Introduction to Signature Variability Analysis with sigvar

Maike Morrison

Contents

1 Introduction

The R package sigvar implements signature variability analysis, a framework for the analysis of mutational signature activities within and across cancer samples. This R package accompanies the paper “Variability of mutational signatures is a footprint of carcinogens’’ by Morrison et al.; please refer to the paper for more details on the methods presented in this package.

The sigvar package contains two core functions to perform signature variability analysis:

• sigvar: Compute the within-sample diversity and across-sample heterogeneity of mutational signature activity in one or multiple populations of samples

• sigboot: Use bootstrapping to statistically compare the within-sample diversity and across-sample heterogeneity of the mutational signature activity between two or more groups of samples

sigvar also includes accessory functions for the visualization of mutational signature data, such as:

• plot_SBS_spectrum: Plot the SBS mutational spectrum of one or more samples of mutational signatures

• plot_signature_prop: Plot the relative activities of mutational signatures in each sample as a stacked bar plot

• plot_dots: Plot the mean mutational signature contributions of one or more groups of samples

1.1 Installation

You can install the development version of sigvar from GitHub with:

install.packages("devtools") # run only if devtools not already installed
devtools::install_github("MaikeMorrison/sigvar",
                         dependencies = TRUE, build_vignettes = TRUE)

After installation, you can access this introductory vignette at an time by running:

vignette("tutorial", package = "sigvar")

1.2 Overview

We begin the tutorial by explaining what types of data are required to use the sigvar R package (Data specifications). We then conduct an example of signature variability analysis (Example analysis), using data from Moody et al. (2021) to explore the within-sample diversity and across-sample heterogeneity of mutational signature activities for esophageal squamous cell carcinoma (ESCC) samples from eight countries which vary dramatically in their ESCC incidences. We begin our example analysis by introducing the data (Example data) and demonstrating the data visualization functions of sigvar (Visualize mutational signature activities). We then perform Signature variability analysis, visualize the results, and use Bootstrapping to determine the statistical significance of differences in SVA results between countries.

2 Data specifications

To use the sigvar package, your data must be in the form of a data frame, matrix, or tibble with rows corresponding to samples and columns corresponding to either metadata (on the left-hand side) or relative activities of mutational signatures (on the right hand side). You can read the data into R using functions such as read.csv, data.table::fread, or readxl::read_xlsx, depending on the file type.

Each row of your matrix must correspond to one sample. If your matrix contains metadata, you must specify K, the number of signatures, and the signatures must all be on the right-hand side of the matrix. The relative activities of all K signatures must sum to 1 for each sample.

Your matrix may contain samples from multiple groups you would like to analyze separately. In this case, you must provide the name of the column specifying the group each sample belongs to as the group parameter of the sigvar or sigboot functions. For example, to separately analyze the smokers and non-smokers in the matrix pictured above, we would specify group = "Smoking".

2.1 Optional: signature similarity matrix

If you would like to perform SVA while accounting for the similarity among mutational signatures, you must provide the sigvar or sigboot functions with a similarity matrix, S, which must also be a data frame, matrix, or tibble. Entry S[i,j] corresponds to the similarity of the mutational spectra of signatures i and j. All diagonal elements must equal 1 (i.e., S[i,j]=1 when i==j), since each signature is identical to itself. All off-diagonal elements must be between 0 and 1, inclusive. In our analyses, we use cosine similarity matrices, where we define entry S[i,j] as the cosine similarity between the mutational spectra of signatures i and j.

As an example, we present below the definitions of the 79 COSMIC (v3.1) SBS mutational signatures, which are included in the sigvar package under the file name COSMIC3.3.1_SBS. The first column of this data frame, Type, specifies the single-base substitution type defining each row, while the other columns (SBS1, SBS2, …, SBS95) contain the relative abundance of each substitution type, which together determine the mutational spectrum of each SBS signature.

