Motif comparisons and P-values
## Warning: replacing previous import 'S4Arrays::read_block' by
## 'DelayedArray::read_block' when loading 'SummarizedExperiment'Introduction
This vignette covers motif comparisons (including metrics, parameters and clustering) and P-values. For an introduction to sequence motifs, see the introductory vignette. For a basic overview of available motif-related functions, see the motif manipulation vignette. For sequence-related utilities, see the sequences vignette.
Motif comparisons
There a couple of functions available in other Bioconductor packages
which allow for motif comparison, such as PWMSimlarity()
(TFBSTools) and motifSimilarity()
(PWMEnrich). Unfortunately these functions are not designed
for comparing large numbers of motifs. Furthermore they are restrictive
in their option range. The universalmotif package aims to
fix this by providing the compare_motifs() function.
Several other functions also make use of the core
compare_motifs() functionality, including
merge_motifs() and view_motifs().
An overview of available comparison metrics
This function has been written to allow comparisons using any of the following metrics:
- Euclidean distance (
EUCL) - Weighted Euclidean distance (
WEUCL) - Kullback-Leibler divergence (
KL) (Kullback and Leibler 1951; Roepcke et al. 2005) - Hellinger distance (
HELL) (Hellinger 1909) - Squared Euclidean distance (
SEUCL) - Manhattan distance (
MAN) - Pearson correlation coefficient (
PCC) - Weighted Pearson correlation coefficient (
WPCC) - Sandelin-Wasserman similarity (
SW; or sum of squared distances) (Sandelin and Wasserman 2004) - Average log-likelihood ratio (
ALLR) (Wang and Stormo 2003) - Lower limit average log-likelihood ratio (
ALLR_LL; minimum column score of -2) (Mahony, Auron, and Benos 2007) - Bhattacharyya coefficient (
BHAT) (Bhattacharyya 1943)
For clarity, here are the R implementations of these
metrics:
EUCL <- function(c1, c2) {
sqrt( sum( (c1 - c2)^2 ) )
}
WEUCL <- function(c1, c2, bkg1, bkg2) {
sqrt( sum( (bkg1 + bkg2) * (c1 - c2)^2 ) )
}
KL <- function(c1, c2) {
( sum(c1 * log(c1 / c2)) + sum(c2 * log(c2 / c1)) ) / 2
}
HELL <- function(c1, c2) {
sqrt( sum( ( sqrt(c1) - sqrt(c2) )^2 ) ) / sqrt(2)
}
SEUCL <- function(c1, c2) {
sum( (c1 - c2)^2 )
}
MAN <- function(c1, c2) {
sum ( abs(c1 - c2) )
}
PCC <- function(c1, c2) {
n <- length(c1)
top <- n * sum(c1 * c2) - sum(c1) * sum(c2)
bot <- sqrt( ( n * sum(c1^2) - sum(c1)^2 ) * ( n * sum(c2^2) - sum(c2)^2 ) )
top / bot
}
WPCC <- function(c1, c2, bkg1, bkg2) {
weights <- bkg1 + bkg2
mean1 <- sum(weights * c1)
mean2 <- sum(weights * c2)
var1 <- sum(weights * (c1 - mean1)^2)
var2 <- sum(weights * (c2 - mean2)^2)
cov <- sum(weights * (c1 - mean1) * (c2 - mean2))
cov / sqrt(var1 * var2)
}
SW <- function(c1, c2) {
2 - sum( (c1 - c2)^2 )
}
ALLR <- function(c1, c2, bkg1, bkg2, nsites1, nsites2) {
left <- sum( c2 * nsites2 * log(c1 / bkg1) )
right <- sum( c1 * nsites1 * log(c2 / bkg2) )
( left + right ) / ( nsites1 + nsites2 )
}
BHAT <- function(c1, c2) {
sum( sqrt(c1 * c2) )
}Motif comparison involves comparing a single column from each motif individually, and adding up the scores from all column comparisons. Since this causes the score to be highly dependent on motif length, the scores can instead be averaged using the arithmetic mean, geometric mean, median, or Fisher Z-transform.
If you’re curious as to how the comparison metrics perform, two
columns can be compared individually using
compare_columns():
c1 <- c(0.7, 0.1, 0.1, 0.1)
c2 <- c(0.5, 0.0, 0.2, 0.3)
compare_columns(c1, c2, "PCC")
#> [1] 0.8006408
compare_columns(c1, c2, "EUCL")
#> [1] 0.3162278Note that some metrics do not work with zero values, and small pseudocounts are automatically added to motifs for the following:
KLALLRALLR_LL
As seen in figure , the distributions for random individual column comparisons tend to be very skewed. This is usually remedied when comparing the entire motif, though some metrics still perform poorly in this regard.
