# planet

## Installation

    if(!requireNamespace("BiocManager", quietly = TRUE))
        install.packages("BiocManager")
    BiocManager::install("planet")

## Cell composition

To infer cell composition on placental villi DNAm samples, we can need to use placental reference cpgs (Yuan 2021). These are provided in this package as `plCellCpGsThird` and `plCellCpGsFirst` for third trimester (term) and first trimester samples, respectively.

In this example we are using term villi DNAm data, so we first load the reference cpgs `plCellCpGsThird`. This is a data frame of 600 cpgs, with mean methylation levels for each cell type.

    # cell deconvolution packages
    library(minfi)
    library(EpiDISH)

    # data wrangling and plotting
    library(dplyr)
    library(ggplot2)
    library(tidyr)
    library(planet)

    # load example data
    data("plBetas")
    data("plCellCpGsThird")
    head(plCellCpGsThird)

    ##            Trophoblasts   Stromal  Hofbauer Endothelial      nRBC
    ## cg10590657    0.1014098 0.9345796 0.8655285   0.8963641 0.8448382
    ## cg14923398    0.1282030 0.8902107 0.9036769   0.9383641 0.9508709
    ## cg05348366    0.1305697 0.9519820 0.8803082   0.9065136 0.9278057
    ## cg17907628    0.1215249 0.9278777 0.8727841   0.8914412 0.9143601
    ## cg26799656    0.1259953 0.9482014 0.8803863   0.8791004 0.9010419
    ## cg11862144    0.1561991 0.9430855 0.9114967   0.9341671 0.9647331
    ##            Syncytiotrophoblast
    ## cg10590657          0.05460441
    ## cg14923398          0.05383193
    ## cg05348366          0.06546727
    ## cg17907628          0.05325227
    ## cg26799656          0.06823985
    ## cg11862144          0.06044207

After our reference cpg data is loaded, we can estimate cell composition by applying either the Constrained Projection approach implemented by the R packages minfi or EpiDISH, or a non-constrained approach by EpiDish. I demonstrate how to do both.

#### Minfi

    houseman_estimates <- minfi:::projectCellType(
        plBetas[rownames(plCellCpGsThird), ],
        plCellCpGsThird,
        lessThanOne = FALSE
    )

    head(houseman_estimates)

    ##            Trophoblasts    Stromal      Hofbauer Endothelial       nRBC
    ## GSM1944936    0.1091279 0.04891919  0.000000e+00  0.08983998 0.05294062
    ## GSM1944939    0.2299918 0.00000000 -1.806592e-19  0.07888007 0.03374149
    ## GSM1944942    0.1934287 0.03483540  0.000000e+00  0.09260353 0.02929310
    ## GSM1944944    0.2239896 0.06249135  1.608645e-03  0.11040693 0.04447951
    ## GSM1944946    0.1894152 0.07935955  0.000000e+00  0.10587439 0.05407587
    ## GSM1944948    0.2045124 0.07657717  0.000000e+00  0.09871149 0.02269798
    ##            Syncytiotrophoblast
    ## GSM1944936           0.6979477
    ## GSM1944939           0.6377822
    ## GSM1944942           0.6350506
    ## GSM1944944           0.5467642
    ## GSM1944946           0.6022329
    ## GSM1944948           0.6085825

#### EpiDISH

    # robust partial correlations
    epidish_RPC <- epidish(
        beta.m = plBetas[rownames(plCellCpGsThird), ],
        ref.m = plCellCpGsThird,
        method = "RPC"
    )

    # CIBERSORT
    epidish_CBS <- epidish(
        beta.m = plBetas[rownames(plCellCpGsThird), ],
        ref.m = plCellCpGsThird,
        method = "CBS"
    )

    ## 1

    ## 2

    ## 3

    # constrained projection (houseman 2012)
    epidish_CP <- epidish(
        beta.m = plBetas[rownames(plCellCpGsThird), ],
        ref.m = plCellCpGsThird,
        method = "CP"
    )

    ## 1

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    ## 24

#### Compare

Below, I demonstrate how we can visually compare the different cell composition estimates.

    data("plColors")