Type	SBS1	SBS2	SBS3	SBS4	SBS5	SBS6	SBS7a	SBS7b	SBS7c	SBS7d	SBS8	SBS9	SBS10a	SBS10b	SBS10c	SBS10d	SBS11	SBS12	SBS13	SBS14	SBS15	SBS16	SBS17a	SBS17b	SBS18	SBS19	SBS20	SBS21	SBS22	SBS23	SBS24	SBS25	SBS26	SBS27	SBS28	SBS29	SBS30	SBS31	SBS32	SBS33	SBS34	SBS35	SBS36	SBS37	SBS38	SBS39	SBS40	SBS41	SBS42	SBS43	SBS44	SBS45	SBS46	SBS47	SBS48	SBS49	SBS50	SBS51	SBS52	SBS53	SBS54	SBS55	SBS56	SBS57	SBS58	SBS59	SBS60	SBS84	SBS85	SBS86	SBS87	SBS88	SBS89	SBS90	SBS91	SBS92	SBS93	SBS94	SBS95
A[C>A]A	0.001	0.000	0.021	0.042	0.012	0.000	0.000	0.002	0.005	0.000	0.044	0.001	0.002	0.000	0.004	0.010	0.000	0.005	0.002	0.001	0.001	0.016	0.002	0.001	0.052	0.001	0.001	0.000	0.006	0.001	0.037	0.010	0.001	0.005	0.001	0.063	0.002	0.010	0.022	0.003	0.005	0.009	0.025	0.004	0.013	0.012	0.028	0.002	0.001	0.030	0.000	0.009	0.004	0.068	0.001	0.025	0.119	0.141	0.015	0.005	0.002	0.006	0.013	0.012	0.059	0.004	0.006	0.004	0.006	0.003	0.009	0.000	0.032	0.002	0.003	0.011	0.012	0.016	0.039
A[C>A]C	0.002	0.000	0.016	0.033	0.009	0.001	0.000	0.000	0.001	0.001	0.047	0.004	0.002	0.006	0.015	0.018	0.001	0.001	0.001	0.013	0.000	0.003	0.001	0.000	0.016	0.001	0.001	0.002	0.000	0.000	0.026	0.007	0.001	0.005	0.002	0.051	0.001	0.018	0.019	0.002	0.007	0.046	0.008	0.001	0.010	0.007	0.013	0.001	0.020	0.006	0.000	0.003	0.005	0.029	0.001	0.007	0.125	0.002	0.006	0.002	0.001	0.002	0.015	0.001	0.007	0.002	0.001	0.005	0.001	0.004	0.004	0.000	0.017	0.001	0.052	0.010	0.008	0.025	0.017
A[C>A]G	0.000	0.000	0.002	0.016	0.002	0.000	0.000	0.000	0.000	0.000	0.005	0.000	0.000	0.000	0.001	0.001	0.000	0.001	0.000	0.000	0.000	0.001	0.000	0.000	0.003	0.000	0.000	0.000	0.001	0.000	0.015	0.001	0.000	0.001	0.000	0.020	0.000	0.002	0.005	0.000	0.000	0.001	0.002	0.001	0.002	0.003	0.003	0.000	0.000	0.001	0.000	0.002	0.000	0.001	0.000	0.007	0.006	0.005	0.004	0.039	0.002	0.000	0.000	0.003	0.001	0.000	0.000	0.000	0.000	0.000	0.006	0.000	0.010	0.000	0.000	0.005	0.002	0.002	0.009
A[C>A]T	0.001	0.000	0.012	0.030	0.007	0.000	0.000	0.001	0.002	0.004	0.047	0.003	0.017	0.000	0.013	0.014	0.000	0.001	0.000	0.083	0.001	0.011	0.000	0.000	0.021	0.001	0.001	0.001	0.002	0.000	0.022	0.005	0.001	0.003	0.004	0.036	0.001	0.006	0.015	0.002	0.001	0.022	0.018	0.002	0.009	0.007	0.015	0.001	0.008	0.002	0.006	0.010	0.003	0.012	0.000	0.008	0.083	0.021	0.009	0.000	0.000	0.001	0.023	0.009	0.006	0.015	0.000	0.009	0.003	0.004	0.005	0.002	0.021	0.002	0.000	0.008	0.008	0.011	0.023
A[C>G]A	0.002	0.000	0.020	0.007	0.010	0.000	0.000	0.000	0.001	0.001	0.004	0.005	0.000	0.000	0.000	0.000	0.000	0.001	0.004	0.000	0.000	0.002	0.001	0.000	0.002	0.003	0.009	0.000	0.002	0.000	0.003	0.008	0.000	0.001	0.000	0.003	0.001	0.008	0.002	0.001	0.001	0.004	0.002	0.034	0.003	0.047	0.012	0.005	0.000	0.000	0.003	0.003	0.001	0.001	0.000	0.000	0.004	0.004	0.002	0.007	0.002	0.003	0.000	0.015	0.021	0.003	0.000	0.007	0.007	0.053	0.008	0.000	0.015	0.001	0.000	0.003	0.009	0.007	0.004
A[C>G]C	0.001	0.000	0.012	0.003	0.006	0.000	0.000	0.000	0.000	0.003	0.003	0.002	0.000	0.000	0.000	0.000	0.000	0.001	0.001	0.000	0.000	0.002	0.001	0.000	0.003	0.002	0.001	0.001	0.000	0.000	0.004	0.002	0.000	0.002	0.000	0.003	0.000	0.003	0.002	0.001	0.001	0.005	0.002	0.011	0.002	0.022	0.009	0.003	0.001	0.000	0.000	0.002	0.001	0.001	0.000	0.000	0.001	0.004	0.001	0.005	0.000	0.000	0.000	0.003	0.002	0.002	0.001	0.011	0.003	0.077	0.004	0.004	0.006	0.000	0.000	0.002	0.005	0.004	0.003
A[C>G]G	0.000	0.000	0.000	0.001	0.002	0.000	0.000	0.001	0.000	0.001	0.000	0.001	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.001	0.000	0.000	0.002	0.001	0.000	0.000	0.000	0.000	0.001	0.000	0.000	0.000	0.000	0.001	0.000	0.003	0.001	0.000	0.000	0.000	0.001	0.006	0.001	0.014	0.002	0.001	0.000	0.005	0.000	0.000	0.000	0.000	0.000	0.000	0.001	0.002	0.001	0.003	0.002	0.001	0.000	0.003	0.002	0.001	0.000	0.000	0.000	0.005	0.031	0.000	0.006	0.000	0.000	0.001	0.002	0.001	0.001
A[C>G]T	0.001	0.000	0.017	0.004	0.010	0.000	0.000	0.000	0.002	0.003	0.006	0.006	0.000	0.000	0.001	0.000	0.001	0.001	0.004	0.000	0.000	0.012	0.000	0.000	0.004	0.002	0.004	0.000	0.000	0.000	0.003	0.003	0.001	0.001	0.000	0.003	0.001	0.004	0.002	0.001	0.002	0.010	0.002	0.019	0.002	0.044	0.015	0.008	0.000	0.000	0.003	0.001	0.002	0.002	0.001	0.000	0.002	0.001	0.002	0.003	0.005	0.001	0.000	0.005	0.002	0.004	0.000	0.030	0.013	0.092	0.010	0.004	0.011	0.001	0.000	0.004	0.014	0.007	0.001
A[C>T]A	0.025	0.000	0.014	0.009	0.033	0.064	0.000	0.001	0.001	0.003	0.007	0.008	0.001	0.001	0.005	0.001	0.026	0.006	0.007	0.012	0.005	0.002	0.001	0.001	0.009	0.028	0.050	0.002	0.002	0.023	0.012	0.021	0.003	0.014	0.001	0.005	0.107	0.018	0.087	0.005	0.004	0.002	0.006	0.006	0.003	0.010	0.020	0.010	0.023	0.000	0.090	0.001	0.015	0.004	0.000	0.001	0.004	0.036	0.003	0.007	0.001	0.005	0.007	0.033	0.081	0.004	0.003	0.054	0.001	0.009	0.009	0.010	0.050	0.001	0.016	0.024	0.009	0.008	0.013
A[C>T]C	0.006	0.001	0.012	0.004	0.018	0.023	0.017	0.038	0.008	0.001	0.003	0.005	0.001	0.005	0.005	0.002	0.146	0.002	0.000	0.010	0.007	0.004	0.002	0.000	0.005	0.014	0.013	0.004	0.000	0.081	0.011	0.013	0.003	0.005	0.003	0.004	0.090	0.009	0.122	0.002	0.004	0.004	0.003	0.003	0.002	0.004	0.012	0.003	0.040	0.000	0.017	0.001	0.009	0.001	0.000	0.000	0.002	0.006	0.002	0.002	0.001	0.000	0.001	0.013	0.007	0.003	0.001	0.042	0.001	0.002	0.003	0.000	0.013	0.000	0.001	0.014	0.005	0.014	0.010
A[C>T]G	0.371	0.000	0.003	0.001	0.006	0.123	0.000	0.001	0.001	0.001	0.003	0.005	0.006	0.000	0.013	0.001	0.008	0.000	0.000	0.027	0.077	0.003	0.000	0.000	0.013	0.002	0.014	0.008	0.001	0.009	0.001	0.020	0.005	0.007	0.007	0.021	0.015	0.008	0.038	0.007	0.000	0.001	0.007	0.007	0.005	0.006	0.000	0.004	0.000	0.001	0.015	0.006	0.005	0.003	0.001	0.002	0.014	0.006	0.004	0.007	0.000	0.005	0.002	0.018	0.009	0.003	0.004	0.003	0.000	0.025	0.125	0.013	0.006	0.000	0.002	0.010	0.018	0.024	0.004
A[C>T]T	0.009	0.002	0.012	0.004	0.022	0.021	0.007	0.011	0.007	0.033	0.013	0.007	0.003	0.008	0.006	0.003	0.110	0.002	0.000	0.016	0.002	0.006	0.001	0.000	0.008	0.037	0.013	0.000	0.001	0.035	0.009	0.006	0.003	0.005	0.005	0.002	0.029	0.010	0.152	0.003	0.004	0.001	0.004	0.006	0.002	0.013	0.017	0.006	0.036	0.001	0.030	0.001	0.007	0.003	0.000	0.000	0.007	0.003	0.002	0.002	0.005	0.001	0.003	0.015	0.007	0.003	0.000	0.055	0.005	0.005	0.011	0.000	0.030	0.001	0.002	0.017	0.007	0.017	0.005
A[T>A]A	0.001	0.000	0.005	0.009	0.007	0.000	0.002	0.002	0.012	0.000	0.019	0.013	0.000	0.002	0.004	0.002	0.000	0.005	0.000	0.001	0.002	0.023	0.001	0.000	0.002	0.001	0.001	0.003	0.064	0.000	0.002	0.020	0.002	0.143	0.000	0.001	0.000	0.008	0.001	0.001	0.103	0.005	0.002	0.002	0.002	0.005	0.006	0.007	0.013	0.015	0.005	0.001	0.003	0.137	0.001	0.000	0.005	0.112	0.002	0.006	0.001	0.004	0.000	0.003	0.024	0.002	0.004	0.002	0.111	0.002	0.010	0.048	0.011	0.061	0.000	0.010	0.010	0.010	0.003
A[T>A]C	0.002	0.001	0.007	0.004	0.007	0.001	0.001	0.000	0.003	0.001	0.021	0.003	0.000	0.000	0.014	0.003	0.000	0.003	0.000	0.000	0.001	0.008	0.001	0.000	0.001	0.000	0.005	0.006	0.007	0.000	0.002	0.009	0.004	0.001	0.000	0.002	0.000	0.006	0.001	0.001	0.045	0.007	0.001	0.002	0.002	0.003	0.004	0.004	0.004	0.004	0.003	0.000	0.002	0.007	0.000	0.000	0.004	0.001	0.001	0.003	0.003	0.001	0.004	0.006	0.001	0.003	0.000	0.000	0.013	0.004	0.001	0.024	0.003	0.000	0.000	0.004	0.006	0.006	0.003
A[T>A]G	0.001	0.000	0.010	0.012	0.009	0.000	0.001	0.002	0.000	0.004	0.023	0.003	0.000	0.000	0.001	0.001	0.000	0.003	0.001	0.000	0.001	0.009	0.000	0.000	0.003	0.001	0.000	0.000	0.060	0.000	0.002	0.006	0.001	0.001	0.000	0.002	0.001	0.005	0.001	0.001	0.035	0.001	0.003	0.002	0.001	0.007	0.007	0.003	0.005	0.001	0.000	0.002	0.000	0.007	0.000	0.000	0.001	0.037	0.001	0.003	0.004	0.008	0.001	0.006	0.005	0.002	0.000	0.000	0.021	0.002	0.004	0.001	0.011	0.000	0.000	0.007	0.005	0.004	0.006
A[T>A]T	0.000	0.000	0.006	0.004	0.006	0.002	0.004	0.008	0.050	0.041	0.025	0.019	0.007	0.003	0.071	0.022	0.000	0.003	0.001	0.002	0.009	0.011	0.001	0.001	0.008	0.001	0.026	0.002	0.003	0.000	0.002	0.015	0.003	0.003	0.002	0.002	0.000	0.004	0.001	0.005	0.316	0.007	0.003	0.003	0.003	0.006	0.011	0.069	0.007	0.004	0.031	0.001	0.002	0.067	0.001	0.000	0.002	0.029	0.001	0.005	0.000	0.004	0.029	0.006	0.009	0.003	0.001	0.011	0.070	0.007	0.004	0.043	0.004	0.023	0.000	0.009	0.013	0.016	0.003
A[T>C]A	0.001	0.000	0.016	0.008	0.046	0.002	0.000	0.001	0.000	0.004	0.010	0.020	0.001	0.009	0.018	0.005	0.001	0.073	0.002	0.013	0.004	0.241	0.000	0.000	0.002	0.003	0.011	0.017	0.025	0.001	0.006	0.021	0.084	0.005	0.001	0.002	0.001	0.020	0.001	0.012	0.005	0.002	0.005	0.073	0.003	0.011	0.019	0.014	0.012	0.000	0.019	0.001	0.018	0.003	0.001	0.001	0.003	0.003	0.004	0.011	0.029	0.004	0.012	0.026	0.080	0.010	0.001	0.000	0.029	0.004	0.009	0.111	0.016	0.006	0.000	0.089	0.034	0.014	0.002
A[T>C]C	0.003	0.000	0.008	0.001	0.013	0.001	0.000	0.000	0.001	0.004	0.005	0.011	0.004	0.001	0.009	0.003	0.001	0.040	0.000	0.006	0.002	0.013	0.013	0.000	0.002	0.001	0.005	0.010	0.001	0.000	0.002	0.010	0.026	0.002	0.000	0.002	0.000	0.007	0.001	0.004	0.001	0.001	0.002	0.038	0.002	0.006	0.007	0.006	0.004	0.031	0.003	0.000	0.048	0.006	0.000	0.000	0.009	0.019	0.002	0.004	0.008	0.000	0.006	0.010	0.005	0.003	0.000	0.000	0.020	0.002	0.003	0.046	0.003	0.000	0.000	0.005	0.003	0.005	0.004
A[T>C]G	0.000	0.000	0.012	0.005	0.038	0.004	0.000	0.001	0.001	0.009	0.007	0.014	0.002	0.001	0.017	0.008	0.000	0.070	0.000	0.012	0.000	0.064	0.000	0.000	0.002	0.002	0.019	0.004	0.008	0.000	0.002	0.020	0.078	0.006	0.000	0.003	0.000	0.008	0.001	0.073	0.000	0.001	0.004	0.017	0.002	0.010	0.015	0.008	0.009	0.001	0.027	0.002	0.033	0.002	0.000	0.000	0.001	0.004	0.003	0.008	0.201	0.004	0.009	0.010	0.007	0.004	0.000	0.000	0.005	0.004	0.001	0.006	0.015	0.000	0.001	0.040	0.006	0.003	0.003
A[T>C]T	0.006	0.000	0.017	0.002	0.038	0.000	0.001	0.002	0.003	0.051	0.015	0.029	0.003	0.000	0.013	0.006	0.001	0.064	0.001	0.006	0.001	0.136	0.008	0.001	0.004	0.002	0.000	0.026	0.001	0.000	0.002	0.014	0.037	0.003	0.001	0.001	0.001	0.008	0.001	0.009	0.002	0.002	0.005	0.048	0.003	0.014	0.019	0.018	0.003	0.009	0.000	0.001	0.030	0.001	0.001	0.000	0.005	0.002	0.005	0.007	0.008	0.014	0.012	0.018	0.008	0.011	0.000	0.009	0.059	0.004	0.006	0.169	0.012	0.003	0.000	0.029	0.011	0.023	0.003
A[T>G]A	0.000	0.000	0.004	0.001	0.004	0.000	0.000	0.000	0.012	0.004	0.002	0.045	0.002	0.000	0.003	0.007	0.000	0.002	0.000	0.000	0.000	0.011	0.001	0.001	0.001	0.002	0.000	0.000	0.002	0.000	0.003	0.005	0.003	0.001	0.010	0.000	0.000	0.004	0.000	0.001	0.003	0.003	0.001	0.022	0.001	0.004	0.007	0.007	0.001	0.003	0.000	0.000	0.000	0.000	0.001	0.000	0.001	0.001	0.002	0.005	0.000	0.000	0.000	0.007	0.013	0.002	0.025	0.000	0.024	0.005	0.008	0.008	0.005	0.005	0.002	0.002	0.025	0.004	0.000
A[T>G]C	0.000	0.000	0.003	0.000	0.003	0.000	0.000	0.001	0.001	0.003	0.001	0.007	0.001	0.000	0.001	0.001	0.000	0.001	0.000	0.003	0.000	0.004	0.001	0.004	0.001	0.002	0.000	0.000	0.000	0.000	0.001	0.000	0.003	0.001	0.017	0.001	0.000	0.002	0.000	0.000	0.000	0.001	0.001	0.011	0.001	0.003	0.007	0.002	0.000	0.004	0.000	0.001	0.001	0.002	0.000	0.000	0.001	0.004	0.001	0.003	0.002	0.001	0.000	0.001	0.001	0.002	0.011	0.001	0.003	0.000	0.001	0.005	0.001	0.000	0.000	0.000	0.002	0.001	0.000
A[T>G]G	0.000	0.000	0.006	0.001	0.008	0.000	0.000	0.001	0.001	0.011	0.005	0.005	0.000	0.001	0.001	0.001	0.000	0.001	0.000	0.001	0.000	0.005	0.001	0.001	0.002	0.002	0.000	0.000	0.003	0.000	0.002	0.007	0.002	0.000	0.008	0.000	0.000	0.004	0.001	0.000	0.000	0.004	0.001	0.017	0.002	0.008	0.008	0.003	0.001	0.035	0.000	0.001	0.002	0.004	0.000	0.000	0.004	0.028	0.001	0.005	0.001	0.144	0.000	0.001	0.003	0.002	0.026	0.001	0.004	0.002	0.003	0.002	0.005	0.000	0.001	0.001	0.005	0.002	0.000
