# Using BuildTrees¶

IgPhyML is easiest to use when run indirectly through the Change-O program BuildTrees by specifying the --igphyml option. Most of these instructions require Change-O 0.4.6 or higher, Alakazam 0.3.0 or higher, and IgPhyML to be installed, with the executable in your PATH variable. If these aren’t possible, see IgPhyML standalone operation

To view all options for BuildTrees , run the command:

BuildTrees.py --help

# Controlling input¶

The process begins with an AIRR formatted data file, in which each sequence has been clustered into a clonal group, which has subsequently had its unmutated V and J sequence predicted germlines. The following column names are required in the input file: fields: sequence_id, sequence, sequence_alignment, germline_alignment_d_mask, v_call, j_call, and clone_id. productive is recommended.

Use BuildTrees.py to break this file into separate sequence alignment files that can be used with IgPhyML. This program will:

1. Filter out nonfunctional sequences.
2. Mask codons split by insertions.
3. Separate clonal groups into separate alignment files (aligned by IMGT site) and information files
4. Create the repertoire files for this dataset.

Create IgPhyML input files from examples/example.tsv without running IgPhyML:

cd examples
BuildTrees.py -d example.tsv --outname ex --log ex.log --collapse

Here the --collapse flag is used to collapse identical sequences. This is highly recommended because identical sequences slow down calculations without actually affecting likelihood values in IgPhyML.

Note

IgPhyML requires at least three sequences in a lineage, so in the case that there is only one observed sequence within a clone, that sequence is duplicated. This will not affect the likelihood calculation because these sequences will have a branch length of zero, but it will affect metrics that take sequence frequency into account.

## Subsampling AIRR datasets¶

IgPhyML runs slowly with more than a few thousand sequences. You can subsample your dataset using the --sample and --minseq options, which will subsample your dataset to the specified depth and then remove all clones below the specified size cutoff:

BuildTrees.py -d example.tsv --collapse --sample 5 --minseq 2 --igphyml

## Selecting individual clones¶

Often only particular clones are of interest for lineage tree analysis. To restrict IgPhyML analysis to particular clones, use the --clones option:

#Only use sequences from clone 2:
BuildTrees.py -d example.tsv --collapse --clone 2 --igphyml

#Only use sequences from clones 1 and 2:
BuildTrees.py -d example.tsv --collapse --clone 1 2 --igphyml

## Cleaning up intermediates¶

BuildTrees produces many intermediate files that are usually not useful to the user. To delete them after IgPhyML is run, use --clean all:

BuildTrees.py -d example.tsv --log ex.log --collapse --igphyml --clean all

## Removing the CDR3 region¶

If you plan to analyze model parameters to study things such as SHM and selection, it’s important to remove the CDR3 region to avoid known model biases in estimating $$\omega$$. To do this, use --ncdr3:

BuildTrees.py -d example.tsv --log ex.log --collapse --ncdr3 --igphyml

# Building lineage trees¶

If you’re simply interested in getting some tree topologies, the fastest option is to just use the GY94 and not estimate any parameters under HLP19. This is done using the --optimize n option:

BuildTrees.py -d example.tsv --collapse --igphyml --clean all --optimize n

The trees can then be visualized using igraph and Alakazam. Open an R session:

library(alakazam)
library(igraph)

#plot largest lineage tree
plot(db$trees[[1]],layout=layout_as_tree) Graph-formatted lineage tree of example clone 1. Run dev.off() after plotting if using the Docker image to create a pdf. In these plots, edge labels represent the expected number of substitutions between nodes in the tree. See the Alakazam documentation for plotting this style of trees. Alternatively, more traditional bifurcating tree topologies can be used: library(alakazam) library(ape) db = readIgphyml("example_igphyml-pass.tab",format="phylo") #plot largest lineage tree plot(db$trees[[1]])

Phylo-formatted lineage tree of example clone 1.

