scMitoMut demo: CRC dataset
26 July 2024
Package
scMitoMut 1.1.1
Install {scMitoMut} from Biocondcutor:
if (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("scMitoMut")or from github:
if (!requireNamespace("remotes", quietly = TRUE))
install.packages("remotes")
remotes::install_github("wenjie1991/scMitoMut")Load required packages.
library(scMitoMut)
library(data.table)
library(ggplot2)
library(rhdf5)1 Overview
1.1 Key functions
Following are the key functions used in the scMitoMut package:
parse_mgatk(): Parses the mgatk output and saves the result in an H5 file.open_h5_file(): Opens the H5 file and returns a “mtmutObj” object.subset_cell(): Subsets the cells in the mtmutObj object.run_model_fit(): Runs the model fitting and saves the results in the H5 file.filter_loc(): Filters the mutations based on criterias.plot_heatmap(): Plots a heatmap for p-values, allele frequencies, or binary mutation status.export_df(),export_af(),export_pval(), andexport_binary(): Export the mutation data indata.frameformat and the allele frequency, p-value, and binary mutation status indata.matrixformat.
1.2 Key conceptions
- Somatic mutation: the mutation that occurs in cells after fertilizatoin.
- AF or VAF: allele frequency (AF) or variant allele frequency (VAF). It is the ratio of the number of reads supporting the variant allele to the total number of reads covering that locus.
IMPORTANT: In this vignette, I used the term “mutation” to refer to the lineage-related somatic mutation. For each mutation, I used the dominant allele as the reference allele. If the reference allele frequency is significantly (FDR < 0.05) lower, I will call the locus a mutation.
1.3 Background
Single-cell genomics technology serves as a powerful tool for gaining insights into cellular heterogeneity and diversity within complex tissues.
Mitochondrial DNA (mtDNA) is characterized by its small size and the presence of multiple copies within a cell. These attributes contribute to achieving robust mtDNA sequencing coverage and depth in single-cell sequencing data, thereby enhancing the detection of somatic mutations without dropout.
In this vignette, the scMitoMut package is used to identify and visualize lineage-related mtDNA single nucleic somatic mutations.
In the following analysis, scMitoMut was used to analyze the allele count data, which is the output of mgatk. Only a few loci have been selected for demonstration purposes to reduce file size and run time. The full dataset can be access from {Signac} vignette.
2 Loading data
We load the allele count table with the parse_table function. The allele count table consists with following columns:
loc: the locus namecell_barcode: the cell barcode in single-cell sequencing datafwd_depth: the forward read count of the allelerev_depth: the reverse read count of the allelealt: the allele namecoverage: the read count covering the locusref: the reference allele name
Instead of using the table above as input, the output from mgatk can also be directly read using the parse_mgatk function.
Using the parse_table function or parse_mgatk function, the allele count data are read and saved into an H5 file.
The H5 file works as a database, which does not occupy memory, and data can be randomly accessed by querying.
It helps with better memory usage and faster loading.
The process may take some minutes. The return value is the H5 file path.
## Load the allele count table
f <- system.file("extdata", "mini_dataset.tsv.gz", package = "scMitoMut")
f_h5_tmp <- tempfile(fileext = ".h5")
f_h5 <- parse_table(f, h5_file = f_h5_tmp)f_h5## [1] "/tmp/Rtmpvh6fgr/file1d4fa139219ae7.h5"After obtaining the H5 file, the open_h5_file function can be utilized to load it, resulting in an object referred to as “mtmutObj”.
Detail: On this step, the mtmutObj has 6 slots
- h5f is the H5 file handle
- mut_table is the allele count table
- loc_list is a list of available loci
- loc_selected is the selected loci
- cell_list is a list of available cell ids
- cell_selected is the selected cell ids
## Open the h5 file as a scMitoMut object
x <- open_h5_file(f_h5)
str(x)## List of 9
## $ file : chr "/tmp/Rtmpvh6fgr/file1d4fa139219ae7.h5"
## $ h5f :Formal class 'H5IdComponent' [package "rhdf5"] with 2 slots
## .. ..@ ID : chr "72057594037927938"
## .. ..@ native: logi FALSE
## $ mut_table :Formal class 'H5IdComponent' [package "rhdf5"] with 2 slots
## .. ..@ ID : chr "144115188075855873"
## .. ..@ native: logi FALSE
## $ loc_list : chr [1:16(1d)] "chrM.200" "chrM.204" "chrM.310" "chrM.824" ...
## $ loc_selected : chr [1:16(1d)] "chrM.200" "chrM.204" "chrM.310" "chrM.824" ...
## $ cell_list : chr [1:1359(1d)] "AAACGAAAGAACCCGA-1" "AAACGAAAGTACCTCA-1" "AAACGAACAGAAAGAG-1" "AAACGAAGTGGTCGAA-1" ...