A[T>G]T	0.000	0.000	0.004	0.000	0.005	0.000	0.000	0.001	0.000	0.014	0.001	0.024	0.009	0.000	0.013	0.005	0.000	0.001	0.000	0.011	0.004	0.008	0.012	0.069	0.002	0.002	0.000	0.000	0.000	0.000	0.005	0.000	0.006	0.000	0.114	0.001	0.001	0.001	0.000	0.001	0.001	0.003	0.001	0.025	0.003	0.006	0.018	0.008	0.001	0.006	0.001	0.000	0.001	0.008	0.000	0.000	0.000	0.002	0.002	0.002	0.000	0.002	0.006	0.006	0.002	0.003	0.016	0.002	0.003	0.001	0.002	0.007	0.004	0.002	0.000	0.004	0.008	0.002	0.000
C[C>A]A	0.000	0.000	0.022	0.080	0.007	0.002	0.000	0.001	0.000	0.014	0.040	0.005	0.003	0.001	0.017	0.010	0.001	0.005	0.001	0.016	0.041	0.010	0.000	0.000	0.073	0.001	0.037	0.000	0.002	0.000	0.025	0.015	0.001	0.005	0.002	0.054	0.001	0.011	0.001	0.003	0.002	0.030	0.055	0.003	0.380	0.010	0.021	0.002	0.012	0.007	0.000	0.237	0.015	0.024	0.001	0.228	0.022	0.007	0.057	0.332	0.000	0.006	0.002	0.009	0.006	0.002	0.001	0.000	0.004	0.006	0.008	0.000	0.024	0.001	0.008	0.018	0.006	0.080	0.058
C[C>A]C	0.002	0.000	0.025	0.079	0.006	0.004	0.000	0.002	0.003	0.003	0.038	0.002	0.000	0.003	0.010	0.015	0.001	0.004	0.001	0.019	0.005	0.009	0.001	0.000	0.019	0.001	0.098	0.003	0.004	0.001	0.034	0.008	0.001	0.002	0.000	0.036	0.000	0.011	0.005	0.002	0.000	0.033	0.020	0.001	0.069	0.009	0.015	0.001	0.033	0.000	0.030	0.149	0.007	0.005	0.000	0.028	0.003	0.003	0.019	0.004	0.003	0.004	0.007	0.004	0.002	0.006	0.000	0.005	0.000	0.003	0.006	0.000	0.014	0.000	0.004	0.014	0.005	0.075	0.038
C[C>A]G	0.000	0.000	0.003	0.025	0.003	0.002	0.000	0.000	0.002	0.000	0.003	0.001	0.000	0.000	0.002	0.001	0.000	0.001	0.000	0.001	0.000	0.002	0.000	0.000	0.012	0.000	0.007	0.002	0.000	0.000	0.016	0.001	0.000	0.000	0.000	0.015	0.000	0.000	0.000	0.000	0.000	0.002	0.007	0.001	0.051	0.006	0.003	0.000	0.002	0.000	0.004	0.068	0.005	0.000	0.135	0.516	0.000	0.004	0.014	0.068	0.006	0.001	0.000	0.001	0.001	0.001	0.000	0.000	0.000	0.001	0.020	0.000	0.003	0.000	0.002	0.005	0.002	0.006	0.015
C[C>A]T	0.000	0.000	0.015	0.069	0.006	0.009	0.000	0.001	0.013	0.000	0.033	0.003	0.017	0.008	0.044	0.018	0.000	0.001	0.000	0.258	0.028	0.002	0.000	0.000	0.036	0.000	0.251	0.019	0.000	0.001	0.017	0.013	0.007	0.003	0.000	0.052	0.000	0.028	0.003	0.003	0.001	0.082	0.060	0.002	0.183	0.006	0.015	0.003	0.021	0.000	0.086	0.193	0.008	0.001	0.001	0.049	0.001	0.005	0.019	0.005	0.005	0.000	0.071	0.008	0.002	0.002	0.000	0.006	0.001	0.003	0.005	0.000	0.017	0.001	0.003	0.012	0.010	0.040	0.055
C[C>G]A	0.000	0.000	0.019	0.008	0.007	0.000	0.000	0.000	0.000	0.001	0.005	0.001	0.000	0.000	0.000	0.000	0.000	0.001	0.006	0.000	0.000	0.004	0.001	0.000	0.001	0.002	0.000	0.000	0.001	0.000	0.003	0.007	0.000	0.005	0.000	0.003	0.001	0.002	0.001	0.001	0.000	0.004	0.001	0.000	0.002	0.029	0.008	0.002	0.004	0.000	0.000	0.001	0.000	0.001	0.000	0.000	0.000	0.003	0.001	0.003	0.001	0.003	0.000	0.001	0.001	0.002	0.000	0.000	0.001	0.021	0.007	0.005	0.015	0.000	0.000	0.002	0.003	0.009	0.004
C[C>G]C	0.000	0.000	0.014	0.007	0.009	0.000	0.000	0.001	0.002	0.000	0.005	0.002	0.000	0.000	0.000	0.000	0.000	0.001	0.001	0.000	0.000	0.004	0.000	0.000	0.002	0.002	0.000	0.000	0.001	0.000	0.005	0.005	0.000	0.001	0.000	0.006	0.001	0.002	0.001	0.000	0.000	0.006	0.001	0.000	0.001	0.037	0.009	0.001	0.007	0.000	0.000	0.001	0.000	0.000	0.000	0.000	0.000	0.000	0.001	0.006	0.001	0.003	0.000	0.003	0.001	0.002	0.000	0.003	0.000	0.058	0.003	0.001	0.010	0.000	0.000	0.004	0.003	0.015	0.004
C[C>G]G	0.000	0.000	0.002	0.003	0.002	0.000	0.000	0.001	0.000	0.001	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.001	0.000	0.000	0.004	0.000	0.000	0.000	0.000	0.000	0.001	0.001	0.000	0.000	0.000	0.001	0.000	0.001	0.001	0.000	0.000	0.001	0.000	0.000	0.001	0.012	0.002	0.000	0.000	0.003	0.000	0.001	0.000	0.001	0.000	0.000	0.001	0.002	0.001	0.002	0.000	0.000	0.000	0.001	0.000	0.001	0.000	0.000	0.000	0.003	0.086	0.000	0.004	0.000	0.000	0.002	0.001	0.002	0.001
C[C>G]T	0.000	0.000	0.022	0.007	0.009	0.000	0.000	0.001	0.004	0.000	0.005	0.003	0.000	0.000	0.000	0.000	0.000	0.002	0.006	0.000	0.000	0.010	0.000	0.000	0.004	0.003	0.000	0.000	0.000	0.000	0.005	0.000	0.001	0.000	0.000	0.007	0.001	0.003	0.000	0.001	0.000	0.016	0.002	0.001	0.001	0.040	0.014	0.005	0.007	0.001	0.000	0.002	0.001	0.000	0.000	0.000	0.001	0.003	0.001	0.001	0.001	0.003	0.001	0.003	0.001	0.003	0.000	0.013	0.000	0.040	0.009	0.003	0.016	0.000	0.000	0.005	0.006	0.009	0.004
C[C>T]A	0.002	0.004	0.016	0.013	0.019	0.013	0.050	0.106	0.003	0.035	0.003	0.007	0.000	0.000	0.004	0.001	0.004	0.007	0.000	0.003	0.011	0.007	0.001	0.000	0.012	0.119	0.011	0.001	0.001	0.062	0.005	0.014	0.002	0.017	0.000	0.008	0.109	0.041	0.017	0.005	0.005	0.005	0.007	0.004	0.004	0.009	0.019	0.007	0.019	0.000	0.004	0.002	0.028	0.001	0.000	0.000	0.004	0.003	0.003	0.015	0.003	0.007	0.000	0.015	0.008	0.003	0.002	0.018	0.002	0.004	0.008	0.000	0.009	0.001	0.036	0.036	0.009	0.014	0.015
C[C>T]C	0.000	0.000	0.020	0.011	0.019	0.015	0.054	0.180	0.006	0.003	0.004	0.009	0.000	0.000	0.007	0.002	0.125	0.000	0.001	0.001	0.011	0.003	0.002	0.000	0.007	0.091	0.013	0.005	0.003	0.188	0.009	0.006	0.003	0.007	0.000	0.002	0.102	0.162	0.028	0.002	0.001	0.082	0.007	0.002	0.005	0.004	0.017	0.002	0.048	0.000	0.003	0.000	0.013	0.005	0.000	0.000	0.004	0.005	0.002	0.005	0.000	0.004	0.001	0.012	0.010	0.000	0.002	0.040	0.000	0.005	0.008	0.000	0.011	0.000	0.000	0.034	0.004	0.018	0.016
C[C>T]G	0.193	0.003	0.002	0.003	0.018	0.122	0.007	0.022	0.009	0.003	0.001	0.007	0.000	0.004	0.009	0.001	0.001	0.000	0.002	0.012	0.025	0.001	0.002	0.001	0.017	0.016	0.017	0.006	0.000	0.014	0.006	0.012	0.003	0.008	0.003	0.015	0.017	0.008	0.011	0.006	0.010	0.003	0.005	0.003	0.004	0.008	0.002	0.010	0.002	0.000	0.016	0.002	0.008	0.002	0.000	0.001	0.001	0.021	0.002	0.014	0.028	0.000	0.008	0.006	0.008	0.001	0.004	0.000	0.000	0.006	0.186	0.000	0.002	0.000	0.001	0.011	0.007	0.019	0.006
C[C>T]T	0.000	0.001	0.023	0.016	0.023	0.014	0.056	0.125	0.020	0.000	0.014	0.011	0.000	0.001	0.010	0.003	0.065	0.003	0.001	0.001	0.015	0.019	0.002	0.000	0.007	0.257	0.012	0.000	0.002	0.156	0.004	0.013	0.002	0.007	0.003	0.001	0.046	0.144	0.042	0.003	0.000	0.047	0.006	0.006	0.004	0.007	0.021	0.008	0.033	0.001	0.005	0.003	0.006	0.003	0.000	0.001	0.012	0.004	0.003	0.016	0.004	0.001	0.001	0.032	0.027	0.002	0.002	0.042	0.002	0.002	0.007	0.004	0.013	0.000	0.001	0.045	0.007	0.025	0.009
C[T>A]A	0.000	0.000	0.008	0.014	0.004	0.000	0.000	0.001	0.003	0.002	0.019	0.005	0.000	0.000	0.001	0.001	0.000	0.001	0.000	0.000	0.000	0.003	0.000	0.000	0.001	0.000	0.000	0.000	0.128	0.000	0.001	0.026	0.001	0.331	0.000	0.002	0.000	0.021	0.000	0.001	0.024	0.053	0.001	0.002	0.001	0.005	0.001	0.004	0.013	0.001	0.000	0.001	0.004	0.014	0.000	0.000	0.000	0.006	0.001	0.004	0.000	0.000	0.001	0.006	0.002	0.000	0.000	0.000	0.010	0.002	0.008	0.000	0.031	0.102	0.000	0.007	0.011	0.012	0.003
C[T>A]C	0.000	0.000	0.018	0.012	0.005	0.001	0.000	0.001	0.013	0.000	0.029	0.003	0.000	0.000	0.003	0.001	0.000	0.006	0.000	0.003	0.000	0.006	0.040	0.002	0.003	0.000	0.007	0.001	0.055	0.000	0.002	0.016	0.001	0.003	0.001	0.001	0.001	0.021	0.001	0.001	0.000	0.033	0.001	0.001	0.002	0.006	0.008	0.003	0.002	0.000	0.003	0.001	0.003	0.001	0.000	0.000	0.001	0.001	0.000	0.005	0.001	0.000	0.001	0.007	0.022	0.002	0.001	0.000	0.001	0.001	0.005	0.003	0.018	0.000	0.000	0.004	0.009	0.009	0.006
C[T>A]G	0.000	0.000	0.013	0.033	0.005	0.000	0.000	0.001	0.001	0.001	0.028	0.005	0.000	0.000	0.001	0.001	0.000	0.001	0.000	0.000	0.000	0.001	0.012	0.001	0.003	0.000	0.000	0.000	0.168	0.000	0.005	0.063	0.001	0.004	0.001	0.002	0.001	0.019	0.001	0.000	0.001	0.058	0.002	0.002	0.002	0.007	0.003	0.003	0.015	0.000	0.001	0.003	0.004	0.000	0.000	0.000	0.001	0.007	0.001	0.003	0.002	0.016	0.002	0.001	0.001	0.000	0.000	0.000	0.000	0.003	0.003	0.000	0.037	0.000	0.000	0.010	0.007	0.008	0.011
C[T>A]T	0.000	0.000	0.013	0.012	0.004	0.000	0.000	0.000	0.049	0.001	0.042	0.004	0.000	0.000	0.003	0.002	0.000	0.002	0.001	0.001	0.001	0.007	0.072	0.008	0.002	0.000	0.000	0.000	0.061	0.000	0.005	0.037	0.001	0.005	0.001	0.002	0.001	0.031	0.001	0.001	0.000	0.067	0.002	0.002	0.002	0.008	0.009	0.009	0.008	0.000	0.000	0.001	0.004	0.000	0.000	0.000	0.000	0.004	0.001	0.009	0.000	0.005	0.000	0.011	0.001	0.004	0.000	0.000	0.011	0.003	0.010	0.000	0.020	0.102	0.000	0.006	0.025	0.015	0.007
C[T>C]A	0.000	0.000	0.009	0.005	0.013	0.002	0.000	0.002	0.008	0.010	0.002	0.014	0.000	0.001	0.012	0.003	0.000	0.045	0.000	0.003	0.001	0.011	0.011	0.001	0.000	0.000	0.012	0.011	0.007	0.000	0.002	0.009	0.052	0.005	0.001	0.001	0.001	0.008	0.001	0.015	0.001	0.001	0.001	0.050	0.002	0.007	0.011	0.010	0.004	0.003	0.015	0.001	0.022	0.002	0.000	0.000	0.001	0.004	0.002	0.010	0.011	0.004	0.003	0.012	0.004	0.002	0.001	0.002	0.007	0.001	0.005	0.000	0.005	0.005	0.000	0.031	0.030	0.006	0.004
C[T>C]C	0.002	0.000	0.015	0.002	0.010	0.001	0.000	0.001	0.004	0.000	0.007	0.015	0.000	0.000	0.010	0.002	0.001	0.050	0.000	0.005	0.002	0.003	0.173	0.006	0.002	0.000	0.005	0.003	0.001	0.000	0.002	0.011	0.031	0.006	0.003	0.002	0.001	0.010	0.001	0.030	0.000	0.002	0.002	0.022	0.002	0.008	0.010	0.005	0.003	0.105	0.003	0.003	0.083	0.006	0.000	0.000	0.018	0.071	0.001	0.004	0.017	0.005	0.003	0.005	0.009	0.006	0.003	0.000	0.006	0.003	0.002	0.002	0.004	0.000	0.000	0.004	0.008	0.015	0.008
C[T>C]G	0.000	0.000	0.010	0.006	0.021	0.005	0.000	0.001	0.002	0.001	0.001	0.015	0.001	0.000	0.015	0.003	0.000	0.037	0.000	0.004	0.001	0.010	0.087	0.004	0.002	0.000	0.026	0.014	0.002	0.000	0.004	0.013	0.079	0.006	0.002	0.004	0.001	0.004	0.002	0.040	0.001	0.002	0.002	0.010	0.003	0.008	0.010	0.006	0.004	0.009	0.034	0.002	0.050	0.003	0.000	0.000	0.002	0.003	0.001	0.008	0.193	0.001	0.000	0.008	0.006	0.002	0.001	0.004	0.001	0.001	0.001	0.000	0.008	0.000	0.000	0.014	0.002	0.007	0.015
C[T>C]T	0.000	0.000	0.016	0.003	0.015	0.001	0.002	0.006	0.043	0.040	0.006	0.033	0.000	0.001	0.019	0.005	0.000	0.046	0.000	0.002	0.002	0.016	0.427	0.018	0.003	0.000	0.007	0.022	0.001	0.000	0.004	0.009	0.056	0.004	0.000	0.002	0.004	0.014	0.001	0.003	0.002	0.003	0.002	0.051	0.004	0.009	0.014	0.008	0.003	0.009	0.008	0.001	0.096	0.005	0.000	0.000	0.001	0.007	0.002	0.005	0.027	0.001	0.000	0.024	0.028	0.012	0.002	0.001	0.017	0.002	0.009	0.000	0.004	0.006	0.000	0.014	0.015	0.016	0.006
C[T>G]A	0.000	0.000	0.004	0.001	0.003	0.000	0.000	0.000	0.003	0.001	0.001	0.026	0.000	0.000	0.011	0.006	0.000	0.001	0.000	0.000	0.000	0.004	0.000	0.002	0.000	0.001	0.001	0.000	0.005	0.000	0.001	0.000	0.001	0.000	0.015	0.001	0.000	0.002	0.000	0.001	0.001	0.004	0.000	0.003	0.001	0.005	0.003	0.003	0.001	0.003	0.000	0.000	0.000	0.000	0.000	0.000	0.001	0.003	0.001	0.001	0.004	0.002	0.006	0.000	0.001	0.001	0.003	0.001	0.004	0.004	0.008	0.000	0.007	0.009	0.001	0.001	0.009	0.002	0.000
C[T>G]C	0.000	0.000	0.007	0.000	0.005	0.001	0.000	0.001	0.006	0.000	0.002	0.007	0.000	0.001	0.002	0.000	0.000	0.001	0.000	0.001	0.000	0.001	0.001	0.031	0.002	0.002	0.002	0.000	0.001	0.000	0.001	0.003	0.005	0.000	0.022	0.000	0.000	0.001	0.000	0.001	0.000	0.001	0.001	0.002	0.001	0.007	0.006	0.002	0.001	0.007	0.003	0.000	0.002	0.000	0.000	0.000	0.000	0.003	0.001	0.004	0.019	0.000	0.000	0.001	0.001	0.003	0.004	0.002	0.002	0.000	0.001	0.002	0.003	0.000	0.000	0.000	0.006	0.002	0.001
C[T>G]G	0.000	0.000	0.011	0.004	0.006	0.001	0.000	0.001	0.001	0.000	0.005	0.014	0.000	0.000	0.007	0.003	0.000	0.001	0.000	0.000	0.000	0.002	0.000	0.014	0.003	0.001	0.003	0.000	0.008	0.000	0.003	0.012	0.006	0.000	0.015	0.000	0.000	0.002	0.001	0.001	0.000	0.006	0.001	0.004	0.002	0.010	0.006	0.003	0.006	0.046	0.003	0.001	0.002	0.004	0.000	0.000	0.004	0.006	0.001	0.003	0.022	0.113	0.004	0.002	0.001	0.002	0.007	0.001	0.001	0.002	0.002	0.002	0.009	0.000	0.000	0.002	0.003	0.003	0.001
C[T>G]T	0.000	0.000	0.007	0.001	0.007	0.001	0.000	0.000	0.003	0.000	0.001	0.057	0.001	0.005	0.030	0.006	0.000	0.000	0.000	0.001	0.002	0.003	0.005	0.550	0.004	0.002	0.005	0.000	0.001	0.000	0.005	0.009	0.008	0.001	0.101	0.001	0.000	0.001	0.001	0.000	0.000	0.003	0.001	0.004	0.007	0.005	0.013	0.011	0.004	0.000	0.000	0.001	0.002	0.000	0.000	0.000	0.000	0.002	0.001	0.003	0.001	0.001	0.000	0.005	0.000	0.021	0.000	0.000	0.002	0.002	0.003	0.000	0.004	0.010	0.000	0.001	0.038	0.003	0.001