    # bind estimate data frames and reshape for plotting
    bind_rows(
        houseman_estimates %>% as.data.frame() %>% mutate(algorithm = "CP (Houseman)"),
        epidish_RPC$estF %>% as.data.frame() %>% mutate(algorithm = "RPC"),
        epidish_CBS$estF %>% as.data.frame() %>% mutate(algorithm = "CBS"),
        epidish_CP$estF %>% as.data.frame() %>% mutate(algorithm = "CP (EpiDISH)")
    ) %>%
        mutate(sample = rep(rownames(houseman_estimates), 4)) %>%
        as_tibble() %>%
        pivot_longer(
            cols = -c(algorithm, sample),
            names_to = "component",
            values_to = "estimate"
        ) %>%

        # relevel for plot
        mutate(component = factor(component,
            levels = c(
                "nRBC", "Endothelial", "Hofbauer",
                "Stromal", "Trophoblasts",
                "Syncytiotrophoblast"
            )
        )) %>%

        # plot
        ggplot(aes(x = sample, y = estimate, fill = component)) +
        geom_bar(stat = "identity") +
        facet_wrap(~algorithm, ncol = 1) +
        scale_fill_manual(values = plColors) +
        scale_y_continuous(
            limits = c(-0.1, 1.1), breaks = c(0, 0.5, 1),
            labels = scales::percent
        ) +
        theme(axis.text.x = element_text(angle = 90, vjust = 0.5)) +
        coord_cartesian(ylim = c(0, 1)) +
        labs(x = "", fill = "")

Some notes:

- Normalize your data with `minfi::preprocessNoob` and BMIQ
- Use all cell CpGs - if some are missing, estimates may vary
- If your samples have been processed in a particular manner, (e.g. sampling from maternal side) expect cell composition to reflect that

## Gestational age

#### Example Data

For demonstration, I use 24 samples from a placental DNAm dataset from GEO, (GSE7519), which contains samples collected in an Australian population. The DNA methylation data (in betas) can be accessed with `data(plBetas)` and corresponding sample information from `data(plPhenoData)`. Note that for demonstration purposes, the cpgs have been filtered to a random ~10,000 CpGs, plus the CpGs used in all of the functions from this package.

    # load example data
    data(plBetas)
    data(plPhenoData)

    dim(plBetas)

    ## [1] 13918    24

    #> [1] 13918    24
    head(plPhenoData)

sample_id | sex | disease | gestation_wk | ga_RPC | ga_CPC | ga_RRPC
--- | --- | --- | --- | --- | --- | ---
GSM1944936 | Male | preeclampsia | 36 | 38.46528 | 38.72867 | 38.65396
GSM1944939 | Male | preeclampsia | 32 | 33.09680 | 34.21252 | 32.62763
GSM1944942 | Female | preeclampsia | 32 | 34.32520 | 35.09565 | 33.32502
GSM1944944 | Male | preeclampsia | 35 | 35.50937 | 36.71512 | 35.51295
GSM1944946 | Female | preeclampsia | 38 | 37.63910 | 37.57931 | 36.61721
GSM1944948 | Female | preeclampsia | 36 | 36.77051 | 38.42632 | 36.72150

    #> # A tibble: 6 x 7
    #>   sample_id  sex   disease   gestation_wk ga_RPC ga_CPC ga_RRPC
    #>   <fct>      <chr> <chr>            <dbl>  <dbl>  <dbl>   <dbl>
    #> 1 GSM1944936 Male  preeclam~           36   38.5   38.7    38.7
    #> 2 GSM1944939 Male  preeclam~           32   33.1   34.2    32.6
    #> 3 GSM1944942 Fema~ preeclam~           32   34.3   35.1    33.3
    #> 4 GSM1944944 Male  preeclam~           35   35.5   36.7    35.5
    #> 5 GSM1944946 Fema~ preeclam~           38   37.6   37.6    36.6
    #> 6 GSM1944948 Fema~ preeclam~           36   36.8   38.4    36.7

There are 3 gestational age clocks for placental DNA methylation data from (Lee 2019):

1. Robust Placental Clock (RPC)
2. Control Placental Clock (CPC)
3. Refined Robust Placental Clock (RRPC)

To predict gestational, we load the example data:

- `plBetas` - DNAm data for 24 placental samples
- `plPhenoData` - Matching sample information

#### Predict Gestational Age

To select the type of clock, we can specify the `type` argument in `predictAge`.