## $ cell_selected: chr [1:1359(1d)] "AAACGAAAGAACCCGA-1" "AAACGAAAGTACCTCA-1" "AAACGAACAGAAAGAG-1" "AAACGAAGTGGTCGAA-1" ...
## $ loc_pass : NULL
## $ loc_filter :List of 5
## ..$ min_cell : num 1
## ..$ model : chr "bb"
## ..$ p_threshold : num 0.05
## ..$ alt_count_threshold: num 0
## ..$ p_adj_method : chr "fdr"
## - attr(*, "class")= chr "mtmutObj"## Show what's in the h5 file
h5ls(x$h5f, recursive = FALSE)## group name otype dclass dim
## 0 / cell_list H5I_DATASET STRING 1359
## 1 / cell_selected H5I_DATASET STRING 1359
## 2 / loc_list H5I_DATASET STRING 16
## 3 / loc_selected H5I_DATASET STRING 16
## 4 / mut_table H5I_GROUP3 Selecting cells
We are only interested in annotated good-quality cells.
So we will select the cells with annotation, which are good quality cells.
f <- system.file("extdata", "mini_dataset_cell_ann.csv", package = "scMitoMut")
cell_ann <- read.csv(f, row.names = 1)
## Subset the cells, the cell id can be found by colnames() for the Seurat object
x <- subset_cell(x, rownames(cell_ann))After subsetting the cells, the cell_selected slot will be updated.
Only the selected cells will be used in the following p-value calculation.
head(x$cell_selected)## [1] "AAACGAAAGAACCCGA-1" "AAACGAAAGTACCTCA-1" "AAACGAACAGAAAGAG-1"
## [4] "AAACGAAGTGGTCGAA-1" "AAACGAATCAATCGTG-1" "AAACGAATCCCACGGA-1"Similarly, we can select loci by using the subset_locus function. It saves time when we only focus on a few loci.
4 Calculating mutation p-value
We built an null-hypothesis that there are not lineage-related mutation for specific locus in all cells. Then we fit the allele frequency distribution with beta-binomial distribution and calculate the probability of observing allele frequency for a specific locus in a cell. If the probability is small, we can reject the null hypothesis and conclude that there is a mutation for that locus in the cell.
To calculate the probability value (p-value), we run run_calling function, which has 2 arguments:
- mtmutObj is the scMitoMut object
- mc.cores is the number of CPU threads to be used
The process will take some time.
The output will be stored in the pval group of the H5 file.
The result is stored in the hard drive, instead of in memory.
We don’t need to re-run the mutation calling when loading the H5 file next time.
The mutation calling is based on beta-binomial distribution. The mutation p-value is the probability that with the null hypothesis: there are no mutations for that locus in the cell.
Detail: For each locus, we calculate the p-value using the following steps. 1. Defining the wild-type allele as the allele with the highest median allele frequency among cells. 2. Fitting a 2 components binomial-mixture distribution as classifier to select the likely wild-type cells. We define the likely wild-type cells if it has a probability >= 0.001 to be the wild type. 3. Using those likely wild-type cells, we fit the beta-binomial model. 4. At last, based on the model, we calculate the p-value of observing the allele frequency of the wild-type allele in specific cell.
## Run the model fitting
run_model_fit(x, mc.cores = 1)## chrM.200## chrM.204## chrM.310## chrM.824## chrM.1000## chrM.1001## chrM.1227## chrM.2285## chrM.6081## chrM.9429## chrM.9728## chrM.9804## chrM.9840## chrM.12889## chrM.16093## chrM.16147## used (Mb) gc trigger (Mb) max used (Mb)
## Ncells 1126122 60.2 2116188 113.1 2116188 113.1
## Vcells 2168958 16.6 8388608 64.0 5676045 43.4## The p-value is kept in the pval group of H5 file
h5ls(x$h5f, recursive = FALSE)## group name otype dclass dim
## 0 / cell_list H5I_DATASET STRING 1359
## 1 / cell_selected H5I_DATASET STRING 1359
## 2 / loc_list H5I_DATASET STRING 16
## 3 / loc_selected H5I_DATASET STRING 16
## 4 / model_par_bb H5I_DATASET COMPOUND 16
## 5 / model_par_bm H5I_DATASET COMPOUND 16
## 6 / mut_table H5I_GROUP
## 7 / pval H5I_GROUP5 Filter mutations
Then we will filter the mutations by using the mut_filter function with the following criteria:
- The mutation has at least 5 cells mutant.