G[C>A]A	0.002	0.000	0.007	0.032	0.010	0.000	0.000	0.001	0.001	0.000	0.024	0.008	0.001	0.001	0.008	0.002	0.000	0.002	0.001	0.002	0.002	0.009	0.002	0.001	0.109	0.001	0.011	0.002	0.002	0.000	0.062	0.013	0.000	0.011	0.000	0.097	0.001	0.013	0.005	0.004	0.003	0.010	0.068	0.002	0.012	0.007	0.014	0.003	0.008	0.009	0.000	0.008	0.007	0.011	0.001	0.008	0.014	0.004	0.008	0.000	0.005	0.001	0.000	0.002	0.003	0.103	0.001	0.003	0.001	0.005	0.007	0.009	0.007	0.001	0.003	0.009	0.002	0.014	0.067
G[C>A]C	0.000	0.000	0.011	0.034	0.008	0.001	0.000	0.000	0.001	0.000	0.026	0.007	0.000	0.002	0.007	0.025	0.000	0.000	0.000	0.022	0.002	0.004	0.001	0.000	0.017	0.000	0.006	0.007	0.002	0.001	0.127	0.006	0.001	0.012	0.001	0.089	0.000	0.021	0.004	0.001	0.000	0.050	0.019	0.001	0.006	0.007	0.010	0.001	0.027	0.002	0.005	0.006	0.003	0.003	0.000	0.002	0.007	0.003	0.007	0.003	0.009	0.000	0.007	0.001	0.001	0.128	0.001	0.010	0.000	0.004	0.005	0.000	0.006	0.000	0.006	0.008	0.002	0.030	0.045
G[C>A]G	0.001	0.000	0.001	0.015	0.002	0.000	0.000	0.000	0.000	0.000	0.003	0.000	0.000	0.000	0.001	0.000	0.000	0.000	0.000	0.001	0.000	0.001	0.000	0.000	0.008	0.000	0.002	0.002	0.001	0.000	0.067	0.001	0.000	0.002	0.000	0.023	0.000	0.001	0.001	0.000	0.000	0.001	0.005	0.001	0.001	0.005	0.003	0.000	0.001	0.000	0.001	0.002	0.000	0.001	0.001	0.007	0.000	0.004	0.004	0.051	0.006	0.000	0.000	0.000	0.003	0.012	0.000	0.001	0.000	0.000	0.008	0.001	0.002	0.000	0.000	0.005	0.000	0.002	0.016
G[C>A]T	0.000	0.000	0.010	0.021	0.007	0.001	0.000	0.000	0.000	0.000	0.027	0.009	0.011	0.003	0.012	0.010	0.000	0.000	0.000	0.126	0.018	0.006	0.000	0.001	0.062	0.000	0.002	0.009	0.001	0.000	0.054	0.010	0.001	0.007	0.000	0.084	0.001	0.004	0.003	0.003	0.000	0.023	0.069	0.001	0.009	0.006	0.011	0.002	0.018	0.001	0.011	0.007	0.002	0.001	0.000	0.006	0.002	0.003	0.004	0.001	0.007	0.001	0.016	0.002	0.000	0.498	0.000	0.010	0.001	0.008	0.001	0.003	0.007	0.000	0.001	0.007	0.003	0.011	0.064
G[C>G]A	0.000	0.000	0.013	0.005	0.005	0.000	0.000	0.000	0.000	0.000	0.003	0.001	0.000	0.000	0.000	0.000	0.000	0.001	0.001	0.000	0.001	0.001	0.001	0.000	0.001	0.001	0.001	0.000	0.001	0.000	0.003	0.004	0.000	0.001	0.000	0.002	0.001	0.002	0.001	0.001	0.000	0.003	0.001	0.001	0.001	0.024	0.005	0.002	0.002	0.014	0.000	0.000	0.000	0.002	0.000	0.000	0.027	0.003	0.001	0.001	0.002	0.001	0.000	0.004	0.002	0.001	0.000	0.011	0.002	0.006	0.003	0.000	0.007	0.000	0.000	0.002	0.004	0.003	0.003
G[C>G]C	0.000	0.000	0.009	0.006	0.005	0.001	0.000	0.000	0.000	0.000	0.002	0.003	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.001	0.003	0.001	0.000	0.001	0.001	0.003	0.001	0.001	0.000	0.006	0.002	0.001	0.001	0.000	0.002	0.000	0.007	0.001	0.001	0.000	0.021	0.001	0.001	0.001	0.021	0.005	0.003	0.001	0.015	0.001	0.001	0.001	0.002	0.000	0.000	0.033	0.001	0.001	0.084	0.000	0.001	0.000	0.003	0.001	0.002	0.000	0.016	0.005	0.108	0.001	0.002	0.005	0.000	0.000	0.002	0.005	0.007	0.002
G[C>G]G	0.000	0.000	0.002	0.003	0.002	0.000	0.000	0.000	0.000	0.001	0.000	0.001	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.001	0.001	0.001	0.000	0.000	0.000	0.003	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.001	0.000	0.001	0.001	0.010	0.001	0.000	0.000	0.013	0.000	0.001	0.000	0.000	0.000	0.000	0.003	0.000	0.001	0.003	0.002	0.006	0.000	0.002	0.002	0.000	0.000	0.002	0.000	0.001	0.005	0.001	0.002	0.000	0.000	0.001	0.001	0.001	0.001
G[C>G]T	0.000	0.000	0.015	0.003	0.007	0.000	0.000	0.000	0.001	0.001	0.005	0.009	0.000	0.000	0.000	0.000	0.000	0.001	0.003	0.000	0.000	0.003	0.001	0.000	0.001	0.000	0.003	0.000	0.000	0.000	0.003	0.002	0.000	0.005	0.000	0.003	0.000	0.003	0.000	0.001	0.000	0.009	0.001	0.001	0.001	0.032	0.007	0.004	0.003	0.018	0.002	0.001	0.001	0.002	0.000	0.000	0.014	0.003	0.001	0.001	0.005	0.000	0.000	0.000	0.001	0.002	0.000	0.076	0.003	0.012	0.004	0.007	0.006	0.000	0.000	0.002	0.006	0.004	0.002
G[C>T]A	0.004	0.000	0.016	0.004	0.019	0.093	0.000	0.001	0.000	0.000	0.000	0.007	0.000	0.000	0.004	0.001	0.020	0.004	0.007	0.017	0.074	0.007	0.002	0.000	0.010	0.061	0.042	0.009	0.002	0.054	0.030	0.010	0.003	0.014	0.003	0.010	0.050	0.007	0.023	0.005	0.000	0.007	0.004	0.003	0.002	0.008	0.014	0.006	0.034	0.001	0.096	0.001	0.013	0.001	0.000	0.000	0.002	0.004	0.002	0.009	0.000	0.006	0.000	0.005	0.007	0.002	0.001	0.090	0.001	0.005	0.002	0.001	0.007	0.000	0.011	0.013	0.006	0.006	0.016
G[C>T]C	0.000	0.000	0.013	0.004	0.021	0.109	0.012	0.026	0.000	0.000	0.000	0.006	0.012	0.015	0.009	0.005	0.114	0.002	0.001	0.008	0.127	0.005	0.000	0.000	0.008	0.039	0.053	0.019	0.002	0.153	0.043	0.007	0.005	0.020	0.000	0.008	0.060	0.011	0.037	0.005	0.000	0.018	0.003	0.002	0.003	0.004	0.011	0.009	0.151	0.000	0.080	0.001	0.004	0.002	0.000	0.000	0.002	0.003	0.001	0.012	0.013	0.001	0.000	0.007	0.003	0.002	0.001	0.111	0.000	0.003	0.003	0.000	0.008	0.000	0.000	0.013	0.005	0.017	0.032
G[C>T]G	0.216	0.000	0.001	0.000	0.012	0.179	0.000	0.000	0.000	0.000	0.002	0.005	0.015	0.013	0.009	0.002	0.002	0.000	0.000	0.000	0.279	0.001	0.002	0.001	0.015	0.003	0.021	0.000	0.000	0.012	0.026	0.011	0.007	0.009	0.000	0.018	0.005	0.002	0.010	0.006	0.017	0.003	0.006	0.004	0.005	0.006	0.000	0.017	0.005	0.000	0.050	0.002	0.002	0.002	0.000	0.001	0.006	0.004	0.003	0.006	0.019	0.012	0.002	0.008	0.007	0.000	0.003	0.009	0.000	0.008	0.018	0.006	0.002	0.000	0.001	0.006	0.009	0.017	0.008
G[C>T]T	0.000	0.000	0.011	0.004	0.019	0.074	0.016	0.005	0.001	0.042	0.005	0.011	0.013	0.031	0.010	0.002	0.070	0.003	0.001	0.010	0.077	0.007	0.000	0.000	0.007	0.109	0.033	0.008	0.002	0.107	0.020	0.010	0.005	0.008	0.000	0.005	0.018	0.006	0.053	0.004	0.001	0.006	0.004	0.002	0.003	0.009	0.013	0.008	0.087	0.002	0.066	0.002	0.004	0.004	0.000	0.000	0.016	0.006	0.001	0.003	0.000	0.005	0.001	0.006	0.005	0.003	0.001	0.180	0.004	0.007	0.001	0.003	0.009	0.001	0.000	0.017	0.004	0.008	0.010
G[T>A]A	0.000	0.000	0.007	0.009	0.004	0.000	0.000	0.001	0.002	0.000	0.013	0.003	0.000	0.000	0.002	0.002	0.000	0.001	0.000	0.000	0.001	0.001	0.000	0.000	0.002	0.000	0.000	0.001	0.053	0.000	0.002	0.036	0.001	0.109	0.000	0.003	0.000	0.002	0.009	0.001	0.019	0.001	0.001	0.001	0.001	0.005	0.003	0.002	0.012	0.006	0.001	0.000	0.006	0.016	0.000	0.000	0.001	0.020	0.001	0.004	0.002	0.002	0.000	0.003	0.002	0.000	0.000	0.001	0.017	0.001	0.009	0.000	0.006	0.039	0.000	0.006	0.006	0.006	0.005
G[T>A]C	0.000	0.000	0.007	0.004	0.003	0.000	0.000	0.000	0.002	0.000	0.013	0.002	0.000	0.000	0.007	0.003	0.000	0.002	0.000	0.003	0.001	0.001	0.003	0.000	0.002	0.000	0.003	0.003	0.010	0.000	0.002	0.006	0.001	0.000	0.000	0.001	0.000	0.003	0.008	0.000	0.002	0.006	0.001	0.001	0.001	0.004	0.003	0.002	0.002	0.001	0.002	0.001	0.001	0.000	0.000	0.000	0.001	0.004	0.000	0.003	0.009	0.000	0.002	0.001	0.001	0.002	0.000	0.000	0.001	0.000	0.003	0.005	0.003	0.000	0.000	0.002	0.004	0.001	0.004
G[T>A]G	0.000	0.000	0.012	0.013	0.004	0.000	0.000	0.001	0.000	0.001	0.016	0.001	0.000	0.000	0.002	0.001	0.000	0.003	0.000	0.000	0.000	0.006	0.001	0.000	0.002	0.000	0.002	0.001	0.038	0.000	0.003	0.038	0.001	0.002	0.000	0.004	0.000	0.006	0.013	0.001	0.007	0.001	0.001	0.001	0.001	0.007	0.004	0.001	0.008	0.005	0.002	0.001	0.002	0.000	0.000	0.000	0.000	0.004	0.001	0.004	0.003	0.013	0.000	0.002	0.020	0.001	0.000	0.000	0.004	0.003	0.007	0.003	0.009	0.000	0.000	0.006	0.004	0.002	0.006
G[T>A]T	0.000	0.000	0.012	0.006	0.003	0.000	0.000	0.000	0.021	0.009	0.028	0.003	0.000	0.000	0.013	0.007	0.000	0.001	0.000	0.001	0.001	0.001	0.005	0.002	0.004	0.000	0.004	0.001	0.009	0.000	0.001	0.020	0.001	0.000	0.000	0.001	0.000	0.001	0.006	0.002	0.008	0.003	0.001	0.002	0.002	0.004	0.006	0.005	0.003	0.001	0.003	0.001	0.001	0.000	0.000	0.000	0.001	0.000	0.001	0.003	0.002	0.005	0.005	0.008	0.003	0.002	0.001	0.000	0.012	0.003	0.007	0.015	0.004	0.017	0.001	0.004	0.005	0.007	0.003
G[T>C]A	0.001	0.000	0.010	0.003	0.014	0.003	0.000	0.000	0.000	0.001	0.003	0.009	0.002	0.014	0.013	0.002	0.001	0.071	0.000	0.011	0.013	0.002	0.000	0.000	0.001	0.001	0.003	0.221	0.001	0.000	0.004	0.017	0.083	0.005	0.000	0.001	0.001	0.005	0.009	0.012	0.000	0.001	0.002	0.030	0.002	0.007	0.006	0.006	0.006	0.000	0.027	0.001	0.022	0.000	0.000	0.000	0.001	0.001	0.002	0.006	0.011	0.003	0.001	0.004	0.002	0.001	0.001	0.000	0.021	0.004	0.003	0.000	0.005	0.003	0.000	0.029	0.011	0.004	0.003
G[T>C]C	0.002	0.000	0.006	0.001	0.008	0.001	0.000	0.000	0.000	0.002	0.003	0.010	0.006	0.005	0.006	0.003	0.001	0.030	0.000	0.007	0.012	0.001	0.015	0.001	0.002	0.000	0.002	0.066	0.001	0.000	0.004	0.004	0.025	0.002	0.001	0.004	0.001	0.007	0.008	0.008	0.001	0.002	0.001	0.018	0.002	0.007	0.005	0.004	0.003	0.022	0.001	0.001	0.057	0.001	0.000	0.000	0.005	0.002	0.002	0.007	0.004	0.003	0.000	0.002	0.001	0.003	0.001	0.001	0.012	0.007	0.004	0.003	0.002	0.000	0.000	0.003	0.003	0.001	0.005
G[T>C]G	0.001	0.000	0.011	0.002	0.015	0.002	0.000	0.001	0.000	0.003	0.003	0.006	0.000	0.001	0.013	0.004	0.001	0.042	0.000	0.011	0.006	0.004	0.004	0.001	0.004	0.001	0.004	0.117	0.001	0.000	0.003	0.013	0.071	0.001	0.001	0.003	0.001	0.005	0.010	0.207	0.000	0.000	0.002	0.010	0.002	0.006	0.006	0.004	0.003	0.001	0.017	0.001	0.035	0.003	0.000	0.000	0.004	0.005	0.001	0.003	0.121	0.006	0.002	0.005	0.011	0.001	0.001	0.000	0.006	0.003	0.004	0.001	0.007	0.000	0.000	0.009	0.000	0.001	0.007
G[T>C]T	0.000	0.000	0.010	0.001	0.014	0.001	0.001	0.007	0.007	0.321	0.006	0.010	0.005	0.012	0.011	0.002	0.000	0.047	0.000	0.011	0.006	0.002	0.005	0.001	0.003	0.002	0.001	0.135	0.001	0.001	0.004	0.014	0.056	0.004	0.001	0.002	0.000	0.005	0.013	0.010	0.000	0.000	0.003	0.040	0.003	0.008	0.007	0.004	0.002	0.001	0.011	0.001	0.061	0.003	0.000	0.000	0.001	0.004	0.003	0.007	0.005	0.013	0.003	0.005	0.002	0.001	0.001	0.004	0.055	0.009	0.007	0.013	0.004	0.001	0.000	0.013	0.006	0.011	0.003
G[T>G]A	0.000	0.000	0.004	0.001	0.003	0.000	0.000	0.000	0.002	0.000	0.001	0.008	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.001	0.000	0.000	0.001	0.001	0.000	0.000	0.002	0.000	0.001	0.006	0.001	0.000	0.001	0.000	0.000	0.000	0.001	0.001	0.000	0.002	0.000	0.012	0.001	0.003	0.002	0.002	0.001	0.061	0.000	0.000	0.000	0.022	0.000	0.000	0.009	0.002	0.001	0.004	0.000	0.006	0.000	0.001	0.008	0.001	0.028	0.000	0.008	0.001	0.003	0.000	0.013	0.006	0.000	0.001	0.004	0.001	0.000
G[T>G]C	0.000	0.000	0.002	0.000	0.002	0.000	0.000	0.000	0.002	0.000	0.001	0.006	0.001	0.001	0.001	0.002	0.000	0.000	0.000	0.002	0.001	0.001	0.000	0.006	0.000	0.001	0.000	0.001	0.000	0.000	0.002	0.000	0.001	0.000	0.002	0.000	0.000	0.001	0.001	0.001	0.000	0.005	0.000	0.007	0.001	0.006	0.002	0.001	0.000	0.075	0.000	0.001	0.000	0.010	0.000	0.000	0.010	0.005	0.001	0.002	0.002	0.003	0.000	0.000	0.003	0.002	0.029	0.001	0.001	0.001	0.001	0.002	0.001	0.000	0.001	0.000	0.003	0.001	0.000
G[T>G]G	0.000	0.000	0.011	0.002	0.005	0.000	0.000	0.002	0.006	0.004	0.004	0.004	0.000	0.002	0.001	0.000	0.000	0.000	0.000	0.001	0.000	0.001	0.001	0.001	0.006	0.001	0.000	0.000	0.000	0.000	0.003	0.009	0.002	0.005	0.003	0.001	0.001	0.003	0.003	0.005	0.000	0.004	0.002	0.010	0.002	0.008	0.004	0.001	0.000	0.241	0.000	0.000	0.001	0.027	0.000	0.000	0.035	0.076	0.001	0.005	0.000	0.300	0.000	0.001	0.023	0.002	0.663	0.000	0.002	0.000	0.003	0.003	0.049	0.000	0.000	0.002	0.001	0.002	0.000
G[T>G]T	0.000	0.000	0.006	0.000	0.002	0.001	0.001	0.002	0.007	0.007	0.001	0.020	0.002	0.005	0.008	0.001	0.000	0.000	0.000	0.002	0.001	0.002	0.007	0.115	0.002	0.000	0.004	0.003	0.000	0.000	0.003	0.003	0.006	0.002	0.021	0.001	0.000	0.003	0.001	0.000	0.000	0.003	0.000	0.017	0.003	0.006	0.006	0.004	0.001	0.075	0.003	0.001	0.001	0.090	0.000	0.000	0.037	0.002	0.001	0.002	0.000	0.001	0.000	0.003	0.017	0.008	0.048	0.001	0.003	0.002	0.002	0.013	0.004	0.003	0.000	0.000	0.006	0.002	0.000
