BUSTED[S] with Multiple Hits

Analysis suggested by Ray Cui [email protected]

This modification of BUSTED[S], which we call which we call BUSTED[SMSH], allows for a gene-wide branch-site test for episodic selection that also allows for site-to-site synonymous rate variation and multiple simultaneous hits. We combine the framework of BUSTED[S] with the MG94 + REV + TRIP codon model that accounts for single, double and triple nucleotide changes.

The core test of BUSTED remains the same, with the analysis fitting an unconstrained model with K rate categories (K = 3 by default) such that ω_i ≤ 1 for i < K, and ω_K ≥ 1 and compares the fit to a constrained model (all ω ≤ 1) using a likelihood ratio test.

Invokation

This analysis has one required argument

• --alignment the alignment file and tree (in FASTA, PHYLIP, MEGA or NEXUS formats)

HyPhy will write Markdown output to the screen and a JSON file with detailed fit results. See example at the end of the document

Complete options list

Available analysis command line options
--------------------------------------
Use --option VALUE syntax to invoke
If a [reqired] option is not provided on the command line, the analysis will prompt for its value
[conditionally required] options may or not be required based on the values of other options

code
        Which genetic code should be used
        default value: Universal

alignment [required]
        An in-frame codon alignment in one of the formats supported by HyPhy

tree [conditionally required]
        A phylogenetic tree (optionally annotated with {})
        applies to: Please select a tree file for the data:
branches
        Branches to test
        default value: All

srv
        Include synonymous rate variation in the model
        default value: Yes

rates
        The number omega rate classes to include in the model [1-10, default 3]
        default value: busted.rate_classes [computed at run time]

syn-rates
        The number alpha rate classes to include in the model [1-10, default 3]
        default value: busted.synonymous_rate_classes [computed at run time]

grid-size
        The number of points in the initial distributional guess for likelihood fitting
        default value: 250 [computed at run time]

starting-points
        The number of initial random guesses to seed rate values optimization
        default value: 1 [computed at run time]

output
        Write the resulting JSON to this file (default is to save to the same path as the alignment file + 'BUSTED.json')
        default value: busted.codon_data_info[terms.json.json] [computed at run time]

save-fit
        Save BUSTED model fit to this file (default is not to save)
        default value: /dev/null

Example run

HYPHYMP  BUSTED-MH.bf --alignment CD2.nex --srv Yes --branches PR

The following data are output to the screen. Analysis Description

BUSTED-SMSH (branch-site unrestricted statistical test of episodic diversification) uses a random effects branch-site model fitted jointly to all or a subset of tree branches in order to test for alignment-wide evidence of episodic diversifying selection and. Assuming there is evidence of positive selection (i.e. there is an omega > 1), BUSTED will also perform a quick evidence-ratio style analysis to explore which individual sites may have been subject to selection. v2.0 adds support for synonymous rate variation, and relaxes the test statistic to 0.5 (chi^2_0 + chi^2_2). Version 2.1 adds a grid search for the initial starting point. Version 2.2 changes the grid search to LHC, and adds an initial search phase to use adaptive Nedler-Mead.

• Requirements: in-frame codon alignment and a phylogenetic tree (optionally annotated with {})

• Citation: Gene-wide identification of episodic selection, Mol Biol Evol. 32(5):1365-71

• Written by: Sergei L Kosakovsky Pond, Sadie Wisotsky

• Contact Information: [email protected]

• Analysis Version: 2.2

code –> Universal Loaded a multiple sequence alignment with 10 sequences, 187 codons, and 1 partitions from /Users/Sadie/bin/hyphy-analyses/BUSTED-MH/CD2.nex

branches –> All

srv –> Yes The number omega rate classes to include in the model (permissible range = [1,10], default value = 3, integer): rates –> 3 The number omega rate classes to include in the model (permissible range = [1,10], default value = 3, integer): syn-rates –> 3 The number of points in the initial distributional guess for likelihood fitting (permissible range = [1,10000], default value = 250, integer): grid-size –> 250 The number of initial random guesses to 'seed' rate values optimization (permissible range = [1,25], default value = 1, integer): starting-points –> 1

Branches to test for selection in the BUSTED analysis

• Selected 16 branches to test in the BUSTED analysis: PIG, COW, Node3, HORSE, CAT, Node2, RHMONKEY, BABOON, Node9, HUMAN, CHIMP, Node12, Node8, Node1, RAT, MOUSE

Obtaining branch lengths and nucleotide substitution biases under the nucleotide GTR model

• Log(L) = -3532.32, AIC-c = 7112.86 (24 estimated parameters)

Improving branch lengths, nucleotide substitution biases, and global dN/dS ratios under a full codon model

• Log(L) = -3467.01, AIC-c = 6997.09 (31 estimated parameters)
• non-synonymous/synonymous rate ratio for test = 0.9975

Fitting MG94 with double instantaneous substitutions

• Log(L) = -3467.01, AIC-c = 6997.09 (31 estimated parameters)
• non-synonymous/synonymous rate ratio for test = 0.9888

Fitting MG94 with double and triple instantaneous substitutions

• Log(L) = -3467.01, AIC-c = 7030.49 (47 estimated parameters)
• non-synonymous/synonymous rate ratio for test = 0.9896

Performing the full (dN/dS > 1 allowed) branch-site model fit

• Log(L) = -3414.49, AIC-c = 6914.96 (42 estimated parameters)
• rate at which 2 nucleotides are changed instantly within a single codon = 0.0000
• rate at which 3 nucleotides are changed instantly within a single codon = 0.0000
• For test branches, the following rate distribution for branch-site combinations was inferred

Selection mode	dN/dS	Proportion, %	Notes
Negative selection	0.000	9.177
Negative selection	0.000	29.502	Collapsed rate class
Diversifying selection	1.878	61.322

• The following rate distribution for site-to-site synonymous rate variation was inferred

Rate	Proportion, %	Notes
0.275	26.571
0.975	63.188
3.038	10.242

Performing the constrained (dN/dS > 1 not allowed) model fit

• Log(L) = -3418.83, AIC-c = 6921.54 (41 estimated parameters)
• rate at which 2 nucleotides are changed instantly within a single codon = 0.0925
• rate at which 3 nucleotides are changed instantly within a single codon = 0.0000
• For test branches under the null (no dN/dS > 1 model), the following rate distribution for branch-site combinations was inferred

Selection mode	dN/dS	Proportion, %	Notes
Negative selection	0.000	2.348
Negative selection	0.000	13.307	Collapsed rate class
Neutral evolution	1.000	84.345

• The following rate distribution for site-to-site synonymous rate variation was inferred

Rate	Proportion, %	Notes
0.327	33.285
1.056	58.091
3.221	8.624

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Branch-site unrestricted statistical test of episodic diversification [BUSTED]

Likelihood ratio test for episodic diversifying positive selection, p = 0.0065.

Check messages.log for diagnostic messages.