#> `summarise()` has grouped output by 'key'. You can override using the `.groups`
#> argument.
Distributions of scores from approximately 500 random motif and individual column comparisons
Comparison parameters
There are several key parameters to keep in mind when comparing motifs. Some of these are:
method: one of the metrics listed previouslytryRC: choose whether to try comparing the reverse complements of each motif as wellmin.overlap: limit the amount of allowed overhang between the two motifsmin.mean.ic,min.position.ic: don’t allow low IC alignments or positions to contribute to the final scorescore.strat: how to combine individual column scores in an alignment
See the following example for an idea as to how some of these settings impact scores:
Example scores from comparing two motifs
| type | method | default | normalised | checkIC |
|---|---|---|---|---|
| similarity | PCC | 0.5145697 | 0.3087418 | 0.9356122 |
| similarity | WPCC | 0.6603253 | 0.5045159 | 0.9350947 |
| distance | EUCL | 0.5489863 | 0.7401466 | 0.2841903 |
| similarity | SW | 1.5579529 | 1.2057098 | 1.8955966 |
| distance | KL | 0.9314823 | 1.2424547 | 0.1975716 |
| similarity | ALLR | -0.3158358 | -0.1895015 | 0.4577374 |
| similarity | BHAT | 0.7533046 | 0.6026437 | 0.9468133 |
| distance | HELL | 0.4154478 | 0.2492687 | 0.2123219 |
| distance | WEUCL | 0.3881919 | 0.5233627 | 0.2009529 |
| distance | SEUCL | 0.4420471 | 0.2652283 | 0.1044034 |
| distance | MAN | 0.8645563 | 0.5187338 | 0.4710645 |
| similarity | ALLR_LL | -0.1706669 | -0.1024001 | 0.4577374 |
Settings used in the previous table:
- normalised:
normalise.scores = TRUE - checkIC:
min.position.ic = 0.25
Comparison P-values
By default, compare_motifs() will compare all motifs
provided and return a matrix. The compare.to will cause
compare_motifs() to return P-values.
library(universalmotif)
library(MotifDb)
motifs <- filter_motifs(MotifDb, organism = "Athaliana")
#> motifs converted to class 'universalmotif'
# Compare the first motif with everything and return P-values
head(compare_motifs(motifs, 1))
#> Warning in compare_motifs(motifs, 1): Some comparisons failed due to low motif
#> IC
#> DataFrame with 6 rows and 8 columns
#> subject subject.i target target.i score logPval
#> <character> <numeric> <character> <integer> <numeric> <numeric>
#> 1 ORA59 1 ERF11 [duplicated #6.. 1371 0.991211 -13.5452
#> 2 ORA59 1 CRF4 [duplicated #566] 1195 0.990756 -13.5247
#> 3 ORA59 1 LOB 1297 0.987357 -13.3725
#> 4 ORA59 1 ERF15 618 0.977213 -12.9254
#> 5 ORA59 1 ERF2 [duplicated #294] 649 0.973871 -12.7804
#> 6 ORA59 1 ERF2 [duplicated #483] 1033 0.973871 -12.7804
#> Pval Eval
#> <numeric> <numeric>
#> 1 1.31042e-06 0.00359318
#> 2 1.33754e-06 0.00366754
#> 3 1.55744e-06 0.00427049
#> 4 2.43548e-06 0.00667809
#> 5 2.81553e-06 0.00772019
#> 6 2.81553e-06 0.00772019P-values are made possible by estimating distribution (usually the
best fitting distribution for motif comparisons) parameters from
randomized motif scores, then using the appropriate
stats::p*() distribution function to return P-values. These
estimated parameters are pre-computed with make_DBscores()
and stored as JASPAR2018_CORE_DBSCORES and
JASPAR2018_CORE_DBSCORES_NORM. Since changing any of the
settings and motif sizes will affect the estimated distribution
parameters, estimated parameters have been pre-computed for a variety of
these. See ?make_DBscores if you would like to generate
your own set of pre-computed scores using your own parameters and
motifs.
Motif trees with ggtree
Using motif_tree()
Additionally, this package introduces the motif_tree()
function for generating basic tree-like diagrams for comparing motifs.