T[C>A]A	0.000	0.001	0.008	0.040	0.008	0.000	0.000	0.003	0.003	0.011	0.030	0.017	0.094	0.000	0.060	0.259	0.000	0.003	0.079	0.000	0.017	0.011	0.000	0.000	0.074	0.001	0.002	0.001	0.001	0.002	0.030	0.007	0.001	0.007	0.014	0.045	0.001	0.002	0.010	0.012	0.004	0.005	0.141	0.002	0.018	0.006	0.026	0.003	0.005	0.006	0.001	0.064	0.012	0.017	0.123	0.033	0.019	0.036	0.464	0.003	0.001	0.002	0.219	0.014	0.007	0.000	0.002	0.002	0.002	0.009	0.004	0.000	0.016	0.002	0.333	0.015	0.010	0.025	0.060
T[C>A]C	0.002	0.000	0.017	0.047	0.009	0.000	0.001	0.004	0.014	0.008	0.025	0.002	0.010	0.004	0.009	0.014	0.001	0.003	0.029	0.024	0.006	0.006	0.000	0.000	0.044	0.001	0.003	0.000	0.002	0.001	0.052	0.010	0.001	0.006	0.000	0.041	0.000	0.009	0.009	0.005	0.001	0.023	0.077	0.002	0.012	0.008	0.021	0.002	0.054	0.000	0.002	0.065	0.007	0.000	0.004	0.005	0.001	0.003	0.047	0.004	0.000	0.000	0.013	0.001	0.000	0.020	0.000	0.000	0.000	0.004	0.004	0.002	0.017	0.001	0.045	0.013	0.004	0.020	0.042
T[C>A]G	0.000	0.000	0.001	0.011	0.002	0.000	0.000	0.000	0.000	0.000	0.002	0.000	0.001	0.001	0.003	0.008	0.000	0.001	0.012	0.000	0.000	0.002	0.000	0.000	0.013	0.000	0.000	0.000	0.000	0.000	0.010	0.000	0.000	0.001	0.000	0.018	0.000	0.001	0.002	0.001	0.000	0.000	0.015	0.001	0.002	0.003	0.003	0.000	0.001	0.000	0.000	0.019	0.003	0.000	0.713	0.041	0.000	0.005	0.136	0.000	0.001	0.000	0.006	0.000	0.003	0.001	0.000	0.000	0.000	0.001	0.003	0.001	0.002	0.000	0.322	0.002	0.000	0.003	0.012
T[C>A]T	0.000	0.000	0.010	0.042	0.013	0.001	0.001	0.001	0.004	0.000	0.032	0.028	0.671	0.081	0.190	0.344	0.000	0.002	0.036	0.152	0.008	0.009	0.000	0.003	0.123	0.001	0.001	0.000	0.000	0.000	0.023	0.025	0.001	0.010	0.001	0.041	0.001	0.005	0.010	0.013	0.000	0.015	0.209	0.002	0.028	0.007	0.024	0.006	0.028	0.000	0.017	0.063	0.003	0.000	0.000	0.009	0.002	0.000	0.061	0.001	0.007	0.004	0.404	0.006	0.015	0.004	0.000	0.001	0.002	0.009	0.004	0.010	0.019	0.001	0.077	0.013	0.009	0.008	0.057
T[C>G]A	0.000	0.000	0.013	0.004	0.008	0.000	0.000	0.000	0.002	0.004	0.001	0.003	0.000	0.000	0.000	0.000	0.000	0.001	0.316	0.000	0.000	0.003	0.004	0.000	0.002	0.002	0.000	0.001	0.004	0.000	0.004	0.010	0.001	0.003	0.000	0.000	0.000	0.000	0.000	0.013	0.000	0.007	0.004	0.002	0.003	0.035	0.013	0.020	0.001	0.000	0.001	0.003	0.003	0.000	0.000	0.000	0.001	0.002	0.002	0.004	0.005	0.007	0.001	0.002	0.002	0.000	0.001	0.000	0.001	0.042	0.001	0.000	0.023	0.001	0.000	0.020	0.013	0.006	0.005
T[C>G]C	0.000	0.000	0.020	0.006	0.008	0.000	0.000	0.002	0.005	0.001	0.001	0.002	0.000	0.000	0.000	0.000	0.000	0.002	0.063	0.000	0.000	0.005	0.001	0.000	0.005	0.003	0.000	0.001	0.002	0.000	0.006	0.003	0.001	0.003	0.000	0.007	0.001	0.003	0.001	0.003	0.000	0.006	0.002	0.001	0.002	0.037	0.014	0.014	0.002	0.001	0.000	0.002	0.002	0.001	0.000	0.000	0.000	0.004	0.001	0.005	0.001	0.003	0.000	0.003	0.002	0.002	0.000	0.000	0.002	0.048	0.003	0.000	0.017	0.000	0.000	0.004	0.007	0.005	0.004
T[C>G]G	0.000	0.000	0.001	0.002	0.002	0.000	0.000	0.000	0.000	0.001	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.014	0.000	0.000	0.000	0.000	0.000	0.001	0.000	0.000	0.000	0.000	0.000	0.001	0.002	0.000	0.000	0.000	0.001	0.000	0.000	0.001	0.000	0.000	0.000	0.000	0.001	0.001	0.010	0.002	0.002	0.000	0.001	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.002	0.001	0.002	0.001	0.000	0.000	0.001	0.001	0.000	0.000	0.000	0.000	0.005	0.026	0.001	0.004	0.000	0.000	0.000	0.001	0.001	0.001
T[C>G]T	0.002	0.001	0.024	0.003	0.013	0.000	0.000	0.001	0.012	0.000	0.000	0.006	0.000	0.000	0.000	0.000	0.000	0.003	0.370	0.000	0.000	0.029	0.000	0.002	0.003	0.002	0.001	0.001	0.000	0.000	0.004	0.017	0.002	0.002	0.000	0.000	0.000	0.004	0.001	0.020	0.002	0.021	0.008	0.003	0.004	0.051	0.024	0.045	0.000	0.000	0.003	0.003	0.005	0.001	0.000	0.001	0.000	0.003	0.002	0.004	0.000	0.002	0.000	0.013	0.018	0.002	0.001	0.005	0.006	0.112	0.013	0.004	0.026	0.001	0.000	0.023	0.026	0.013	0.004
T[C>T]A	0.001	0.535	0.006	0.004	0.020	0.012	0.239	0.007	0.017	0.000	0.005	0.004	0.000	0.003	0.002	0.001	0.010	0.006	0.007	0.004	0.027	0.006	0.001	0.000	0.011	0.044	0.008	0.004	0.002	0.015	0.002	0.011	0.003	0.019	0.005	0.006	0.099	0.008	0.025	0.015	0.002	0.003	0.011	0.004	0.007	0.004	0.030	0.012	0.005	0.000	0.001	0.002	0.019	0.007	0.000	0.000	0.019	0.002	0.001	0.006	0.000	0.000	0.000	0.008	0.004	0.000	0.000	0.000	0.004	0.009	0.000	0.000	0.010	0.002	0.036	0.017	0.009	0.010	0.009
T[C>T]C	0.000	0.097	0.013	0.007	0.023	0.010	0.331	0.212	0.014	0.006	0.000	0.007	0.003	0.048	0.009	0.004	0.159	0.003	0.004	0.000	0.010	0.004	0.000	0.001	0.011	0.025	0.009	0.000	0.001	0.044	0.012	0.006	0.003	0.008	0.004	0.000	0.135	0.025	0.039	0.003	0.000	0.013	0.007	0.003	0.007	0.005	0.023	0.006	0.033	0.000	0.002	0.002	0.013	0.037	0.000	0.001	0.104	0.002	0.002	0.006	0.001	0.004	0.000	0.017	0.015	0.000	0.001	0.027	0.003	0.008	0.006	0.000	0.011	0.001	0.000	0.017	0.008	0.020	0.010
T[C>T]G	0.110	0.045	0.001	0.000	0.016	0.053	0.075	0.059	0.000	0.002	0.000	0.003	0.003	0.441	0.009	0.005	0.000	0.000	0.001	0.005	0.022	0.003	0.000	0.000	0.012	0.004	0.003	0.000	0.000	0.003	0.003	0.014	0.003	0.005	0.002	0.015	0.015	0.009	0.010	0.004	0.000	0.001	0.004	0.003	0.004	0.005	0.002	0.005	0.000	0.000	0.000	0.002	0.004	0.002	0.000	0.001	0.004	0.007	0.002	0.010	0.000	0.005	0.000	0.005	0.003	0.000	0.002	0.000	0.000	0.014	0.060	0.009	0.002	0.000	0.002	0.004	0.010	0.005	0.003
T[C>T]T	0.000	0.301	0.008	0.007	0.021	0.007	0.108	0.103	0.002	0.050	0.005	0.006	0.023	0.128	0.013	0.008	0.121	0.006	0.001	0.000	0.002	0.022	0.000	0.001	0.004	0.064	0.000	0.000	0.000	0.027	0.005	0.012	0.002	0.012	0.002	0.004	0.053	0.020	0.068	0.009	0.001	0.008	0.008	0.006	0.007	0.004	0.023	0.012	0.015	0.001	0.003	0.003	0.004	0.082	0.000	0.000	0.082	0.043	0.002	0.008	0.000	0.032	0.000	0.037	0.180	0.002	0.004	0.017	0.039	0.013	0.011	0.000	0.010	0.003	0.000	0.025	0.011	0.013	0.004
T[T>A]A	0.007	0.000	0.006	0.009	0.006	0.000	0.000	0.003	0.015	0.016	0.011	0.026	0.002	0.003	0.010	0.009	0.000	0.004	0.000	0.000	0.001	0.012	0.000	0.001	0.011	0.000	0.012	0.000	0.071	0.000	0.004	0.021	0.001	0.052	0.000	0.001	0.000	0.007	0.001	0.004	0.270	0.004	0.002	0.002	0.002	0.007	0.007	0.080	0.008	0.000	0.007	0.000	0.004	0.154	0.000	0.000	0.002	0.020	0.001	0.003	0.002	0.004	0.009	0.003	0.012	0.002	0.001	0.007	0.059	0.004	0.008	0.021	0.007	0.354	0.002	0.008	0.020	0.010	0.004
T[T>A]C	0.000	0.000	0.011	0.004	0.004	0.000	0.000	0.002	0.035	0.001	0.020	0.002	0.000	0.001	0.003	0.001	0.000	0.007	0.000	0.002	0.000	0.005	0.006	0.000	0.003	0.000	0.000	0.001	0.024	0.000	0.003	0.003	0.001	0.000	0.001	0.002	0.000	0.004	0.002	0.000	0.000	0.005	0.001	0.001	0.001	0.004	0.010	0.015	0.002	0.001	0.000	0.000	0.004	0.000	0.000	0.000	0.001	0.003	0.000	0.004	0.000	0.000	0.001	0.009	0.001	0.002	0.000	0.001	0.008	0.002	0.004	0.000	0.003	0.001	0.000	0.003	0.011	0.004	0.003
T[T>A]G	0.000	0.000	0.007	0.010	0.005	0.000	0.000	0.002	0.012	0.006	0.013	0.017	0.001	0.000	0.001	0.004	0.000	0.003	0.000	0.000	0.000	0.005	0.002	0.000	0.002	0.000	0.000	0.000	0.074	0.000	0.002	0.010	0.000	0.000	0.000	0.001	0.000	0.003	0.002	0.001	0.008	0.001	0.001	0.001	0.001	0.005	0.005	0.009	0.005	0.000	0.000	0.001	0.001	0.000	0.000	0.000	0.000	0.004	0.001	0.002	0.000	0.010	0.006	0.004	0.001	0.000	0.000	0.001	0.007	0.001	0.005	0.004	0.010	0.001	0.001	0.007	0.010	0.004	0.004
T[T>A]T	0.002	0.000	0.013	0.006	0.009	0.000	0.001	0.000	0.279	0.027	0.038	0.017	0.005	0.002	0.005	0.007	0.001	0.005	0.000	0.002	0.000	0.007	0.002	0.001	0.007	0.001	0.007	0.000	0.030	0.000	0.002	0.015	0.001	0.002	0.006	0.001	0.001	0.009	0.001	0.003	0.046	0.005	0.003	0.002	0.003	0.011	0.021	0.054	0.004	0.000	0.014	0.001	0.003	0.004	0.000	0.000	0.003	0.006	0.001	0.005	0.000	0.002	0.001	0.029	0.015	0.003	0.001	0.007	0.063	0.002	0.005	0.075	0.006	0.164	0.001	0.006	0.031	0.017	0.004
T[T>C]A	0.000	0.000	0.015	0.002	0.018	0.001	0.002	0.006	0.030	0.080	0.002	0.014	0.001	0.009	0.017	0.006	0.000	0.085	0.000	0.006	0.003	0.013	0.002	0.001	0.002	0.001	0.014	0.083	0.005	0.000	0.001	0.014	0.042	0.002	0.001	0.003	0.001	0.005	0.001	0.011	0.001	0.000	0.003	0.047	0.002	0.006	0.010	0.063	0.007	0.000	0.020	0.001	0.009	0.001	0.001	0.000	0.001	0.004	0.003	0.007	0.005	0.005	0.001	0.005	0.003	0.002	0.000	0.000	0.013	0.002	0.010	0.004	0.007	0.010	0.000	0.034	0.093	0.009	0.003
T[T>C]C	0.003	0.000	0.009	0.001	0.010	0.001	0.001	0.002	0.021	0.015	0.003	0.014	0.005	0.000	0.017	0.007	0.000	0.047	0.000	0.003	0.006	0.004	0.016	0.001	0.006	0.001	0.017	0.056	0.000	0.000	0.003	0.008	0.032	0.004	0.002	0.002	0.000	0.011	0.001	0.013	0.006	0.001	0.002	0.046	0.002	0.007	0.008	0.027	0.003	0.036	0.011	0.001	0.055	0.018	0.000	0.000	0.029	0.004	0.002	0.005	0.008	0.001	0.001	0.027	0.020	0.002	0.001	0.001	0.024	0.002	0.007	0.002	0.003	0.000	0.000	0.004	0.014	0.005	0.005
T[T>C]G	0.000	0.000	0.007	0.002	0.013	0.002	0.000	0.004	0.013	0.023	0.000	0.009	0.000	0.001	0.015	0.005	0.000	0.040	0.000	0.004	0.001	0.007	0.002	0.001	0.003	0.001	0.012	0.028	0.001	0.000	0.002	0.005	0.030	0.001	0.001	0.000	0.000	0.004	0.002	0.321	0.001	0.000	0.001	0.010	0.002	0.005	0.006	0.021	0.003	0.000	0.019	0.001	0.024	0.002	0.000	0.000	0.001	0.002	0.001	0.005	0.089	0.004	0.002	0.003	0.002	0.001	0.000	0.000	0.004	0.002	0.002	0.000	0.009	0.000	0.000	0.013	0.011	0.002	0.007
T[T>C]T	0.004	0.000	0.014	0.001	0.019	0.002	0.001	0.003	0.088	0.043	0.005	0.039	0.003	0.000	0.020	0.008	0.000	0.060	0.001	0.003	0.001	0.008	0.021	0.001	0.003	0.001	0.019	0.041	0.000	0.000	0.004	0.017	0.058	0.002	0.002	0.001	0.000	0.012	0.001	0.009	0.007	0.001	0.003	0.062	0.003	0.010	0.010	0.046	0.002	0.002	0.037	0.001	0.037	0.012	0.001	0.000	0.005	0.021	0.002	0.002	0.013	0.001	0.000	0.195	0.032	0.003	0.001	0.010	0.094	0.003	0.007	0.097	0.007	0.004	0.001	0.014	0.014	0.017	0.006
T[T>G]A	0.000	0.000	0.007	0.000	0.005	0.000	0.000	0.001	0.019	0.000	0.000	0.065	0.003	0.011	0.009	0.020	0.000	0.002	0.000	0.000	0.000	0.010	0.000	0.000	0.001	0.002	0.000	0.000	0.005	0.000	0.000	0.002	0.001	0.001	0.042	0.000	0.000	0.002	0.000	0.000	0.000	0.004	0.001	0.018	0.001	0.008	0.008	0.041	0.001	0.000	0.000	0.000	0.001	0.002	0.000	0.000	0.000	0.003	0.001	0.001	0.000	0.006	0.009	0.003	0.001	0.002	0.002	0.002	0.007	0.007	0.008	0.030	0.007	0.018	0.006	0.002	0.053	0.004	0.000
T[T>G]C	0.000	0.000	0.006	0.000	0.007	0.000	0.000	0.002	0.018	0.000	0.001	0.009	0.000	0.006	0.001	0.001	0.000	0.001	0.000	0.001	0.000	0.006	0.000	0.009	0.002	0.002	0.000	0.000	0.001	0.000	0.003	0.001	0.002	0.000	0.036	0.000	0.000	0.002	0.000	0.000	0.000	0.002	0.001	0.020	0.001	0.005	0.012	0.016	0.000	0.008	0.000	0.000	0.001	0.000	0.000	0.000	0.001	0.004	0.001	0.004	0.001	0.006	0.000	0.004	0.002	0.002	0.011	0.001	0.007	0.002	0.003	0.021	0.003	0.000	0.000	0.000	0.014	0.001	0.000
T[T>G]G	0.001	0.000	0.008	0.002	0.007	0.000	0.000	0.001	0.008	0.000	0.003	0.011	0.000	0.003	0.001	0.003	0.000	0.001	0.000	0.000	0.000	0.005	0.001	0.005	0.001	0.002	0.003	0.000	0.006	0.000	0.003	0.009	0.003	0.002	0.014	0.000	0.001	0.005	0.001	0.001	0.002	0.005	0.001	0.030	0.002	0.007	0.009	0.020	0.003	0.023	0.001	0.001	0.001	0.002	0.000	0.000	0.009	0.003	0.001	0.006	0.008	0.080	0.000	0.005	0.007	0.002	0.024	0.000	0.003	0.002	0.002	0.000	0.005	0.000	0.001	0.002	0.013	0.003	0.000
T[T>G]T	0.000	0.000	0.011	0.001	0.014	0.001	0.000	0.002	0.022	0.000	0.001	0.064	0.019	0.090	0.025	0.015	0.000	0.002	0.000	0.001	0.006	0.016	0.005	0.122	0.005	0.004	0.002	0.000	0.001	0.000	0.004	0.008	0.009	0.000	0.479	0.001	0.009	0.007	0.001	0.002	0.000	0.004	0.002	0.029	0.005	0.011	0.025	0.088	0.002	0.001	0.009	0.001	0.001	0.004	0.000	0.000	0.002	0.003	0.003	0.007	0.001	0.041	0.029	0.119	0.022	0.000	0.032	0.007	0.013	0.005	0.006	0.102	0.006	0.008	0.001	0.002	0.055	0.003	0.000