We will apply all three clocks on this data, and add the predicted age to the sample information data.frame, `plPhenoData`.

    plPhenoData <- plPhenoData %>%
        mutate(
            ga_RPC = predictAge(plBetas, type = "RPC"),
            ga_CPC = predictAge(plBetas, type = "CPC"),
            ga_RRPC = predictAge(plBetas, type = "RRPC")
        )

    ## 558 of 558 predictors present.

    ## 546 of 546 predictors present.

    ## 395 of 395 predictors present.

Note that the number of predictors (CpGs) that were used in our data are printed. It’s important to take note if a significant number of predictive CpGs are missing in your data, as this can affect the predicted gestational age accuracy.

Next, I plot the difference between predicted and reported gestational age, for each of the 3 gestational age predictors.

    plPhenoData %>%

        # reshape, to plot
        pivot_longer(
            cols = contains("ga"),
            names_to = "clock_type",
            names_prefix = "ga_",
            values_to = "ga"
        ) %>%

        # plot code
        ggplot(aes(x = gestation_wk, y = ga, col = disease)) +
        geom_point() +
        geom_smooth(method = "lm", se = FALSE) +
        facet_wrap(~clock_type) +
        theme(legend.position = "top") +
        labs(x = "Reported GA (weeks)", y = "Inferred GA (weeks)", col = "")

    ## `geom_smooth()` using formula 'y ~ x'

## Ethnicity

Before predicting ethnicity You can ensure that you have all features using the `ethnicityCpGs` vector:

    data(ethnicityCpGs)
    all(ethnicityCpGs %in% rownames(plBetas))

    ## [1] TRUE

    results <- predictEthnicity(plBetas)

    ## 1860 of 1860 predictors present.

    results %>%
        tail(8)

Sample_ID | Predicted_ethnicity_nothresh | Predicted_ethnicity | Prob_African | Prob_Asian | Prob_Caucasian | Highest_Prob
--- | --- | --- | --- | --- | --- | ---
GSM1944959 | Asian | Asian | 0.0123073 | 0.9523544 | 0.0353383 | 0.9523544
GSM1944960 | Caucasian | Caucasian | 0.0156961 | 0.1595213 | 0.8247827 | 0.8247827
GSM1944961 | Asian | Asian | 0.0208421 | 0.8954518 | 0.0837061 | 0.8954518
GSM1944962 | Caucasian | Caucasian | 0.0009276 | 0.0008801 | 0.9981923 | 0.9981923
GSM1944963 | Caucasian | Caucasian | 0.0022635 | 0.0028007 | 0.9949358 | 0.9949358
GSM1944964 | Caucasian | Caucasian | 0.0065973 | 0.0112013 | 0.9822014 | 0.9822014
GSM1944965 | Caucasian | Caucasian | 0.0021578 | 0.0024196 | 0.9954226 | 0.9954226
GSM1944966 | Caucasian | Caucasian | 0.0011397 | 0.0017651 | 0.9970952 | 0.9970952

`predictEthnicity` returns probabilities corresponding to each ethnicity for each sample (e.g `Prob_Caucasian`, `Prob_African`, `Prob_Asian`). This applies a glmnet model described in (Yuan 2019). A final classification is determined in two ways:

1. `Predicted_ethnicity_nothresh` - returns a classification corresponding to the highest class-specific probability.

