Getting started with cepip

1. What the package does

cepip relates a gene of interest (GOI) to the mean expression of its partner genes, cell by cell, and finds where that relationship changes.

1. A GAM, y ~ x + s(x), is fitted by REML with the basis dimension k chosen by the lowest penalized residual sum of squares (PRSS).
2. Each cell gets an explanatory power (EP). The top round(n x DE) cells by EP are the TREP cells.
3. The inflection point (IP) is the first negative-to-positive zero crossing of a residual GAM R(x) of the top-EP cells, with a 95% CI and an IP reliability score (IPRS).
4. Optional: the Spearman-Kendall cell-level score (SKCS) keeps the cells that carry the co-expression (SKCS-core).
5. Optional: CMRF (copula monotone recovery filter) recovers cells near the module through a tolerance band and a repeated stratified k-fold cross-validation gate (RSk-fCV).

Steps 1 to 3 are the published CEP-IP. The package does not transform the data, so x and y must be on the scale you want to analyse (the SEA-AD analysis used ln(CP10K + 1)).

2. Quick example on simulated data

cepip_toy holds 1,200 simulated cells from 12 donors.

d <- cepip_toy
d$y <- cepip_composite(d, paste0("P", 1:8))
res <- cepip(d$x, d$y, donor = d$donor, id = d$id, cmrf = TRUE, tests = FALSE)
#> CEP-IP: 1200 cells, 12 donors
#>   GAM: k = 7 | DE = 0.7492
#>   IP: x = 2.4135 [2.3525, 2.4756] | y = 1.1938 | IPRS = 0.656 (Strong)
#>   SKCS-core: 665 cells | TREP 574
#>   Band: delta_base = 0.16097 | candidates 190
#>   CMRF: Original + Refined 647 | Recovered 165 | Original only 18 | Excluded 370
#>   Validation1: DE = 0.8804 | agreement 96.0%
#> Done in 2 s.
res
#> CEP-IP analysis of 1200 cells from 12 donors
#>   GAM: k = 7, DE = 0.7492, TREP cells = 895
#>   IP: x = 2.4135 (95% CI 2.3525 to 2.4756), y = 1.1938, IPRS = 0.656 (Strong)
#>   SKCS-core cells: 665
#>   CMRF classes:
#>     Original + Refined   647
#>     Recovered            165
#>     Original only        18
#>     Excluded             370
#>   Module: 812 cells
#>   Validation1: DE = 0.8804, label agreement 96.0%

With donor, the GAMs include a donor random intercept and weight each cell by 1 / (cells of its donor). Without donor, every cell has weight 1, as in the published CEP-IP.

plot_fit(res)

plot_residual(res$ip)

plot_module(res$cmrf)

The Pre-IP and Post-IP labels use the response axis by default (y < IPy is Pre-IP). The published CEP-IP analyses split on the GOI axis, which ip_axis = "x" reproduces.

3. Tuning the CMRF band

The band is widest at the IP. Its half-width is delta_base x (1 + kappa x K(u)), where K is a Gaussian kernel with standard deviation bw centred at the IP. cepip_band() is fast, so several settings can be compared before running the gate. cepip_cmrf() uses kappa = 0.5 and bw = 0.1 when no band is given.

b <- cepip_band(res$skcs, res$ip, kappa = 0.5, bw = 0.1)
plot_band(b)
#> Warning in ggplot2::annotate("label", x = uq[2], y = mean(c(at("a_lo", uq[2]),
#> : Ignoring unknown parameters: `label.size`
#> Warning in ggplot2::annotate("label", x = uq[c(1, 3)] + c(-off, off), y =
#> (at("a_lo", : Ignoring unknown parameters: `label.size`

cm <- cepip_cmrf(band = b, workers = 2)
plot_rskfcv(cm, "first")
plot_rskfcv(cm, "last")