In order to convert a matrix of signature definitions such as this matrix to a pairwise cosine similarity matrix, we recommend using the cossim function in the sigvar R package. Below we provide example code to both generate a similarity matrix from a matrix of mutational signature definitions and verify that its entries are between 0 and 1, and that its diagonal elements equal 1.

# Exclude the first column, "Type", which defines each  mutational type
cosmic_sbs_sim = sigvar::cossim(as.matrix(sigvar::COSMIC3.3.1_SBS[,-1]))

# View the first 10 rows and 5 columns of the similarity matrix
cosmic_sbs_sim[1:10, 1:5]
#>             SBS1       SBS2       SBS3       SBS4      SBS5
#> SBS1  1.00000000 0.02031489 0.05372626 0.02361321 0.1942669
#> SBS2  0.02031489 1.00000000 0.09833748 0.04469572 0.2526434
#> SBS3  0.05372626 0.09833748 1.00000000 0.63777474 0.7928402
#> SBS4  0.02361321 0.04469572 0.63777474 1.00000000 0.3801633
#> SBS5  0.19426687 0.25264337 0.79284023 0.38016329 1.0000000
#> SBS6  0.77021158 0.06403593 0.21684573 0.08186122 0.4138381
#> SBS7a 0.04804510 0.71515949 0.21073504 0.08209717 0.3603178
#> SBS7b 0.06974539 0.26959035 0.34612947 0.14372094 0.4300020
#> SBS7c 0.02369922 0.05861210 0.32534537 0.12121403 0.2550711
#> SBS7d 0.01457751 0.07276100 0.27448994 0.08036521 0.3331761