This allows for a visual result from compare_motifs(). All
options from compare_motifs() are available in
motif_tree(). This function uses the ggtree
package and outputs a ggplot object (from the
ggplot2 package), so altering the look of the trees can be
done easily after motif_tree() has already been run.
library(universalmotif)
library(MotifDb)
motifs <- filter_motifs(MotifDb, family = c("AP2", "B3", "bHLH", "bZIP",
"AT hook"))
#> motifs converted to class 'universalmotif'
motifs <- motifs[sample(seq_along(motifs), 100)]
tree <- motif_tree(motifs, layout = "daylight", linecol = "family")
## Make some changes to the tree in regular ggplot2 fashion:
# tree <- tree + ...
tree
Using compare_motifs() and ggtree()
While motif_tree() works as a quick and convenient
tree-building function, it can be inconvenient when more control is
required over tree construction. For this purpose, the following code
goes through how exactly motif_tree() generates trees.
library(universalmotif)
library(MotifDb)
library(ggtree)
library(ggplot2)
motifs <- convert_motifs(MotifDb)
motifs <- filter_motifs(motifs, organism = "Athaliana")
motifs <- motifs[sample(seq_along(motifs), 25)]
## Step 1: compare motifs
comparisons <- compare_motifs(motifs, method = "PCC", min.mean.ic = 0,
score.strat = "a.mean")
## Step 2: create a "dist" object
# The current metric, PCC, is a similarity metric
comparisons <- 1 - comparisons
comparisons <- as.dist(comparisons)
# We also want to extract names from the dist object to match annotations
labels <- attr(comparisons, "Labels")
## Step 3: get the comparisons ready for tree-building
# The R package "ape" provides the necessary "as.phylo" function
comparisons <- ape::as.phylo(hclust(comparisons))
## Step 4: incorporate annotation data to colour tree lines
family <- sapply(motifs, function(x) x["family"])
family.unique <- unique(family)
# We need to create a list with an entry for each family; within each entry
# are the names of the motifs belonging to that family
family.annotations <- list()
for (i in seq_along(family.unique)) {
family.annotations <- c(family.annotations,
list(labels[family %in% family.unique[i]]))
}
names(family.annotations) <- family.unique
# Now add the annotation data:
comparisons <- ggtree::groupOTU(comparisons, family.annotations)
## Step 5: draw the tree
tree <- ggtree(comparisons, aes(colour = group), layout = "rectangular") +
theme(legend.position = "bottom", legend.title = element_blank())
## Step 6: add additional annotations
# If we wish, we can additional annotations such as tip labelling and size
# Tip labels:
tree <- tree + geom_tiplab()
# Tip size:
tipsize <- data.frame(label = labels,
icscore = sapply(motifs, function(x) x["icscore"]))
tree <- tree %<+% tipsize + geom_tippoint(aes(size = icscore))Plotting motifs alongside trees
Unfortunately, the universalmotif package does not
provide any function to easily plot motifs as part of trees (as is
possible via the motifStack package). However, it can be
done (somewhat roughly) by plotting a tree and a set of motifs side by
side. In the following example, the cowplot package is used
to glue the two plots together, though other packages which perform this
function are available.
library(universalmotif)
library(MotifDb)
library(cowplot)
## Get our starting set of motifs:
motifs <- convert_motifs(MotifDb[1:10])
## Get the tree: make sure it's a horizontal type layout
tree <- motif_tree(motifs, layout = "rectangular", linecol = "none")
## Now, make sure we order our list of motifs to match the order of tips:
mot.names <- sapply(motifs, function(x) x["name"])
names(motifs) <- mot.names
new.order <- tree$data$label[tree$data$isTip]
new.order <- rev(new.order[order(tree$data$y[tree$data$isTip])])
motifs <- motifs[new.order]
## Plot the two together (finessing of margins and positions may be required):
plot_grid(nrow = 1, rel_widths = c(1, -0.15, 1),