- The FDR (false discovery rate) adjusted p-value (mutation quality q-value) is less than 0.05.
The output is a data.frame with 2 columns
- loc is the locus
- mut_cell_n is the cell number
We can see that there are 12 loci after filtering.
Detail: The mut_filter function has 4 arguments:
- mtmutObj is the mtmutObj object
- min_cell is the minimum number of mutant cells
- p_adj_method is the method used to adjust the p-value.
- p_threshold is the adjusted p-value (q-value) threshold
## Filter mutation
x <- filter_loc(
mtmutObj = x,
min_cell = 2,
model = "bb",
p_threshold = 0.01,
p_adj_method = "fdr"
)
x$loc_pass## [1] "chrM.200" "chrM.204" "chrM.310" "chrM.824" "chrM.1227"
## [6] "chrM.2285" "chrM.6081" "chrM.9429" "chrM.9728" "chrM.9804"
## [11] "chrM.9840" "chrM.12889" "chrM.16093" "chrM.16147"6 Visualization
We will visualize the mutation by heatmap using the plot_heatmap function.
It can draw a heatmap of q-value, allele frequency, or binarized mutation status.
Its input is the mtmutObj object.
It will independently apply all the filters we used in the mut_filter function, and select the cells and loci that pass the filter criteria.
In all kinds of figures, the mutation status will be calculated, and the loci and cells are ordered by the mutation status.
Detail: The plot_heatmap arguments.
- mtmutObj is the scMitoMut object.
- pos_list is the list of loci.
- cell_ann is the cell annotation.
- ann_colors is the color of the cell annotation.
- type is the type of the heatmap which can be p, af, or binary.
- p_adj_method is the method used to adjust the p-value.
- p_threshold is the adjusted p-value threshold to determine if a cell has mutation when selecting the cells and loci.
- min_cell_n is the minimum number of cells that have mutation when selecting the cells and loci.
- p_binary is the adjusted p-value threshold to get the binary mutation status.
- percent_interp is the percentage overlap threshold between mutations, to determine if two mutations are correlated for interpolating the mutation status
- n_interp is the number of overlapped cells to determine if two mutations are correlated for interpolating.
The interpolation is based on the assumption that the mutation are unique, it is rare to have two mutation in the same population. Therefore, when two mutations are correlated, one of them is likely a subclone of the other one. The interpolation is utilized primarily for the purpose of ordering cells during visualization.
6.1 Binary heatmap
The binary heatmap displays the mutation status of each cell corresponding to each locus. The color red suggests the presence of a mutant, whereas blue indicates its absence, and white denotes a missing value.
## Prepare the color for cell annotation
colors <- c(
"Cancer Epi" = "#f28482",
Blood = "#f6bd60"
)
ann_colors <- list("SeuratCellTypes" = colors)## binary heatmap
plot_heatmap(x,
cell_ann = cell_ann, ann_colors = ann_colors, type = "binary",
percent_interp = 0.2, n_interp = 3
)
Also we can turn off the interpolation by setting percent_interp = 1.
## binary heatmap
plot_heatmap(x,
cell_ann = cell_ann, ann_colors = ann_colors, type = "binary",
percent_interp = 1, n_interp = 3
)
6.2 P value heatmap
The p-value heatmap illustrates the adjusted p-values for each cell corresponding to each locus. The arrangement of the cells and loci is based on their binary mutation status.
## p value heatmap
plot_heatmap(x,
cell_ann = cell_ann, ann_colors = ann_colors, type = "p",
percent_interp = 0.2, n_interp = 3
)
6.3 AF heatmap
The allele frequency heatmap illustrates the allele frequency of each cell at each locus. The order of the cells and loci are determined by the mutation status too.
## allele frequency heatmap
plot_heatmap(x,
cell_ann = cell_ann, ann_colors = ann_colors, type = "af",
percent_interp = 0.2, n_interp = 3
)
7 Exporting mutation data
We can export the mutation data by using the following functions:
export_dfexport the mutation data as adata.frameexport_afexport the AF data as adata.matrixwith loci as row names and cells as column names.export_pvalexport the p-value data as adata.matrixwith loci as row names and cells as column names.export_binaryexport the mutation status data as adata.matrixwith loci as row names and cells as column names.
