of squares equal to one
range standardize values into range (0,1) (same to minmax(0,1)). If all values are constant, they will be transformed to 0.
rank rank replaces abundance values by their increasing ranks leaving zeros unchanged.
rrank rrank is similar but uses relative ranks with maximum 1.
pa scale x to presence/absence scale (0/1).
standardize scale x to zero mean and unit variance.
hellinger square root of method = "total"
log logarithmic transformation as suggested by Anderson et al. (2006): logb(x)+1 for x>0, where b is the base of the logarithm; zeros are left as zeros.
alr Additive log ratio ('alr') transformation (Aitchison 1986) reduces data skewness and compositionality bias.
clr centered log ratio ('clr') transformation proposed by Aitchison (1986) reduces data skewness and compositionality bias.
rclr robust clr ('rclr') is similar to regular clr (see above) but allows data that contains zeroes. 
trans(otutab, method = "log1") %>% head()
NS1 NS2 NS3 NS4 NS5 NS6 WS1 WS2 WS3 WS4 WS5 WS6 CS1 CS2 CS3 CS4 CS5 CS6
s__un_f__Thermomonosporaceae 6.996682 7.560601 6.698268 7.211557 6.970730 6.976348 7.133296 7.376508 7.193686 6.848005 7.118016 6.919684 7.746733 7.831617 7.444249 7.588830 7.266827 7.331715
s__Pelomonas_puraquae 7.582229 7.118826 7.767687 7.712891 7.973844 7.512071 6.469250 6.206576 7.115582 7.158514 6.860664 6.455199 7.174724 7.324490 6.739337 7.029088 7.302496 7.069023
s__Rhizobacter_bergeniae 6.378426 6.129050 6.791221 6.804614 7.112327 6.749931 6.405228 6.154858 6.976348 6.936343 6.741701 6.508769 6.937314 7.497207 6.910751 7.090910 7.085902 6.637258
s__Flavobacterium_terrae 5.501258 5.459586 7.501634 6.513230 7.276556 6.198479 5.765191 7.563720 7.309212 6.903747 6.359574 5.886104 6.985642 7.105786 6.626718 6.049734 6.940222 7.253470
s__un_g__Rhizobacter 7.267525 6.023448 6.280396 6.633318 7.162397 6.228511 6.222576 6.381816 6.100319 6.431331 6.489205 6.063785 7.032624 7.277939 6.311735 6.369901 7.008505 6.806829
s__un_o__Burkholderiales 6.787845 6.527958 6.715383 6.816736 7.315218 6.937314 5.463832 5.533390 5.886104 5.945421 5.961005 5.863631 6.313548 6.293419 6.169611 6.327937 6.242223 6.208590

rm_low(), guolv() and hebing() functions can help filter or aggregate the omics data.

3.2 Stackplot

# sum the abundance to Phylum level
hebing(otutab, taxonomy$Phylum, margin = 1, act = "sum") -> phylum
stackplot(phylum, metadata, group = "Group", topN = 10) +
  scale_fill_manual(values = get_cols(10))
stackplot(phylum, metadata,
  group = "Group", style = "sample",
  group_order = TRUE, flow = TRUE, relative = FALSE
) +
  scale_fill_manual(values = get_cols(10))
Figure 3.1: Stack plot
Figure 3.2: Stack flow plot with sample style

The stackplot function offers a wide range of parameters to facilitate the adjustment of the final graphical output. For further details and examples on drawing bar plots and their transformations in R, please refer to R Draw the bar plot and its transformation.

3.3 Rarefaction

Rarefaction is a method used to assess the relationship between sample community data sampling effort and species richness within the sample. Commonly used in ecology, it evaluates whether the observed species diversity saturates given a certain number of samples or sequences, and whether additional sampling is needed to fully capture species richness within the sample.

For sample data, Rarefaction curves display the number of observed species at different levels of sampling effort (or sequence numbers). By plotting Rarefaction curves, one can assess how species diversity changes with varying sampling depths and determine if saturation has been reached.