# Confirm that the diagonal elements of the matrix are all equal to 1
all(diag(cosmic_sbs_sim) == 1)
#> [1] TRUE

# Confirm that all elements are non-negative
all(cosmic_sbs_sim >= 0)
#> [1] TRUE

# Confirm that no elements exceed 1
all(cosmic_sbs_sim <= 1)
#> [1] TRUE

When including a cosine similarity matrix in sigvar or sigboot, it is important that the ordering of signatures in the similarity matrix matches the ordering in your signature activity matrix. You should reorder the rows and columns of your similarity matrix to match your activity matrix:

# Suppose this is the list of signatures included 
# in your relative activity matrix:
sigs = c("SBS95", "SBS10a", "SBS5", "SBS1", "SBS17b")

# Re-order the rows and columns of the similarity matrix
sim_reordered = cosmic_sbs_sim[sigs, sigs]

sim_reordered
#>             SBS95     SBS10a       SBS5       SBS1     SBS17b
#> SBS95  1.00000000 0.38204509 0.44330584 0.06556141 0.01485958
#> SBS10a 0.38204509 1.00000000 0.13864361 0.01788389 0.01455829
#> SBS5   0.44330584 0.13864361 1.00000000 0.19426687 0.09887873
#> SBS1   0.06556141 0.01788389 0.19426687 1.00000000 0.00211760
#> SBS17b 0.01485958 0.01455829 0.09887873 0.00211760 1.00000000

3 Example analysis

library(sigvar)
library(dplyr)
library(tidyr)
library(ggplot2)
library(patchwork)

3.1 Example data

In this example, we apply signature variability analysis (SVA) to data from Moody et al. (2021). This data set contains 552 esophageal squamous cell carcinoma (ESCC) samples collected across eight countries which vary dramatically in their incidence of ESCC. Moody et al. (2021) reported the activities of 43 SBS, DBS, and ID mutational signatures for each sample.

The example data set, ESCC_sig_activity, has 552 rows, each corresponding to one sample, and 46 columns:

• Country: the country in which each sample was collected

• Incidence_Level: the ESCC incidence level of the country where the sample was collected

• Sample: the identifier for each sample

• SBS1, SBS2, …, ID17: the relative activities of the 43 mutational signatures reported by Moody et al. 

The rightmost 43 entries of each row sum to 1, since these entries correspond to the relative activities of all mutational signatures.

You can explore the structure of this data using the following functions:

# open the data set in a new window
View(ESCC_sig_activity)

# view the structure of the data set
str(ESCC_sig_activity)

Here are the first 40 rows and the first 20 columns of the relative abundance matrix, ESCC_sig_activity:

Country	Incidence_Level	Sample	SBS1	SBS2	SBS3	SBS4	SBS5	SBS8	SBS10a	SBS10b	SBS12	SBS13	SBS14	SBS15	SBS16	SBS17a	SBS17b	SBS18	SBS20
Kenya	High	PD37727a	0.1479571	0.0000000	0	0	0	0.0000000	0	0	0	0.0000000	0	0.0000000	0.0000000	0.1267247	0.2507703	0.3060281	0
Kenya	High	PD37728a	0.1563596	0.2072368	0	0	0	0.0000000	0	0	0	0.2342105	0	0.0000000	0.0000000	0.0000000	0.0000000	0.3085526	0
Kenya	High	PD37729a	0.0000000	0.1957034	0	0	0	0.0000000	0	0	0	0.2642043	0	0.0000000	0.0000000	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD37730a	0.1645137	0.2344225	0	0	0	0.0000000	0	0	0	0.2621581	0	0.0000000	0.0000000	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD37731a	0.0000000	0.3172216	0	0	0	0.0000000	0	0	0	0.6565411	0	0.0000000	0.0000000	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD37733a	0.1344803	0.2122607	0	0	0	0.0000000	0	0	0	0.3423795	0	0.0000000	0.0000000	0.0000000	0.1097650	0.0000000	0
Kenya	High	PD37734a	0.1736811	0.1416716	0	0	0	0.0000000	0	0	0	0.1926497	0	0.0000000	0.0000000	0.0000000	0.0000000	0.4078245	0
Kenya	High	PD37735a	0.1995916	0.2003573	0	0	0	0.0000000	0	0	0	0.2197550	0	0.0000000	0.0000000	0.0000000	0.0000000	0.2942828	0
Kenya	High	PD37737a	0.1001535	0.0934382	0	0	0	0.0000000	0	0	0	0.2927859	0	0.0000000	0.0000000	0.0000000	0.0000000	0.4173062	0
Kenya	High	PD37738a	0.2138171	0.0000000	0	0	0	0.0000000	0	0	0	0.1813971	0	0.0000000	0.0000000	0.0000000	0.0000000	0.4971054	0
Kenya	High	PD37739a	0.3500224	0.0000000	0	0	0	0.0000000	0	0	0	0.0000000	0	0.0000000	0.0000000	0.0000000	0.0000000	0.4541797	0
Kenya	High	PD37740a	0.0772918	0.4086627	0	0	0	0.0000000	0	0	0	0.4172414	0	0.0000000	0.0000000	0.0000000	0.0530698	0.0000000	0
Kenya	High	PD37741a	0.0926210	0.2165535	0	0	0	0.0000000	0	0	0	0.3831837	0	0.0000000	0.0000000	0.0000000	0.0000000	0.2550909	0
Kenya	High	PD37742a	0.1912434	0.1464098	0	0	0	0.0000000	0	0	0	0.1653240	0	0.0000000	0.0000000	0.0000000	0.0914186	0.3264448	0
Kenya	High	PD37743a	0.1390852	0.2651543	0	0	0	0.0000000	0	0	0	0.5057642	0	0.0000000	0.0000000	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD37745a	0.1999146	0.1255874	0	0	0	0.0000000	0	0	0	0.1631781	0	0.0000000	0.0000000	0.0000000	0.0000000	0.3583938	0
Kenya	High	PD37746a	0.1407636	0.1658311	0	0	0	0.0000000	0	0	0	0.1912842	0	0.0000000	0.0000000	0.0000000	0.0000000	0.4330891	0
Kenya	High	PD37747a	0.0680806	0.0000000	0	0	0	0.0000000	0	0	0	0.0000000	0	0.0000000	0.0000000	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD37748a	0.2138456	0.1366437	0	0	0	0.0000000	0	0	0	0.0855382	0	0.0000000	0.0000000	0.0000000	0.0000000	0.4367524	0
Kenya	High	PD37749a	0.0872017	0.0882863	0	0	0	0.0000000	0	0	0	0.1633406	0	0.0000000	0.0000000	0.0000000	0.0379610	0.5442516	0
Kenya	High	PD37751a	0.4005771	0.0000000	0	0	0	0.0000000	0	0	0	0.1930745	0	0.0000000	0.0000000	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD37752a	0.3857291	0.0000000	0	0	0	0.0000000	0	0	0	0.0000000	0	0.0000000	0.0000000	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD37753a	0.1916347	0.2031583	0	0	0	0.0000000	0	0	0	0.2902262	0	0.0000000	0.0000000	0.0000000	0.1438327	0.0000000	0
Kenya	High	PD37754a	0.0968917	0.0458735	0	0	0	0.0000000	0	0	0	0.0370847	0	0.0000000	0.0000000	0.0000000	0.0000000	0.2006431	0
Kenya	High	PD37756a	0.1114923	0.2620926	0	0	0	0.0000000	0	0	0	0.2631218	0	0.0000000	0.1989708	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD37757a	0.2347097	0.2983545	0	0	0	0.0000000	0	0	0	0.2815896	0	0.0000000	0.0000000	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD39480a	0.1358399	0.2743077	0	0	0	0.0000000	0	0	0	0.4968668	0	0.0000000	0.0000000	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD39481a	0.1284289	0.1825436	0	0	0	0.0000000	0	0	0	0.1920200	0	0.0000000	0.0000000	0.0000000	0.0000000	0.3845387	0
Kenya	High	PD39482a	0.1815166	0.1879147	0	0	0	0.0000000	0	0	0	0.4407583	0	0.0000000	0.0000000	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD39483a	0.2300647	0.2015086	0	0	0	0.0000000	0	0	0	0.4698276	0	0.0000000	0.0000000	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD39484a	0.1351974	0.1105263	0	0	0	0.0000000	0	0	0	0.1815789	0	0.0000000	0.0000000	0.0000000	0.1296053	0.0000000	0
Kenya	High	PD39485a	0.5666401	0.0000000	0	0	0	0.0000000	0	0	0	0.0000000	0	0.0000000	0.0000000	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD39486a	0.1102564	0.1320513	0	0	0	0.0000000	0	0	0	0.3696581	0	0.0000000	0.0000000	0.0000000	0.0000000	0.3444444	0
Kenya	High	PD39487a	0.1019417	0.2097087	0	0	0	0.0000000	0	0	0	0.3742718	0	0.0000000	0.0000000	0.0000000	0.0000000	0.2791262	0
Kenya	High	PD39488a	0.2120519	0.3071447	0	0	0	0.0000000	0	0	0	0.3643529	0	0.0000000	0.0000000	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD39489a	0.2087325	0.2872144	0	0	0	0.0000000	0	0	0	0.3695652	0	0.0000000	0.0000000	0.0000000	0.0000000	0.0000000	0
Kenya	High	PD39490a	0.2713029	0.1199828	0	0	0	0.0000000	0	0	0	0.1412320	0	0.0000000	0.0000000	0.0000000	0.0000000	0.3219575	0
Kenya	High	PD39491a	0.0443322	0.1753370	0	0	0	0.1461065	0	0	0	0.3660536	0	0.0000000	0.0000000	0.0000000	0.0545699	0.0000000	0
Kenya	High	PD39492a	0.3357822	0.0000000	0	0	0	0.0000000	0	0	0	0.0920484	0	0.0000000	0.0000000	0.0000000	0.0000000	0.4624028	0
Iran	High	PD39493a	0.0000000	0.0000000	0	0	0	0.0000000	0	0	0	0.0000000	0	0.6128401	0.0000000	0.0000000	0.0000000	0.0000000	0

We also provide in the sigvar R package a pairwise similarity matrix, ESCC_sig_similarity, which contains the cosine similarity of every pair of mutational signatures in ESCC_sig_activity. Here are the first 20 rows and the first 20 columns of our example pairwise similarity matrix, ESCC_sig_similarity:

	SBS1	SBS2	SBS3	SBS4	SBS5	SBS8	SBS10a	SBS10b	SBS12	SBS13	SBS14	SBS15	SBS16	SBS17a	SBS17b	SBS18	SBS20	SBS22	SBS23	SBS25
SBS1	1.0000000	0.0201805	0.0535556	0.0237961	0.1937252	0.0403272	0.0178153	0.2261521	0.0162946	0.0066415	0.0819253	0.5802505	0.0251488	0.0070674	0.0021315	0.1222348	0.1053953	0.0115136	0.0669358	0.2220882
SBS2	0.0201805	1.0000000	0.0976350	0.0442427	0.2507040	0.0451821	0.0184561	0.2153352	0.0373945	0.0165478	0.0119417	0.0788649	0.0583269	0.0017881	0.0014449	0.0648479	0.0305619	0.0065897	0.1009755	0.1295618
SBS3	0.0535556	0.0976350	1.0000000	0.6388656	0.7931046	0.6899156	0.1231872	0.1094357	0.4705106	0.2872972	0.3065806	0.2116067	0.4007606	0.2460950	0.1101847	0.4407690	0.4064382	0.3208363	0.3903166	0.7065496
SBS4	0.0237961	0.0442427	0.6388656	1.0000000	0.3796886	0.8153682	0.2761284	0.0773892	0.1189756	0.1044626	0.5593881	0.1607519	0.1708463	0.0511886	0.0131611	0.7334002	0.5844836	0.2808917	0.1346847	0.5092766
SBS5	0.1937252	0.2507040	0.7931046	0.3796886	1.0000000	0.4458145	0.1378329	0.2584875	0.6717103	0.1568997	0.2375744	0.3226022	0.6503794	0.2047274	0.0984575	0.3559307	0.3385899	0.1979688	0.4599202	0.6605703
SBS8	0.0403272	0.0451821	0.6899156	0.8153682	0.4458145	1.0000000	0.2513271	0.0688638	0.1914769	0.0692716	0.4632958	0.1260129	0.2505716	0.1301199	0.0192262	0.6358120	0.3742737	0.4599843	0.0950848	0.6575469
SBS10a	0.0178153	0.0184561	0.1231872	0.2761284	0.1378329	0.2513271	1.0000000	0.1917503	0.0270345	0.0934545	0.4707028	0.0676983	0.0493840	0.0030611	0.0146249	0.5996969	0.0374854	0.0052843	0.0216403	0.2074047
SBS10b	0.2261521	0.2153352	0.1094357	0.0773892	0.2584875	0.0688638	0.1917503	1.0000000	0.0481274	0.0173187	0.1153053	0.1239129	0.0706129	0.0057231	0.0547215	0.1720968	0.0511988	0.0087553	0.0849631	0.1970329
SBS12	0.0162946	0.0373945	0.4705106	0.1189756	0.6717103	0.1914769	0.0270345	0.0481274	1.0000000	0.0226970	0.0982441	0.0637591	0.5661923	0.3455623	0.0195125	0.0898237	0.1596766	0.0963924	0.0372668	0.4334025
SBS13	0.0066415	0.0165478	0.2872972	0.1044626	0.1568997	0.0692716	0.0934545	0.0173187	0.0226970	1.0000000	0.0390357	0.0190529	0.0956481	0.0066428	0.0027159	0.1284134	0.0131312	0.0134314	0.0128090	0.1720028
SBS14	0.0819253	0.0119417	0.3065806	0.5593881	0.2375744	0.4632958	0.4707028	0.1153053	0.0982441	0.0390357	1.0000000	0.1572703	0.1010679	0.0206134	0.0123765	0.5477284	0.7153773	0.0187495	0.0549886	0.2760719
SBS15	0.5802505	0.0788649	0.2116067	0.1607519	0.3226022	0.1260129	0.0676983	0.1239129	0.0637591	0.0190529	0.1572703	1.0000000	0.0567792	0.0159662	0.0152175	0.2238023	0.3045854	0.0171900	0.3775605	0.2367056
SBS16	0.0251488	0.0583269	0.4007606	0.1708463	0.6503794	0.2505716	0.0493840	0.0706129	0.5661923	0.0956481	0.1010679	0.0567792	1.0000000	0.0777334	0.0296286	0.1308860	0.1058018	0.1548068	0.0775241	0.3538006
SBS17a	0.0070674	0.0017881	0.2460950	0.0511886	0.2047274	0.1301199	0.0030611	0.0057231	0.3455623	0.0066428	0.0206134	0.0159662	0.0777334	1.0000000	0.0550455	0.0282942	0.0559677	0.0766213	0.0069278	0.1920717
SBS17b	0.0021315	0.0014449	0.1101847	0.0131611	0.0984575	0.0192262	0.0146249	0.0547215	0.0195125	0.0027159	0.0123765	0.0152175	0.0296286	0.0550455	1.0000000	0.0316796	0.0236703	0.0117087	0.0017297	0.0916637
SBS18	0.1222348	0.0648479	0.4407690	0.7334002	0.3559307	0.6358120	0.5996969	0.1720968	0.0898237	0.1284134	0.5477284	0.2238023	0.1308860	0.0282942	0.0316796	1.0000000	0.2926484	0.0649940	0.1061001	0.3962052
SBS20	0.1053953	0.0305619	0.4064382	0.5844836	0.3385899	0.3742737	0.0374854	0.0511988	0.1596766	0.0131312	0.7153773	0.3045854	0.1058018	0.0559677	0.0236703	0.2926484	1.0000000	0.0433629	0.2293898	0.2880163
SBS22	0.0115136	0.0065897	0.3208363	0.2808917	0.1979688	0.4599843	0.0052843	0.0087553	0.0963924	0.0134314	0.0187495	0.0171900	0.1548068	0.0766213	0.0117087	0.0649940	0.0433629	1.0000000	0.0203528	0.7015022
SBS23	0.0669358	0.1009755	0.3903166	0.1346847	0.4599202	0.0950848	0.0216403	0.0849631	0.0372668	0.0128090	0.0549886	0.3775605	0.0775241	0.0069278	0.0017297	0.1061001	0.2293898	0.0203528	1.0000000	0.2141683
SBS25	0.2220882	0.1295618	0.7065496	0.5092766	0.6605703	0.6575469	0.2074047	0.1970329	0.4334025	0.1720028	0.2760719	0.2367056	0.3538006	0.1920717	0.0916637	0.3962052	0.2880163	0.7015022	0.2141683	1.0000000

Here is a heat map plot of the similarity matrix:

Note that:

• The diagonal elements of this matrix are all 1, since each signature is identical to itself

• The columns and rows are in the same order (i.e., column 1 corresponds to the same signature as row 1, etc.)

• The ordering of the signatures in the similarity matrix, ESCC_sig_similarity, matches the ordering in the relative abundance matrix, ESCC_sig_activity.

3.2 Visualize mutational signature activities

We can visualize these signature activities using the function plot_signature_prop, which generates a series of stacked bar plots. Each vertical bar corresponds to one sample (i.e., one row of ESCC_sig_activity).

Because ESCC_sig_activity contains metadata, we specify K=43 so that only the rightmost 43 columns are treated as signature abundances. Specifying group = "Country" creates one panel for each country, while specifying arrange = TRUE reorders signatures vertically from most to least abundant and reorders samples horizontally by the abundances of their most abundant signatures.

Because plot_signature_prop returns a ggplot2 object, it can be modified with ggplot2 functions, such as scale_color_manual and scale_fill_manual.

plot_signature_prop(relab_matrix = ESCC_sig_activity, 
              K = 43, 
              group = "Country",
              arrange = TRUE) + 
  scale_color_manual(values = all_sig_pal)  + 
  scale_fill_manual(values = all_sig_pal)

# all_sig_pal is a vector of hex color codes, each corresponding to 
# one signature used in our paper. 
# Below we show the first 6 entries as an example.
all_sig_pal[1:6]
#>     SBS40    SBS40a    SBS40b    SBS40c      SBS5      SBS1 
#> "#2874A6" "#81A1C8" "#0E4384" "#2874A6" "#FFC642" "#C0392B"

We can also visualize the country-level mean signature activities with the function plot_dots in the sigvar R package.

plot_dots(sig_activity = ESCC_sig_activity, 
          K = 43, group = "Country", 
          facet = "Incidence_Level", 
          pivot = TRUE)

3.3 Signature variability analysis

Signature variability analysis can be conducted in a single line of code:

sva = sigvar(sig_activity = ESCC_sig_activity,
             K = 43, 
             S = ESCC_sig_similarity, 
             group = "Country")

knitr::kable(sva)

Country	across_sample_heterogeneity	mean_within_sample_diversity
Kenya	0.1556304	0.6624312
Iran	0.1835807	0.6197756
Tanzania	0.1482450	0.6651016
China	0.1473591	0.6527459
Japan	0.1971177	0.6105965
Malawi	0.1717769	0.6378888
Brazil	0.1955283	0.6036918
UK	0.1765105	0.5973674

Signature variability analysis quantifies both the mean signature diversity within each sample, as well as the heterogeneity in signature activities across samples. Both variability statistics range between 0 and 1.

If we add to our SVA results a column corresponding to the incidence of each country, we are able to determine if signature heterogeneity or diversity are associated with ESCC incidence.

sva_incidence = ESCC_sig_activity %>% 
  transmute(Country = as.character(Country), 
            Incidence_Level) %>%
  distinct %>%
  right_join(sva)
  
knitr::kable(sva_incidence)

Country	Incidence_Level	across_sample_heterogeneity	mean_within_sample_diversity
Kenya	High	0.1556304	0.6624312
Iran	High	0.1835807	0.6197756
Tanzania	High	0.1482450	0.6651016
China	High	0.1473591	0.6527459
Japan	Low	0.1971177	0.6105965
Malawi	High	0.1717769	0.6378888
Brazil	Low	0.1955283	0.6036918
UK	Low	0.1765105	0.5973674

ggplot(sva_incidence,
       aes(x = mean_within_sample_diversity, 
           y = across_sample_heterogeneity, 
           color = Incidence_Level)) + 
  geom_point(size = 4) +
  ggrepel::geom_text_repel(aes(label = Country)) +
  theme_bw()

ggplot(sva_incidence %>% 
         pivot_longer(cols = c("across_sample_heterogeneity", 
                               "mean_within_sample_diversity"), 
                      names_to = "Statistic", 
                      values_to = "SVA"),
       aes(x = Incidence_Level, y = SVA)) + 
  geom_boxplot(aes(color = Incidence_Level)) +
  geom_point(aes(color = Incidence_Level), size = 4, alpha = 0.6) +
  facet_wrap(~ Statistic, scales = "free_y") + 
  ggpubr::stat_compare_means(method = "t.test") +
  theme_bw() 

We see that high-ESCC-incidence countries have more within-sample signature diversity and less across-sample heterogeneity than low-incidence countries.

3.4 Bootstrapping

When comparing one group of populations to another group of populations, like when comparing high- and low-incidence countries, we can use conventional statistical tests for comparing two distributions, such as Wilcoxon rank-sum tests and T-tests. However, when comparing the SVA results of just one population to another population (say we wanted to compare the SVA results of just Japan to those of just Kenya), we rely on a statistical method known as bootstrapping (implemented as sigboot in the sigvar R package).

We will demonstrate sigboot with an example comparing Japan (\(n=37\) samples) to Kenya (\(m=68\) samples). Bootstrapping proceeds in the following steps:

1. Merge the two relative abundance matrices to generate a single matrix with \(n+m\) samples.

2. Draw \(n\) samples with replacement from this merged matrix. This generates a bootstrap replicate of Japan. Perform SVA on this replicate matrix.

3. Draw \(m\) samples with replacement from the merged matrix to generate a bootstrap replicate of Kenya. Perform SVA on this replicate matrix.

4. Compute the difference between the values of each SVA statistic from steps 3 and 4.

5. Repeat steps 2-4 many times to generate many bootstrapped differences in SVA values between Japan and Kenya under the null hypothesis that there is no true difference in the distribution generating their samples.

6. Compare the true difference in SVA values between Japan and Kenya to the distribution of bootstrapped differences. The fraction of bootstrap replicate differences whose absolute values are greater than the absolute value of the true value is the two-sided P-value for our statistical test. One-sided tests could be performed by computing the fraction of differences greater or less than the true value.

This procedure is implemented in the function sigboot, which can conduct many pairwise comparisons. In the below example, we generate 500 bootstrap replicates (n_replicates = 500) of the difference in SVA values between each country (group = "Country") in our signature activity matrix (relab_matrix = ESCC_sig_activity). We account for the cosine similarity between signatures in each computation (S = ESCC_sig_similarity). Because bootstrapping is a random process, running the code multiple times would give slightly different results. Setting a random seed (seed = 1) makes this result repeatable. If you wish to perform a one-sided statistical test, you can specify alternative = "lesser" or alternative = "greater"; the default value is alternative = "two.sided".

comparison_boot = sigboot(sig_activity = ESCC_sig_activity %>% 
                         filter(Country %in% c("Japan", "Kenya")),
                       K = 43, 
                       group = "Country",
                       S = ESCC_sig_similarity,
                       n_replicates = 500, 
                       seed = 1)

comparison_boot$bootstrap_distribution_plot

We see that, for the SVA statistics across_sample_heterogeneity and mean_within_sample_diversity, the red dots fall far outside of the range of most of the black dots, suggesting that there is a significant difference in SVA results between Kenya and Japan. This difference is reflected by low two-sided P-values:

comparison_boot$P_values
#>      group_1 group_2 across_sample_heterogeneity mean_within_sample_diversity
#> [1,] "Kenya" "Japan" "0.042"                     "0.018"

4 Conclusion

This concludes the tutorial on the sigvar R package for signature variability analysis. We hope you found it helpful! For guidance on specific functions, access the documentation by typing ? into the R console (e.g., ?sigboot). To see all available functions, type ?sigvar::. For more details on signature variability analysis, see the paper “Variability of mutational signatures is a footprint of carcinogens’’ by Morrison et al. If you find a problem with the R package, please open an issue on Github using this link.