tree + xlab(""), NULL,
view_motifs(motifs, names.pos = "right") +
ylab(element_blank()) +
theme(
axis.line.y = element_blank(),
axis.ticks.y = element_blank(),
axis.text.y = element_blank(),
axis.text = element_text(colour = "white")
)
)
Motif P-values
Motif P-values are not usually discussed outside of the bioinformatics literature, but are actually quite a challenging topic. To illustrate this, consider the following example motif:
library(universalmotif)
m <- matrix(c(0.10,0.27,0.23,0.19,0.29,0.28,0.51,0.12,0.34,0.26,
0.36,0.29,0.51,0.38,0.23,0.16,0.17,0.21,0.23,0.36,
0.45,0.05,0.02,0.13,0.27,0.38,0.26,0.38,0.12,0.31,
0.09,0.40,0.24,0.30,0.21,0.19,0.05,0.30,0.31,0.08),
byrow = TRUE, nrow = 4)
motif <- create_motif(m, alphabet = "DNA", type = "PWM")
motif
#>
#> Motif name: motif
#> Alphabet: DNA
#> Type: PWM
#> Strands: +-
#> Total IC: 10.03
#> Pseudocount: 0
#> Consensus: SHCNNNRNNV
#>
#> S H C N N N R N N V
#> A -1.32 0.10 -0.12 -0.40 0.21 0.15 1.04 -1.07 0.44 0.04
#> C 0.53 0.20 1.03 0.60 -0.12 -0.66 -0.54 -0.27 -0.12 0.51
#> G 0.85 -2.34 -3.64 -0.94 0.11 0.59 0.07 0.59 -1.06 0.30
#> T -1.47 0.66 -0.06 0.26 -0.25 -0.41 -2.31 0.25 0.31 -1.66Let us then use this motif with scan_sequences():
data(ArabidopsisPromoters)
res <- scan_sequences(motif, ArabidopsisPromoters, verbose = 0,
calc.pvals = FALSE, threshold = 0.8, threshold.type = "logodds")
head(res)
#> DataFrame with 6 rows and 13 columns
#> motif motif.i sequence sequence.i start stop score
#> <character> <integer> <character> <integer> <integer> <integer> <numeric>
#> 1 motif 1 AT1G08090 21 925 934 5.301
#> 2 motif 1 AT1G49840 27 980 989 5.292
#> 3 motif 1 AT1G76590 19 848 857 5.869
#> 4 motif 1 AT2G15390 6 337 346 5.643
#> 5 motif 1 AT3G57640 33 174 183 5.510
#> 6 motif 1 AT4G14365 35 799 808 5.637
#> match thresh.score min.score max.score score.pct strand
#> <character> <numeric> <numeric> <numeric> <numeric> <character>
#> 1 CTCCAAAGAA 5.2248 -15.4 6.531 81.1667 +
#> 2 CTCTGGATTC 5.2248 -15.4 6.531 81.0289 +
#> 3 CTCTAGAGAC 5.2248 -15.4 6.531 89.8637 +
#> 4 CCCCGGAGAC 5.2248 -15.4 6.531 86.4033 +
#> 5 GCCCAGATAG 5.2248 -15.4 6.531 84.3669 +
#> 6 CTCCAAAGTC 5.2248 -15.4 6.531 86.3114 +Now let us imagine that we wish to rank these matches by P-value. First, we must calculate the match probabilities:
## One of the matches was CTCTAGAGAC, with a score of 5.869 (max possible = 6.531)
bkg <- get_bkg(ArabidopsisPromoters, 1)
bkg <- structure(bkg$probability, names = bkg$klet)
bkg
#> A C G T
#> 0.34768 0.16162 0.15166 0.33904Now, use these to calculate the probability of getting CTCTAGAGAC.
hit.prob <- bkg["A"]^3 * bkg["C"]^3 * bkg["G"]^2 * bkg["T"]^2
hit.prob <- unname(hit.prob)
hit.prob
#> [1] 4.691032e-07Calculating the probability of a single match was easy, but then comes the challenging part: calculating the probability of all possible matches with a score higher than 5.869, and then summing these. This final sum then represents the probability of finding a match which scores at least 5.869. One way is to list all possible sequence combinations, then filtering based on score; however this “brute force” approach is unreasonable for all but the smallest of motifs.
The dynamic programming algorithm for calculating P-values and scores
Instead of trying to find and calculate the probabilities of all matches with a score or higher than the query score, one can use a dynamic programming algorithm to generate a much smaller distribution of probabilities for the possible range of scores using set intervals. This method is implemented by the FIMO tool (Grant, Bailey, and Noble 2011). The theory behind it is also explained in Gupta et al. (2007), though the purpose of the algorithm is for motif comparison instead of motif P-values (however it is the same algorithm). The basic concept will also be briefly explained here.