For species data, Rarefaction curves display the number of observed samples at different levels of sampling effort (or sequence numbers). Through this method, one can evaluate how sample numbers change with different sampling depths and determine if additional samples are necessary to fully capture species richness.

a <- rare_curve_sample(otutab)
plot(a)

a <- rare_curve_species(otutab, mode = 1)
plot(a)
Figure 3.3: Rarefaction curves of sample data
Figure 3.4: Rarefaction curves of species data

3.4 Phylogenetic tree

ann_tree(taxonomy, otutab) -> tree
easy_tree(tree, add_abundance = FALSE)
Figure 3.5: Phylogenetic tree 
easy_tree(tree, add_tiplab = FALSE) -> p
some_tax <- table(taxonomy$Phylum) %>%
  sort(decreasing = TRUE) %>%
  head(5) %>%
  names()
add_strip(p, some_tax)
Figure 3.6: Phylogenetic tree with some strips

You can refer to Basic phylogenetic tree and Advanced phylogenetic tree to plot the phylogenetic tree using ggtree.

In addition, you can also choose to use online platforms like iPhylo and iTOL for interactive plotting.

iPhylo is a free online platform for generating, annotating, and visualizing phylogenetic trees. It allows you to plot various tree diagrams for species, compounds, and other hierarchical structures and conveniently add complex annotation information.

iPhylo Website: https://iphylo.net

For more information, you can check out our Online Guide Book.

sangji_plot(tree, width = 900, height = 700)
k__Bacteria → p__Proteobacteria 11.1k k__Bacteria → p__Actinobacteria 6.89k p__Actinobacteria → c__Actinobacteria 6.88k c__Actinobacteria → o__Actinomycetales 6.63k p__Proteobacteria → c__Betaproteobacteria 4.42k c__Betaproteobacteria → o__Burkholderiales 4.32k p__Proteobacteria → c__Alphaproteobacteria 3.26k p__Proteobacteria → c__Gammaproteobacteria 2.86k o__Burkholderiales → f__Comamonadaceae 2.20k c__Alphaproteobacteria → o__Rhizobiales 2.19k c__Gammaproteobacteria → o__Pseudomonadales 1.96k o__Pseudomonadales → f__Pseudomonadaceae 1.96k o__Actinomycetales → f__Thermomonosporaceae 1.73k o__Actinomycetales → f__Pseudonocardiaceae 1.69k f__Pseudomonadaceae → g__Rhizobacter 1.69k g__un_f__Thermomonosporaceae → s__un_f__Thermomonosporaceae 1.45k f__Thermomonosporaceae → g__un_f__Thermomonosporaceae 1.45k g__Pelomonas → s__Pelomonas_puraquae 1.40k f__Comamonadaceae → g__Pelomonas 1.40k k__Bacteria → p__Bacteroidetes 1.38k p__Bacteroidetes → c__Flavobacteriia 1.13k c__Flavobacteriia → o__Flavobacteriales 1.13k o__Flavobacteriales → f__Flavobacteriaceae 1.09k f__Flavobacteriaceae → g__Flavobacterium 1.06k o__Actinomycetales → g__Streptomyces 984 g__Rhizobacter → s__Rhizobacter_bergeniae 922 g__Flavobacterium → s__Flavobacterium_terrae 916 g__Rhizobacter → s__un_g__Rhizobacter 772 k__Bacteria → p__Firmicutes 578 k__Bacteria → p__Chloroflexi 301 s__un_f__Thermomonosporaceae 1.45k s__un_f__Thermomonosporaceae1.45ks__Pelomonas_puraquae 1.40k s__Pelomonas_puraquae1.40ks__Rhizobacter_bergeniae 922 s__Rhizobacter_bergeniae922s__Flavobacterium_terrae 916 s__Flavobacterium_terrae916s__un_g__Rhizobacter 772 s__un_g__Rhizobacter772k__Bacteria 20.5k k__Bacteria20.5kp__Actinobacteria 6.89k p__Actinobacteria6.89kp__Proteobacteria 11.1k p__Proteobacteria11.1kp__Bacteroidetes 1.38k p__Bacteroidetes1.38kp__Firmicutes 578 p__Firmicutes578p__Chloroflexi 301 p__Chloroflexi301c__Actinobacteria 6.88k c__Actinobacteria6.88kc__Betaproteobacteria 4.42k c__Betaproteobacteria4.42kc__Gammaproteobacteria 2.86k c__Gammaproteobacteria2.86kc__Flavobacteriia 1.13k c__Flavobacteriia1.13kc__Alphaproteobacteria 3.26k c__Alphaproteobacteria3.26ko__Actinomycetales 6.63k o__Actinomycetales6.63ko__Burkholderiales 4.32k o__Burkholderiales4.32ko__Pseudomonadales 1.96k o__Pseudomonadales1.96ko__Flavobacteriales 1.13k o__Flavobacteriales1.13ko__Rhizobiales 2.19k o__Rhizobiales2.19kf__Thermomonosporaceae 1.73k f__Thermomonosporaceae1.73kf__Comamonadaceae 2.20k f__Comamonadaceae2.20kf__Pseudomonadaceae 1.96k f__Pseudomonadaceae1.96kf__Flavobacteriaceae 1.09k f__Flavobacteriaceae1.09kf__Pseudonocardiaceae 1.69k f__Pseudonocardiaceae1.69kg__un_f__Thermomonosporaceae 1.45k g__un_f__Thermomonosporaceae1.45kg__Pelomonas 1.40k g__Pelomonas1.40kg__Rhizobacter 1.69k g__Rhizobacter1.69kg__Flavobacterium 1.06k g__Flavobacterium1.06kg__Streptomyces 984 g__Streptomyces984KingdomPhylumClassOrderFamilyGenusSpecies
Figure 3.7: Sankey plot of taxanomy 
sunburst(tree)
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Figure 3.8: Sunburst plot of taxanomy