4. Downstream analyses

The four groups are Module Post-IP (PostIP), Module Pre-IP (PreIP), non-Module Post-IP (NMPost) and non-Module Pre-IP (NMPre). Without CMRF, the TREP cells take the place of the Module.

dp <- cepip_donor(res)
head(dp)
#>   donor n_total n_PostIP pct_PostIP n_PreIP pct_PreIP n_NMPost pct_NMPost
#> 1   D01     100       30         30      38        38        3          3
#> 2   D02     100       37         37      30        30        5          5
#> 3   D03     100       42         42      37        37        3          3
#> 4   D04     100       39         39      26        26        9          9
#> 5   D05     100       35         35      33        33       10         10
#> 6   D06     100       29         29      37        37        8          8
#>   n_NMPre pct_NMPre pct_module
#> 1      29        29         68
#> 2      28        28         67
#> 3      18        18         79
#> 4      26        26         65
#> 5      22        22         68
#> 6      26        26         66

cepip_deg() compares two groups with a paired design across donors. It needs log1p-normalized expression (genes x cells) with the cell ids as column names.

deg <- cepip_deg(res, expr, contrast = c("PostIP_vs_PreIP", "PostIP_vs_NMPost"))
plot_deg(deg, "PostIP_vs_PreIP")

cepip_associate() relates a donor-level measure to outcomes with ordinary least squares on z-scores, adjusting for covariates.

tab <- merge(dp, donor_metadata, by = "donor")
as <- cepip_associate(tab, "pct_PostIP", outcomes = c("MMSE", "Braak"),
                      covariates = c("age", "sex", "PMI"))
plot_associate(as)

Which groups to compare, the outcomes and the covariates depend on the question and on the data available. The defaults of the DEG step (5% expressing cells, at least 3 cells per donor and group, at least 5 paired donors, FDR < 0.05 and |log2FC| >= 0.25) are those of the SEA-AD analysis.

5. The partner screen

cepip_screen() screens candidate GOIs for partner genes by Spearman rs and Kendall tau-b. By default a GOI is screened when it is expressed in at least 25% of the cells with a mean of at least 0.3 over the expressing cells, and a partner passes at rs >= 0.6 and tau-b >= 0.5. These defaults suit ln(CP10K + 1) data from one cell population; adjust them for other data.

sc <- cepip_screen(expr, donor = donor)
head(sc$ranking)
plot_screen(sc)

6. Reproducing the SEA-AD analysis

The example data of the paper are the Micro-PVM_3-SEAAD cells of the Seattle Alzheimer’s Disease Brain Cell Atlas (SEA-AD) with MT-CO3 > 0, and the cells of the housekeeping-gene control arm. They are downloaded once with cepip_example(). Use of the SEA-AD data is subject to the Allen Institute Terms of Use and Citation Policy.

pv <- cepip_example("pvm3")
MT8 <- c("MT-ATP6", "MT-CO2", "MT-ND3", "MT-ND4", "MT-CYB", "MT-ND1", "MT-ND2", "MT-CO1")
d <- pv[pv$region_set == "NeoC", ]
res <- cepip(d[["MT-CO3"]], cepip_composite(d, MT8), donor = d$donor, id = d$barcode,
             cmrf = TRUE, ip_k = 10, y_spine = d$composite_part8, workers = 4)
cepip_save(list(NeoC = res), "PVM_3")

The script system.file("scripts", "reproduce_seaad.R", package = "cepip") runs both region sets of the PVM_3 arm, the HKG8 control arm and the same-cell housekeeping control, and compares every value with the published one. Expected values for PVM_3 NeoC include IPx = 2.4953, IPRS = 0.791, 8,407 SKCS-core cells, 8,103 OriRef cells, 1,351 Recovered cells and 643 cells removed by the gate.

The published SEA-AD analysis used ip_k = 10 for the IP fit and fitted the CMRF spine on the composite column of its data export, which is stored in single precision (y_spine). The eight partner genes were chosen from the per-region screens of the paper.