For each individual position-letter score in the PWM, the chance of
getting that score from the respective background probability of that
letter is added to the intervals in which getting that specific score
could allow the final score to land. Once this probability distribution
is generated, it can be converted to a cumulative distribution and
re-used for any input P-value/score to output the equivalent
score/P-value. For P-value inputs, it finds the specific score interval
where the accompanying P-value in the cumulative distribution smaller or
equal to it, then reports the score of the previous interval. For score
inputs, the scores are rounded to the nearest interval in the cumulative
distribution and the accompanying P-value retrieved. The major
advantages of this method include only looking for the probabilities of
the range of scores with a set interval, cutting down on needing to find
the probabilities of all actual possible scores (and thus increasing
performance by several orders of magnitude for larger/higher-order
motifs), and being able to re-use the distribution for any number of
query P-value/scores. Although this method involves rounding off scores
to allow a small set interval, in practice in the
universalmotif package it offers the same maximum possible
level of accuracy as the exhaustive method (described in the next
section) as motif PWMs are always internally rounded to a thousandth of
a decimal place for speed. This leaves as the only downside the
inability to allow non-finite values to exist in the PWM (e.g. from
zero-probabilities) since then a known range with set intervals could
not possibly be created.
Going back to our example, we can see this in action using the
motif_pvalue() function:
res <- res[1:6, ]
pvals <- motif_pvalue(motif, res$score, bkg.probs = bkg)
res2 <- data.frame(motif=res$motif,match=res$match,pval=pvals)[order(pvals), ]
knitr::kable(res2, digits = 22, row.names = FALSE, format = "markdown")| motif | match | pval |
|---|---|---|
| motif | CTCTAGAGAC | 0.001495587 |
| motif | CCCCGGAGAC | 0.001947592 |
| motif | CTCCAAAGTC | 0.001962531 |
| motif | GCCCAGATAG | 0.002257443 |
| motif | CTCCAAAGAA | 0.002825922 |
| motif | CTCTGGATTC | 0.002852671 |
To illustrate that we can also do the inverse of this calculation:
res$score
#> [1] 5.301 5.292 5.869 5.643 5.510 5.637
motif_pvalue(motif, pvalue = pvals, bkg.probs = bkg)
#> [1] 5.301 5.292 5.869 5.643 5.510 5.637You may occasionally see slight errors at the last couple of digits.
These are generally unavoidable to the internal rounding mechanisms of
the universalmotif package.
Let us consider more examples, such as the following larger motif:
data(ArabidopsisMotif)
ArabidopsisMotif
#>
#> Motif name: YTTTYTTTTTYTTTY
#> Alphabet: DNA
#> Type: PPM
#> Strands: +-
#> Total IC: 15.99
#> Pseudocount: 1
#> Consensus: YTYTYTTYTTYTTTY
#> Target sites: 617
#> E-value: 2.5e-87
#>
#> Y T Y T Y T T Y T T Y T T T Y
#> A 0.01 0.00 0.00 0.00 0.00 0.06 0.00 0.01 0.00 0.00 0.02 0.00 0.00 0.00 0.00
#> C 0.30 0.17 0.31 0.01 0.54 0.02 0.24 0.25 0.22 0.04 0.39 0.21 0.16 0.18 0.43
#> G 0.16 0.05 0.03 0.01 0.00 0.02 0.11 0.00 0.04 0.05 0.03 0.01 0.02 0.00 0.11
#> T 0.53 0.78 0.66 0.98 0.45 0.90 0.66 0.74 0.74 0.91 0.55 0.77 0.83 0.82 0.46Using the motif_range() utility, we can get an idea of
the possible range of scores:
We can use these ranges to confirm our cumulative distribution of P-values:
(pvals2 <- motif_pvalue(ArabidopsisMotif, score = motif_range(ArabidopsisMotif)))
#> [1] 1.000000e+00 2.143914e-09And again, going back to scores from these P-values:
As a note: if you ever provide scores which are outside the possible ranges, then you will get the following behaviour:
We can also use this function for the higher-order
multifreq motif representation.