Of course, these are quite simple trees. A more interesting tree can be visualized from a different provided dataset:

library(alakazam)
library(ape)

#plot largest lineage tree
plot(ladderize(db$trees[[1]]),cex=0.7,no.margin=TRUE) Phylo-formatted lineage tree of a larger B cell clone. Alternatively, to estimate ML tree topologies using the HLP19 model, use: BuildTrees.py -d example.tsv --collapse --igphyml --clean all --optimize tlr This will be slower than using the GY94 model but does return meaningful HLP19 parameter estimates. These results can be visualized in the same manner using Alakazam. # Evolutionary hypothesis testing¶ ## The HLP19 model¶ The HLP19 model is the heart of IgPhyML and adjusts for features of affinity maturation that violate the assumptions of most other phylogenetic models. It uses four sets of parameters to characterize the types of mutations the occurred over a lineage’s development, and to help build the tree. $$\omega$$: Also called dN/dS, or the ratio of nonsynonymous (amino acid replacement) and synonymous (silent) mutation rates. This parameter generally relates to clonal selection, with totally neutral amino acid evolution having an $$\omega \approx 1$$, negative selection indicated by $$\omega < 1$$ and diversifying selection indicated by $$\omega > 1$$. Generally, we find a lower $$\omega$$ for FWRs than CDRs, presumably because FWRs are more structurally constrained. $$\kappa$$: Ratio of transitions (within purines/pyrimidines) to transversions (between purines/pyrimidines). For normal somatic hypermutation this ratio is usually $$\approx 2$$. Motif mutability (e.g. $$h^{WRC}$$): Mutability parameters for specified hot- and coldspot motifs. These estimates are equivalent to the fold-change in mutability for that motif compared to regular motifs, minus one. So, $$h^{WRC} > 0$$ indicates at hotspot, $$h^{WRC} < 0$$ indicates a coldspot, and $$h^{WRC} = 2$$ indicates a 3x increase in WRC substitution rate. The HLP19 model by default estimates six motif mutability parameters: four hotspots (WRC, GYW, WA, and TW) and two coldspots (SYC and GRS). ## Specifying parameters¶ Substitution parameters are specified using the -t for $$\kappa$$ (transition/transverion rate), --omega for $$\omega$$ (nonsynonymous/synonymous mutation rate), and --motifs and --hotness for specifying the motif mutability models. The default for all of these is to estimate shared parameter values across all lineages, which is also specified by e. Due to default parameter settings, the following two commands are equivalent: BuildTrees.py -d example.tsv --collapse --igphyml BuildTrees.py -d example.tsv --collapse --igphyml -t e --omega e,e \ --motifs WRC_2:0,GYW_0:1,WA_1:2,TW_0:3,SYC_2:4,GRS_0:5 \ --hotness e,e,e,e,e,e --optimize lr Note that here we use --optimize lr, which will keep tree topologies the same and only estimate branch lengths and substitution parameters. This will keep topologies the same as the GY94, but will estimate substitution parameters much more quickly. Using --optimize tlr will also optimize tree topology, using --optimize r will only optimize model parameters, and --optimize n will not optimize topology, branch lengths, or model parameters. The default setting is to estimate a separate $$\omega$$ parameter for FWR and CDR regions. If you want one $$\omega$$ for all regions, use: BuildTrees.py -d example.tsv --collapse --igphyml --omega e You can also constrain motifs to have the same mutabilities by altering the indexes after the ‘:’ in the --motifs option. For motif mutability, each value in the --hotness option corresponds to the index specified in the --motifs option. For example, to estimate a model in which WRC/GYW, WA/TW, and SYC/GRS motifs are respectively constrained to have the same mutabilities, use: BuildTrees.py -d example.tsv --collapse --igphyml \ --motifs WRC_2:0,GYW_0:0,WA_1:1,TW_0:1,SYC_2:2,GRS_0:2 \ --hotness e,e,e ## Confidence interval estimation¶ It is possible to estimate 95% confidence intervals for any of these parameters by adding a ‘c’ to the parameter specification. For example, to estimate a 95% confidence interval for $$\omega _{CDR}$$ but not $$\omega _{FWR}$$, use: BuildTrees.py -d example.tsv --collapse --ncdr3 --clean all --igphyml --omega e,ce To estimate a 95% confidence interval for $$\omega _{FWR}$$ but not $$\omega _{CDR}$$, use: BuildTrees.py -d example.tsv --collapse --ncdr3 --clean all --igphyml --omega ce,e Any combination of confidence interval specifications can be used for the above parameter options. For instance, to estimate confidence intervals for GYW mutability, use: BuildTrees.py -d example.tsv --collapse --ncdr3 --clean all --igphyml --hotness e,ce,e,e,e,e which is equivalent to: BuildTrees.py -d example.tsv --collapse --ncdr3 --clean all --igphyml \ --motifs WRC_2:0,GYW_0:1,WA_1:2,TW_0:3,SYC_2:4,GRS_0:5 \ --hotness e,ce,e,e,e,e Remember it is important to remove the CDR3 region for this kind of analysis. You can find further explanation of the different options in the commandline help page of BuildTrees, including controlling output directories and file names. ## Visualizing results¶ Model hypothesis testing can be easily accomplished with the Alakazam functions readIgphyml and combineIgphyml. In this example, we first run IgPhyML on an example file and estimate confidence intervals on $$\omega _{CDR}$$: BuildTrees.py -d example.tsv --collapse --nproc 2 --ncdr3 --clean all --igphyml --omega e,ce Then, open an R session, where we load the example result and two other samples. To compare maximum likelihood parameter estimates for all samples, use (run dev.off() after plotting if using the Docker image to create a pdf): #!/usr/bin/R library(alakazam) library(ggplot2) #read in three different samples ex = readIgphyml("example_igphyml-pass.tab",id="EX") s1 = readIgphyml("sample1_igphyml-pass.tab",id="S1") s2 = readIgphyml("sample2_igphyml-pass.tab",id="S2") #print out parameter values print(ex$param[1,])