Those functions have the same filtering options as the plot_heatmap function.
## Export the mutation data as data.frame
m_df <- export_df(x)
m_df[1:10, ]## loc cell_barcode alt_depth fwd_depth rev_depth coverage pval
## 1 chrM.1227 AAACGAAAGAACCCGA-1 90 48 42 91 1
## 2 chrM.1227 AAACGAAAGTACCTCA-1 49 23 26 49 1
## 3 chrM.1227 AAACGAACAGAAAGAG-1 60 25 35 60 1
## 4 chrM.1227 AAACGAAGTGGTCGAA-1 52 25 27 52 1
## 5 chrM.1227 AAACTCGAGGTCGGTA-1 25 15 10 25 1
## 6 chrM.1227 AAACTCGCAGTGGTCC-1 36 21 15 37 1
## 7 chrM.1227 AAACTCGTCGGGCTCA-1 49 24 25 49 1
## 8 chrM.1227 AAACTGCGTGAGGGTT-1 54 27 27 54 1
## 9 chrM.1227 AAAGATGAGTCGCCTG-1 77 32 45 77 1
## 10 chrM.1227 AAAGATGCAGGAGCAT-1 27 15 12 27 1
## af alt_count mut_status
## 1 0.989011 1 FALSE
## 2 1.000000 0 FALSE
## 3 1.000000 0 FALSE
## 4 1.000000 0 FALSE
## 5 1.000000 0 FALSE
## 6 0.972973 1 FALSE
## 7 1.000000 0 FALSE
## 8 1.000000 0 FALSE
## 9 1.000000 0 FALSE
## 10 1.000000 0 FALSE## Export allele frequency data as data.matrix
export_af(x)[1:5, 1:5]## CATTCCGGTACCCACG-1 AAACGAAAGAACCCGA-1 CAAAGCTAGGTCGTTT-1
## chrM.16147 0.8974359 0.3606557 0.8695652
## chrM.310 0.2500000 0.5434783 0.4000000
## chrM.12889 1.0000000 0.9821429 1.0000000
## chrM.9728 1.0000000 1.0000000 1.0000000
## chrM.1227 1.0000000 0.9890110 1.0000000
## GTCACGGAGCTGCCAC-1 GTCTACCTCTCTATTG-1
## chrM.16147 0.5000000 0.7954545
## chrM.310 0.5405405 0.5102041
## chrM.12889 1.0000000 1.0000000
## chrM.9728 1.0000000 1.0000000
## chrM.1227 1.0000000 1.0000000## Export p-value data as data.matrix
export_pval(x)[1:5, 1:5]## CATTCCGGTACCCACG-1 AAACGAAAGAACCCGA-1 CAAAGCTAGGTCGTTT-1
## chrM.16147 0.0099457441 2.972594e-10 0.008205852
## chrM.310 0.0001327973 2.889483e-03 0.004930106
## chrM.12889 1.0000000000 3.559443e-01 1.000000000
## chrM.9728 1.0000000000 1.000000e+00 1.000000000
## chrM.1227 1.0000000000 1.000000e+00 1.000000000
## GTCACGGAGCTGCCAC-1 GTCTACCTCTCTATTG-1
## chrM.16147 1.253323e-06 0.0006184542
## chrM.310 5.137155e-03 0.0008829350
## chrM.12889 1.000000e+00 1.0000000000
## chrM.9728 1.000000e+00 1.0000000000
## chrM.1227 1.000000e+00 1.0000000000## Export binary mutation status data as data.matrix
export_binary(x)[1:5, 1:5]## CATTCCGGTACCCACG-1 AAACGAAAGAACCCGA-1 CAAAGCTAGGTCGTTT-1
## chrM.16147 1 1 1
## chrM.310 1 1 1
## chrM.12889 0 0 0
## chrM.9728 0 0 0
## chrM.1227 0 0 0
## GTCACGGAGCTGCCAC-1 GTCTACCTCTCTATTG-1
## chrM.16147 1 1
## chrM.310 1 1
## chrM.12889 0 0
## chrM.9728 0 0
## chrM.1227 0 08 Show the p value, af plot versus cell types
Lastly, we try to show the distribution of p value and allele frequency value versus cell types.