data(examplemotif2)
examplemotif2["multifreq"]["2"]
#> $`2`
#> 1 2 3 4 5 6
#> AA 0.0 0.5 0.5 0.5 0.0 0
#> AC 0.0 0.0 0.0 0.0 0.5 0
#> AG 0.0 0.0 0.0 0.0 0.0 0
#> AT 0.0 0.0 0.0 0.0 0.0 0
#> CA 0.5 0.0 0.0 0.0 0.0 0
#> CC 0.0 0.0 0.0 0.0 0.0 1
#> CG 0.0 0.0 0.0 0.0 0.0 0
#> CT 0.5 0.0 0.0 0.0 0.0 0
#> GA 0.0 0.0 0.0 0.0 0.0 0
#> GC 0.0 0.0 0.0 0.0 0.0 0
#> GG 0.0 0.0 0.0 0.0 0.0 0
#> GT 0.0 0.0 0.0 0.0 0.0 0
#> TA 0.0 0.0 0.0 0.0 0.0 0
#> TC 0.0 0.0 0.0 0.0 0.5 0
#> TG 0.0 0.0 0.0 0.0 0.0 0
#> TT 0.0 0.5 0.5 0.5 0.0 0
motif_range(examplemotif2, use.freq = 2)
#> min max
#> -39.948 18.921
motif_pvalue(examplemotif2, score = 15, use.freq = 2)
#> [1] 1.907349e-06
motif_pvalue(examplemotif2, pvalue = 0.00001, use.freq = 2)
#> [1] 9.276Feel free to use this function with any alphabets, such as amino acid motifs or even made up ones!
(m <- create_motif(alphabet = "QWERTY"))
#>
#> Motif name: motif
#> Alphabet: EQRTWY
#> Type: PPM
#> Total IC: 13.74
#> Pseudocount: 0
#>
#> [,1] [,2] [,3] [,4] [,5] [,6] [,7] [,8] [,9] [,10]
#> E 0.01 0.00 0.40 0.00 0.54 0.01 0.82 0.01 0.13 0.04
#> Q 0.63 0.62 0.01 0.03 0.26 0.11 0.15 0.00 0.00 0.68
#> R 0.00 0.01 0.00 0.00 0.05 0.01 0.00 0.63 0.00 0.23
#> T 0.00 0.00 0.00 0.49 0.07 0.00 0.01 0.24 0.02 0.00
#> W 0.35 0.02 0.44 0.39 0.05 0.00 0.01 0.03 0.03 0.00
#> Y 0.00 0.35 0.15 0.08 0.03 0.87 0.01 0.10 0.82 0.05
motif_pvalue(m, pvalue = c(1, 0.1, 0.001, 0.0001, 0.00001))
#> [1] -149.603 -17.999 4.541 9.975 13.928The branch-and-bound algorithm for calculating P-values from scores
The alternative to the dynamic programming algorithm is to
exhaustively find all actual possible hits with a score equal to or
greater than the input score. Generally there is no advantage to solving
this exhaustively, with the exception that it allows non-finite values
to be present (i.e., zero-probability letters which were not
pseudocount-adjusted during the calculation of the PWM). A few
algorithms have been proposed to make solving this problem exhaustively
more efficient, but the method adopted by the
universalmotif package is that of Luehr, Hartmann, and Söding (2012). The authors
propose using a branch-and-bound2 algorithm (with a few tricks) alongside a
certain approximation. Briefly: motifs are first reorganized so that the
highest scoring positions and letters are considered first in the
branch-and-bound algorithm. Then, motifs past a certain width (in the
original paper, 10) are split in sub-motifs. All possible combinations
are found in these sub-motifs using the branch-and-bound algorithm, and
P-values calculated for the sub-motifs. Finally, the P-values are
combined.
The motif_pvalue() function modifies this process
slightly by allowing the size of the sub-motifs to be specified via the
k parameter; and additionally, whereas the original
implementation can only calculate P-values for motifs with a maximum of
17 positions (and motifs can only be split in at most two), the
universalmotif implementation allows for any length of
motif to be used (and motifs can be split any number of times). Changing
k allows one to decide between speed and accuracy; smaller
k leads to faster but worse approximations, and larger
k leads to slower but better approximations. If
k is equal to the width of the motif, then the calculation
is exact. Is it important to note however that this is is still
a computationally intenstive task for larger motifs unless it is broken
up into several sub-motifs, though at this point significant accuracy is
lost due to the high level of approximation.
Now, let us return to our original example, and this time for the
branch-and-bound algorithm set method = "exhaustive":
res <- res[1:6, ]
pvals <- motif_pvalue(motif, res$score, bkg.probs = bkg, method = "e")
res2 <- data.frame(motif=res$motif,match=res$match,pval=pvals)[order(pvals), ]
knitr::kable(res2, digits = 22, row.names = FALSE, format = "markdown")| motif | match | pval |
|---|---|---|
| motif | CTCTAGAGAC | 0.001494052 |
| motif | CCCCGGAGAC | 0.001944162 |
| motif | CTCCAAAGTC | 0.001960741 |
| motif | GCCCAGATAG | 0.002255555 |
| motif | CTCCAAAGAA | 0.002823098 |
| motif | CTCTGGATTC | 0.002848363 |
The default k in motif_pvalue() is 8. I
have found this to be a good tradeoff between speed and P-value
correctness.