3.5 Rtaxonkit

Taxonkit is a Practical and Efficient NCBI Taxonomy Toolkit ().

We recommend you download this excellent software to help next analysis. Or you can use Taxonkit in R by pctax interface as followed:

# 1. This function help you install suitable version taxonkit
install_taxonkit()
# taxonkit has been successfully installed!

# 2. Then download the NCBI taxonomy database.
download_taxonkit_dataset()
# Taxonkit files downloaded and copied successfully.

# 3. Check whether taxonkit is ready
check_taxonkit()
# ==============Taxonkit is available if there is help message above==============
# =========================Taxonkit dataset is available!=========================

Then you can use taxonkit in R just like in terminal.

?taxonkit_lineage

# taxonkit_list
# taxonkit_reformat
# taxonkit_name2taxid
# taxonkit_filter
# taxonkit_lca
lineage <- taxonkit_lineage("9606\n63221", show_name = TRUE, show_rank = TRUE, text = T)
lineage
[1] "9606\tcellular organisms;Eukaryota;Opisthokonta;Metazoa;Eumetazoa;Bilateria;Deuterostomia;Chordata;Craniata;Vertebrata;Gnathostomata;Teleostomi;Euteleostomi;Sarcopterygii;Dipnotetrapodomorpha;Tetrapoda;Amniota;Mammalia;Theria;Eutheria;Boreoeutheria;Euarchontoglires;Primates;Haplorrhini;Simiiformes;Catarrhini;Hominoidea;Hominidae;Homininae;Homo;Homo sapiens\tHomo sapiens\tspecies"                                                   
[2] "63221\tcellular organisms;Eukaryota;Opisthokonta;Metazoa;Eumetazoa;Bilateria;Deuterostomia;Chordata;Craniata;Vertebrata;Gnathostomata;Teleostomi;Euteleostomi;Sarcopterygii;Dipnotetrapodomorpha;Tetrapoda;Amniota;Mammalia;Theria;Eutheria;Boreoeutheria;Euarchontoglires;Primates;Haplorrhini;Simiiformes;Catarrhini;Hominoidea;Hominidae;Homininae;Homo;Homo sapiens;Homo sapiens neanderthalensis\tHomo sapiens neanderthalensis\tsubspecies"