#combine objects into a dataframe
comb = combineIgphyml(list(ex,s1,s2),format="long")

ggplot(comb[grepl("MLE",comb\$variable),],
aes(x=ID,y=variable,fill=value)) + geom_tile() +
theme_bw() + scale_fill_distiller(palette="RdYlBu")

Maximum likelihood HLP19 parameter estimates for three samples.

Maximum likelihood point estimates of each parameter are specified with “_MLE”, while upper and lower confidence interval bounds of a parameter are specified with “_UCI” and “_LCI” respectively. Which estimates are available is controlled by the model specified and whether confidence intervals were estimated when running IgPhyML.

properly test the hypothesis that $$\omega _{CDR}$$ parameter estimates are significantly different among these datasets, use:

#!/usr/bin/R
library(alakazam)
library(ggplot2)

#combine objects into a dataframe
comb = combineIgphyml(list(ex,s1,s2),format="wide")

#compare CDR dN/dS for three samples
ggplot(comb,aes(x=ID,y=OMEGA_CDR_MLE, ymin=OMEGA_CDR_LCI,
ymax=OMEGA_CDR_UCI)) + geom_point() +
geom_errorbar(width=0.1) + theme_bw()

95% confidence intervals for $$\omega _{CDR}$$ of three samples.

Where we can see that the confidence interval for Sample 1 does not overlap with the confidence interval for Sample 2, meanging we conclude Sample 1 has significantly lower $$\omega _{CDR}$$ than Sample 2. However, the confidence intervals for our example file (ex) are too wide to reach any firm conclusion.

# Optimizing performance¶

IgPhyML is a computationally intensive program. There are some ways to make calculations more practical, detailed below.

Data subsampling: IgPhyML runs slowly with more than a few thousand sequences. You can subsample your dataset using the --sample and --minseq options in BuildTrees.py, which will subsample your dataset to the specified depth and then remove all clones below the specified size cutoff (see Subsampling Change-O datasets).

Analyzing specific clones: The --clone option can be used to analyze only the specified clones.

Parallelizing computations: It is possible to parallelize likelihood calculations by splitting computations across multiple cores using the --nproc option. Currently, calculations are parallelized by tree, so there is no use in using more threads than lineages.

Enforcing minimum lineage size: Many repertoires often contain huge numbers of small lineages that can make computations impractical. To limit the size of lineages being analyzed, specify a cutoff with --minseq when running BuildTrees.py.