To demonstrate the effect that k has on the output
P-value, consider the following (and also note that for this motif
k = 10 represents an exact calculation):
scores <- c(-6, -3, 0, 3, 6)
k <- c(2, 4, 6, 8, 10)
out <- data.frame(k = c(2, 4, 6, 8, 10),
score.minus6 = rep(0, 5),
score.minus3 = rep(0, 5),
score.0 = rep(0, 5),
score.3 = rep(0, 5),
score.6 = rep(0, 5))
for (i in seq_along(scores)) {
for (j in seq_along(k)) {
out[j, i + 1] <- motif_pvalue(motif, scores[i], k = k[j], bkg.probs = bkg,
method = "e")
}
}
knitr::kable(out, format = "markdown", digits = 10)| k | score.minus6 | score.minus3 | score.0 | score.3 | score.6 |
|---|---|---|---|---|---|
| 2 | 0.9692815 | 0.6783292 | 0.2241568 | 0.01809649 | 0.0000000000 |
| 4 | 0.8516271 | 0.4945960 | 0.1581260 | 0.02271134 | 0.0009788176 |
| 6 | 0.7647867 | 0.4298417 | 0.1418337 | 0.02291211 | 0.0012812392 |
| 8 | 0.7647867 | 0.4298417 | 0.1418337 | 0.02291211 | 0.0012812392 |
| 10 | 0.7649169 | 0.4299862 | 0.1419202 | 0.02293202 | 0.0012830021 |
For this particular motif, while the approximation worsens slightly
as k decreases, it is still quite accurate when the number
of motif subsets is limited to two. Usually, you should only have to
worry about k for longer motifs (such as those sometimes
generated by MEME), where the number of sub-motifs
increases.
The random subsetting algorithm for calculating scores from P-values
Similarly to calculating P-values, exact scores can be calculated
from small motifs, and approximate scores from big motifs using
subsetting. When an exact calculation is performed, all possible scores
are extracted and a quantile function extracts the appropriate score.
For approximate calculations, the overall set of scores are approximate
several times by randomly adding up all possible scores from each
k subset before a quantile function is used.
Starting from a set of P-values and setting
method = "exhaustive":
bkg <- c(A=0.25, C=0.25, G=0.25, T=0.25)
pvals <- c(0.1, 0.01, 0.001, 0.0001, 0.00001)
scores <- motif_pvalue(motif, pvalue = pvals, bkg.probs = bkg, k = 10,
method = "e")
scores.approx6 <- motif_pvalue(motif, pvalue = pvals, bkg.probs = bkg, k = 6,
method = "e")
scores.approx8 <- motif_pvalue(motif, pvalue = pvals, bkg.probs = bkg, k = 8,
method = "e")
pvals.exact <- motif_pvalue(motif, score = scores, bkg.probs = bkg, k = 10,
method = "e")
pvals.approx6 <- motif_pvalue(motif, score = scores, bkg.probs = bkg, k = 6,
method = "e")
pvals.approx8 <- motif_pvalue(motif, score = scores, bkg.probs = bkg, k = 8,
method = "e")
res <- data.frame(pvalue = pvals, score = scores,
pvalue.exact = pvals.exact,
pvalue.k6 = pvals.approx6,
pvalue.k8 = pvals.approx8,
score.k6 = scores.approx6,
score.k8 = scores.approx8)
knitr::kable(res, format = "markdown", digits = 22)| pvalue | score | pvalue.exact | pvalue.k6 | pvalue.k8 | score.k6 | score.k8 |
|---|---|---|---|---|---|---|
| 1e-01 | 1.324 | 1.000299e-01 | 9.995747e-02 | 9.995747e-02 | 1.3344 | 1.3280 |
| 1e-02 | 3.596 | 1.000309e-02 | 9.991646e-03 | 9.991646e-03 | 3.5901 | 3.5975 |
| 1e-03 | 4.858 | 1.000404e-03 | 9.984970e-04 | 9.984970e-04 | 4.8156 | 4.8869 |
| 1e-04 | 5.647 | 1.001358e-04 | 9.727478e-05 | 9.727478e-05 | 5.6724 | 5.7134 |
| 1e-05 | 6.182 | 1.049042e-05 | 9.536743e-06 | 9.536743e-06 | 5.5744 | 6.1119 |
Starting from a set of scores:
bkg <- c(A=0.25, C=0.25, G=0.25, T=0.25)