2. `Predicted_ethnicity` - if the highest class-specific probability is below `0.75`, then the the sample is assigned an `Amibiguous` label. This threshold can be adjusted with the `threshold` argument. Samples with this label might require special attention in downstream analyses.

    results %>%
        ggplot(aes(
            x = Prob_Caucasian, y = Prob_African,
            col = Predicted_ethnicity
        )) +
        geom_point(alpha = 0.7) +
        coord_cartesian(xlim = c(0, 1), ylim = c(0, 1)) +
        scale_x_continuous(labels = scales::percent) +
        scale_y_continuous(labels = scales::percent) +
        labs(x = "P(Caucasian)", y = "P(African)")

    results %>%
        ggplot(aes(
            x = Prob_Caucasian, y = Prob_Asian,
            col = Predicted_ethnicity
        )) +
        geom_point(alpha = 0.7) +
        coord_cartesian(xlim = c(0, 1), ylim = c(0, 1)) +
        scale_x_continuous(labels = scales::percent) +
        scale_y_continuous(labels = scales::percent) +
        labs(x = "P(Caucasian)", y = "P(Asian)")

We can’t compare this to self-reported ethnicity as it is unavailable. But we know these samples were collected in Sydney, Australia, and are therefore likely mostly European with some East Asian participants.

    table(results$Predicted_ethnicity)

    ##
    ##     Asian Caucasian
    ##         2        22

A note on adjustment in differential methylation analysis

Because ‘Ambiguous’ samples might have different mixtures of ancestries, it might be inadequate to adjust for them as one group in an analysis of admixed populations (e.g. 50/50 Asian/African should not be considered the same group as 50/50 Caucasian/African). One solution would be to simply remove these samples. Another would be to adjust for the raw probabilities-in this case, use only two of the three probabilities, since the third will be redundant (probabilities sum to 1). If sample numbers are large enough in each group, stratifying downstream analyses by ethnicity might also be a valid option.

## References

Yuan V, Hui D, Yin Y, Peñaherrera MS, Beristain AG, Robinson WP. Cell-specific characterization of the placental methylome. BMC Genomics. 2021 Jan 6;22(1):6.

Yuan V, Price EM, Del Gobbo G, Mostafavi S, Cox B, Binder AM, et al. Accurate ethnicity prediction from placental DNA methylation data. Epigenetics & Chromatin. 2019 Aug 9;12(1):51.

Lee Y, Choufani S, Weksberg R, Wilson SL, Yuan V, Burt A, et al. Placental epigenetic clocks: estimating gestational age using placental DNA methylation levels. Aging (Albany NY). 2019 Jun 24;11(12):4238–53.

## Session Info

    sessionInfo()