## The `m_df` is exported using the `export_df` function above.
m_dt <- data.table(m_df)
m_dt[, cell_type := cell_ann[as.character(m_dt$cell_barcode), "SeuratCellTypes"]]
m_dt_sub <- m_dt[loc == "chrM.1227"]
m_dt_sub[, sum((pval) < 0.01, na.rm = TRUE), by = cell_type]## cell_type V1
## <char> <int>
## 1: Cancer Epi 28
## 2: Blood 0m_dt_sub[, sum((1 - af) > 0.05, na.rm = TRUE), by = cell_type]## cell_type V1
## <char> <int>
## 1: Cancer Epi 42
## 2: Blood 2## The p value versus cell types
ggplot(m_dt_sub) +
aes(x = cell_type, y = -log10(pval), color = cell_type) +
geom_jitter() +
scale_color_manual(values = colors) +
theme_bw() +
geom_hline(yintercept = -log10(0.01), linetype = "dashed") +
ylab("-log10(FDR)")## Warning in FUN(X[[i]], ...): NaNs produced## Warning: Removed 4 rows containing missing values or values outside the scale range
## (`geom_point()`).
## The allele frequency versus cell types
ggplot(m_dt_sub) +
aes(x = cell_type, y = 1 - af, color = factor(cell_type)) +
geom_jitter() +
scale_color_manual(values = colors) +
theme_bw() +
geom_hline(yintercept = 0.05, linetype = "dashed") +
ylab("1 - Dominant Allele Frequency")
## The p value versus allele frequency
ggplot(m_dt_sub) +
aes(x = -log10(pval), y = 1 - af, color = factor(cell_type)) +
geom_point() +
scale_color_manual(values = colors) +
theme_bw() +
geom_hline(yintercept = 0.05, linetype = "dashed") +
geom_vline(xintercept = -log10(0.01), linetype = "dashed") +
xlab("-log10(FDR)") +
ylab("1 - Dominant Allele Frequency")## Warning in FUN(X[[i]], ...): NaNs produced
## Warning in FUN(X[[i]], ...): Removed 4 rows containing missing values or values outside the scale range
## (`geom_point()`).
9 Session Info
sessionInfo()## R version 4.4.1 (2024-06-14)
## Platform: x86_64-pc-linux-gnu
## Running under: Ubuntu 22.04.4 LTS
##
## Matrix products: default
## BLAS: /home/biocbuild/bbs-3.20-bioc/R/lib/libRblas.so
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0
##
## locale:
## [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
## [3] LC_TIME=en_GB LC_COLLATE=C
## [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
## [7] LC_PAPER=en_US.UTF-8 LC_NAME=C
## [9] LC_ADDRESS=C LC_TELEPHONE=C
## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
##
## time zone: America/New_York
## tzcode source: system (glibc)
##
## attached base packages:
## [1] stats graphics grDevices utils datasets methods base
##
## other attached packages:
## [1] rhdf5_2.49.0 ggplot2_3.5.1 data.table_1.15.4 scMitoMut_1.1.1
## [5] knitr_1.48 BiocStyle_2.33.1
##
## loaded via a namespace (and not attached):
## [1] sass_0.4.9 utf8_1.2.4 generics_0.1.3
## [4] stringi_1.8.4 hms_1.1.3 digest_0.6.36
## [7] magrittr_2.0.3 RColorBrewer_1.1-3 evaluate_0.24.0
## [10] grid_4.4.1 bookdown_0.40 fastmap_1.2.0
## [13] R.oo_1.26.0 plyr_1.8.9 jsonlite_1.8.8
## [16] R.utils_2.12.3 tinytex_0.52 BiocManager_1.30.23
## [19] fansi_1.0.6 scales_1.3.0 jquerylib_0.1.4
## [22] cli_3.6.3 rlang_1.1.4 R.methodsS3_1.8.2
## [25] munsell_0.5.1 withr_3.0.0 cachem_1.1.0
## [28] yaml_2.3.10 tools_4.4.1 parallel_4.4.1
## [31] tzdb_0.4.0 dplyr_1.1.4 colorspace_2.1-1
## [34] Rhdf5lib_1.27.0 vctrs_0.6.5 R6_2.5.1
## [37] magick_2.8.4 lifecycle_1.0.4 zlibbioc_1.51.1
## [40] stringr_1.5.1 pkgconfig_2.0.3 pillar_1.9.0
## [43] bslib_0.7.0 gtable_0.3.5 glue_1.7.0
## [46] Rcpp_1.0.13 highr_0.11 xfun_0.46
## [49] tibble_3.2.1 tidyselect_1.2.1 rhdf5filters_1.17.0
## [52] farver_2.1.2 htmltools_0.5.8.1 labeling_0.4.3
## [55] rmarkdown_2.27 readr_2.1.5 pheatmap_1.0.12
## [58] compiler_4.4.1