scores <- -2:6
pvals <- motif_pvalue(motif, score = scores, bkg.probs = bkg, k = 10,
method = "e")
scores.exact <- motif_pvalue(motif, pvalue = pvals, bkg.probs = bkg, k = 10,
method = "e")
scores.approx6 <- motif_pvalue(motif, pvalue = pvals, bkg.probs = bkg, k = 6,
method = "e")
scores.approx8 <- motif_pvalue(motif, pvalue = pvals, bkg.probs = bkg, k = 8,
method = "e")
pvals.approx6 <- motif_pvalue(motif, score = scores, bkg.probs = bkg, k = 6,
method = "e")
pvals.approx8 <- motif_pvalue(motif, score = scores, bkg.probs = bkg, k = 8,
method = "e")
res <- data.frame(score = scores, pvalue = pvals,
pvalue.k6 = pvals.approx6,
pvalue.k8 = pvals.approx8,
score.exact = scores.exact,
score.k6 = scores.approx6,
score.k8 = scores.approx8)
knitr::kable(res, format = "markdown", digits = 22)| score | pvalue | pvalue.k6 | pvalue.k8 | score.exact | score.k6 | score.k8 |
|---|---|---|---|---|---|---|
| -2 | 4.627047e-01 | 4.625721e-01 | 4.625721e-01 | -2.000 | -2.0098 | -2.0009 |
| -1 | 3.354645e-01 | 3.353453e-01 | 3.353453e-01 | -1.000 | -1.0093 | -0.9986 |
| 0 | 2.185555e-01 | 2.184534e-01 | 2.184534e-01 | 0.000 | 0.0075 | -0.0004 |
| 1 | 1.244183e-01 | 1.243525e-01 | 1.243525e-01 | 1.000 | 0.9856 | 0.9936 |
| 2 | 5.911160e-02 | 5.907822e-02 | 5.907822e-02 | 2.000 | 2.0026 | 2.0017 |
| 3 | 2.163410e-02 | 2.160931e-02 | 2.160931e-02 | 3.000 | 3.0264 | 2.9962 |
| 4 | 5.360603e-03 | 5.347252e-03 | 5.347252e-03 | 4.000 | 3.9513 | 4.0001 |
| 5 | 7.162094e-04 | 7.152557e-04 | 7.152557e-04 | 5.000 | 4.9858 | 5.0140 |
| 6 | 2.193451e-05 | 2.193451e-05 | 2.193451e-05 | 6.057 | 5.6564 | 6.0238 |
As you may have noticed, results from exact calculations are not
quite exact. This is due to the universalmotif
package rounding off values internally for speed.
Session info
#> R version 4.4.2 (2024-10-31)
#> Platform: x86_64-pc-linux-gnu
#> Running under: Ubuntu 24.04.1 LTS
#>
#> Matrix products: default
#> BLAS: /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3
#> LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.26.so; LAPACK version 3.12.0
#>
#> locale:
#> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
#> [3] LC_TIME=en_US.UTF-8 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: Etc/UTC
#> tzcode source: system (glibc)
#>
#> attached base packages:
#> [1] stats4 stats graphics grDevices utils datasets methods
#> [8] base
#>
#> other attached packages:
#> [1] cowplot_1.1.3 dplyr_1.1.4 ggtree_3.15.0
#> [4] ggplot2_3.5.1 MotifDb_1.49.0 GenomicRanges_1.59.0
#> [7] Biostrings_2.75.1 GenomeInfoDb_1.43.0 XVector_0.47.0
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#>
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#> [23] S4Arrays_1.7.1 curl_6.0.0
#> [25] splitstackshape_1.4.8 DelayedArray_0.33.1
#> [27] aplot_0.2.3 abind_1.4-8
#> [29] BiocParallel_1.41.0 withr_3.0.2
#> [31] purrr_1.0.2 sys_3.4.3
#> [33] grid_4.4.2 fansi_1.0.6
#> [35] colorspace_2.1-1 scales_1.3.0
#> [37] MASS_7.3-61 SummarizedExperiment_1.37.0
#> [39] cli_3.6.3 rmarkdown_2.29
#> [41] crayon_1.5.3 treeio_1.31.0
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#> [61] glue_1.8.0 codetools_0.2-20
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#> [69] htmltools_0.5.8.1 GenomeInfoDbData_1.2.13
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#> [73] Biobase_2.67.0 lattice_0.22-6
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