Nephele uses the ampvis2 R package v2.3.2 for statistical computation. We also make use of the Plotly R interface for the plotly.js charting library. The plots can be minimally edited interactively. However, they also have a link to export the data to the Plotly Chart Studio which allows for making a variety of charts. We have a tutorial for making simple edits to the plots here.

The rarefaction curves are made with the amp_rarecurve function. The table is written to *rarecurve.txt* and the plot to *rarecurve.html*.

`rarecurve <- amp_rarecurve(amp, color_by = "TreatmentGroup")`

The ** sampling depth** is used to filter out samples with few counts. Samples with counts which fall below the

`sampling depth`

are removed from the OTU table using amp_subset_samples. The names of samples that are removed are output to Additionally, the `sampling depth`

is used to rarefy the counts for the Bray-Curtis PCoA and the alpha diversity plots. The OTU table is rarefied using rrarefy from the vegan R package. The table of the filtered, rarefied counts is saved as *rarefied_OTU_table.txt*.

If you do not provide a `sampling depth`

, please see FAQ:How is the sampling depth calculated? for more information.

```
amp <- amp_subset_samples(amp, minreads = sampdepth)
otu <- rrarefy(t(amp$abund), sampdepth)
amprare <- ampsub
amprare$abund <- t(otu)
```

Principal coordinates analysis using binomial and Bray-Curtis distances is carried out using amp_ordinate. For more information on the formulae for the distance measures, see `vegdist`

.

The binomial distance is able to handle varying sample sizes, so the raw counts from the OTU table are used. For the Bray-Curtis distance, the rarefied counts are used. The coordinates from the plots are written to *pcoa_binomial.txt* and *pcoa_bray.txt* and the plots to *pcoa_binomial.html* and *pcoa_bray.html*. At least 3 samples are needed for these plots.

```
pcoa_binomial <- amp_ordinate(amp, filter_species = 0.01, type = "PCOA",
distmeasure = "binomial", sample_color_by = "TreatmentGroup",
detailed_output = TRUE, transform = "none")
pcoa_bray <- amp_ordinate(amprare, filter_species = 0.01, type = "PCOA",
distmeasure = "bray", sample_color_by = "TreatmentGroup",
detailed_output = TRUE, transform = "none")
```

The Shannon diversity and Chao species richness are computed using amp_alphadiv. The rarefied counts are used for this computation. The diversity values are saved in *alphadiv.txt*, and boxplots are output to *alphadiv.html*. At least 3 samples are needed to produce these plots.

`alphadiv <- amp_alphadiv(amprare, measure = "shannon", richness = TRUE, rarefy = sampdepth)`

The interactive heatmap is implemented using the morpheus R API developed at the Broad Institute. Documentation for how to use the heatmap can be found on the morpheus website.

The heatmap, *seq_heatmap.html*, is made from the raw OTU table with the counts normalized to 100 to represent the relative abundances using amp_subset_samples before the heatmap is made with morpheus. If there are too many OTUs or sequence variants, then the heatmap is made at the species level instead. A heatmap is also made from the rarefied counts, *seq_heatmap_rarefied.html*.

```
amptax <-amp_subset_samples(amp, normalise = TRUE)
heatmap <- morpheus(amp$abund, columns = columns, rows = rows,
columnColorModel = list(type = as.list(colors)),
colorScheme = list(scalingMode = "fixed", stepped = FALSE),
columnAnnotations = amptax$metadata, rowAnnotations = amptax$tax)
```

Complete descriptions of the output files can be found in the Plots section above. To learn how to edit the plots, see the visualization tutorial.

*rarecurve.html*: rarefaction curve plot*rarecurve.txt*: tabular data used to make the rarefaction curve plot*samples_being_ignored.txt*: list of samples removed from the analysis*pcoa_*.html*: PCoA plots*pcoa_*.txt*: tabular data used to make the PCoA plots*rarefied_OTU_table.txt*: rarefied OTU table used for Bray-Curtis PCoA and alpha diversity plots*alphadiv.html*: alpha diversity boxplots*alphadiv.txt*: tabular data used to make the alpha diversity plots*seq_heatmap.html*: heatmap of OTU/sequence variant abundances

- M A, SM K, AS Z, RH K and PH N (2015). "Back to Basics - The Influence of DNA Extraction and Primer Choice on Phylogenetic Analysis of Activated Sludge Communities."
*PLoS ONE*,**10**(7), pp. e0132783. http://dx.plos.org/10.1371/journal.pone.0132783. - Gould J (2018).
*morpheus: Interactive heat maps using 'morpheus.js' and 'htmlwidgets'*. R package version 0.1.1.1, https://github.com/cmap/morpheus.R. - Sievert C, Parmer C, Hocking T, Chamberlain S, Ram K, Corvellec M and Despouy P (2017).
*plotly: Create Interactive Web Graphics via 'plotly.js'*. R package version 4.7.1, https://CRAN.R-project.org/package=plotly. - McMurdie PJ and Paulson JN (2016).
*biomformat: An interface package for the BIOM file format*. https://github.com/joey711/biomformat/. - Oksanen J, Blanchet FG, Friendly M, Kindt R, Legendre P, McGlinn D, Minchin PR, O'Hara RB, Simpson GL, Solymos P, Stevens MHH, Szoecs E, Wagner H (2019).
*vegan: Community Ecology Package*. R package version 2.5-4, https://CRAN.R-project.org/package=vegan.