    ## R version 4.2.0 RC (2022-04-19 r82224 ucrt)
    ## Platform: x86_64-w64-mingw32/x64 (64-bit)
    ## Running under: Windows Server x64 (build 20348)
    ##
    ## Matrix products: default
    ##
    ## locale:
    ## [1] LC_COLLATE=C
    ## [2] LC_CTYPE=English_United States.utf8
    ## [3] LC_MONETARY=English_United States.utf8
    ## [4] LC_NUMERIC=C
    ## [5] LC_TIME=English_United States.utf8
    ##
    ## attached base packages:
    ## [1] parallel  stats4    stats     graphics  grDevices utils     datasets
    ## [8] methods   base
    ##
    ## other attached packages:
    ##  [1] planet_1.4.0                tidyr_1.2.0
    ##  [3] ggplot2_3.3.5               dplyr_1.0.8
    ##  [5] EpiDISH_2.12.0              minfi_1.42.0
    ##  [7] bumphunter_1.38.0           locfit_1.5-9.5
    ##  [9] iterators_1.0.14            foreach_1.5.2
    ## [11] Biostrings_2.64.0           XVector_0.36.0
    ## [13] SummarizedExperiment_1.26.0 Biobase_2.56.0
    ## [15] MatrixGenerics_1.8.0        matrixStats_0.62.0
    ## [17] GenomicRanges_1.48.0        GenomeInfoDb_1.32.0
    ## [19] IRanges_2.30.0              S4Vectors_0.34.0
    ## [21] BiocGenerics_0.42.0
    ##
    ## loaded via a namespace (and not attached):
    ##   [1] BiocFileCache_2.4.0       plyr_1.8.7
    ##   [3] splines_4.2.0             BiocParallel_1.30.0
    ##   [5] digest_0.6.29             htmltools_0.5.2
    ##   [7] fansi_1.0.3               magrittr_2.0.3
    ##   [9] memoise_2.0.1             tzdb_0.3.0
    ##  [11] limma_3.52.0              readr_2.1.2
    ##  [13] annotate_1.74.0           askpass_1.1
    ##  [15] siggenes_1.70.0           prettyunits_1.1.1
    ##  [17] colorspace_2.0-3          blob_1.2.3
    ##  [19] rappdirs_0.3.3            xfun_0.30
    ##  [21] crayon_1.5.1              RCurl_1.98-1.6
    ##  [23] jsonlite_1.8.0            genefilter_1.78.0
    ##  [25] GEOquery_2.64.0           survival_3.3-1
    ##  [27] glue_1.6.2                gtable_0.3.0
    ##  [29] zlibbioc_1.42.0           DelayedArray_0.22.0
    ##  [31] Rhdf5lib_1.18.0           HDF5Array_1.24.0
    ##  [33] scales_1.2.0              DBI_1.1.2
    ##  [35] rngtools_1.5.2            Rcpp_1.0.8.3
    ##  [37] xtable_1.8-4              progress_1.2.2
    ##  [39] bit_4.0.4                 proxy_0.4-26
    ##  [41] mclust_5.4.9              preprocessCore_1.58.0
    ##  [43] httr_1.4.2                RColorBrewer_1.1-3
    ##  [45] ellipsis_0.3.2            farver_2.1.0
    ##  [47] pkgconfig_2.0.3           reshape_0.8.9
    ##  [49] XML_3.99-0.9              sass_0.4.1
    ##  [51] dbplyr_2.1.1              utf8_1.2.2
    ##  [53] labeling_0.4.2            tidyselect_1.1.2
    ##  [55] rlang_1.0.2               AnnotationDbi_1.58.0
    ##  [57] munsell_0.5.0             tools_4.2.0
    ##  [59] cachem_1.0.6              cli_3.3.0
    ##  [61] generics_0.1.2            RSQLite_2.2.12
    ##  [63] evaluate_0.15             stringr_1.4.0
    ##  [65] fastmap_1.1.0             yaml_2.3.5
    ##  [67] knitr_1.38                bit64_4.0.5
    ##  [69] beanplot_1.3.1            scrime_1.3.5
    ##  [71] purrr_0.3.4               KEGGREST_1.36.0
    ##  [73] nlme_3.1-157              doRNG_1.8.2
    ##  [75] sparseMatrixStats_1.8.0   nor1mix_1.3-0
    ##  [77] xml2_1.3.3                biomaRt_2.52.0
    ##  [79] compiler_4.2.0            filelock_1.0.2
    ##  [81] curl_4.3.2                png_0.1-7
    ##  [83] e1071_1.7-9               tibble_3.1.6
    ##  [85] bslib_0.3.1               stringi_1.7.6
    ##  [87] highr_0.9                 GenomicFeatures_1.48.0
    ##  [89] lattice_0.20-45           Matrix_1.4-1
    ##  [91] multtest_2.52.0           vctrs_0.4.1
    ##  [93] pillar_1.7.0              lifecycle_1.0.1
    ##  [95] rhdf5filters_1.8.0        jquerylib_0.1.4
    ##  [97] data.table_1.14.2         bitops_1.0-7
    ##  [99] rtracklayer_1.56.0        R6_2.5.1
    ## [101] BiocIO_1.6.0              codetools_0.2-18
    ## [103] MASS_7.3-57               assertthat_0.2.1
    ## [105] rhdf5_2.40.0              openssl_2.0.0
    ## [107] rjson_0.2.21              withr_2.5.0
    ## [109] GenomicAlignments_1.32.0  Rsamtools_2.12.0
    ## [111] GenomeInfoDbData_1.2.8    locfdr_1.1-8
    ## [113] mgcv_1.8-40               hms_1.1.1
    ## [115] quadprog_1.5-8            grid_4.2.0
    ## [117] base64_2.0                class_7.3-20
    ## [119] rmarkdown_2.14            DelayedMatrixStats_1.18.0
    ## [121] illuminaio_0.38.0         restfulr_0.0.13