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library(tidyverse)
library(ggridges)
library(coxme)
library(lme4)
library(nlme)
library(brms)
library(tidybayes)
library(kableExtra)
library(knitrhooks) # install with devtools::install_github("nathaneastwood/knitrhooks")
output_max_height() # a knitrhook option
options(stringsAsFactors = FALSE)
# load eclosion data
eclosion.dat <- read.csv("data/1.eclosion_times.csv")
# remove vials seeded with more than 100 larvae
unique(eclosion.dat[which(eclosion.dat$eclosing < 0), "ID"]) # 4 vials overseeded
eclosion.dat.trim <- eclosion.dat %>%
filter(ID %in% eclosion.dat[which(eclosion.dat$eclosing < 0), "ID"] == FALSE)
# expand data frame so each row is a single fly
ecl.dat <- reshape::untable(eclosion.dat.trim[ ,c(1:7, 9, 10)],
num = eclosion.dat.trim[, 8])
# load wing length data
wing_length <- read.csv("data/1.wing_length.csv") %>%
# scale wing vein length to make effect size comparisons with other data sets?
mutate(Length = as.numeric(scale(Length)))
# add replicate
wing_length$LINE <- paste0(wing_length$Treatment, substr(wing_length$Rep, 2, 2))
cols <- c("M females" = "pink",
"P females" = "red",
"M males" = "skyblue",
"P males" = "blue")
eplot <- ecl.dat %>%
filter(EVENT == 1) %>%
mutate(var = paste(TRT, SEX)) %>%
ggplot(aes(x = DAY, y = SEX, fill = var)) +
geom_boxplot() +
scale_fill_manual(values = c("pink", "skyblue", "red", "blue"), name = "") +
theme_bw() +
theme(legend.position = 'non') +
NULL
lplot <- wing_length %>%
mutate(var = paste(Treatment, Sex)) %>%
ggplot(aes(x = Sex, y = Length)) +
geom_violin(aes(fill = var), alpha = .5) +
geom_boxplot(aes(fill = var), width = .1, position = position_dodge(width = .9)) +
scale_colour_manual(values = c("pink", "skyblue", "red", "blue"), name = "") +
scale_fill_manual(values = c("pink", "skyblue", "red", "blue"), name = "") +
theme_bw() +
theme() +
NULL
gridExtra::grid.arrange(eplot, lplot, ncol = 2)
Figure 1: Time to eclosion in days for flies in each treatment split by sex. Wing vein IV length has been scaled (subtracted the mean and divided by the standard deviation). We see one Monogamy male with an unusually small value for wing length - possible error in data entry or measurement error?
Here are some useful links for understanding survival analyis. For instance, this link provides an explanation of how to interpret hazard ratios.
The exponents of the coefficients from a Cox model give the hazard ratio, an estimate of the effect size of covariates. In short, hazard ratios give the probability of the event occurring compared to ‘control’ in our case compared to Monogamy females.
coxme
We will model time to eclosion (in days) using survival analysis. We measured the time in days from 1st instar until eclosion (EVENT
= 1) upon which flies were stored in ethanol before counting. Of the initially seeded 100 flies per vial, the remaining flies not emerging after two consecutive days of no observed eclosions were right censored (EVENT
= 0) on the last observation day. In total 14400 larvae were seeded (100 larvae x 2 Treatment
x 4 LINE
x 6 VIAL
x 3 SEED
). For P1 only three vials were seeded on day B so we seeded 3 additional vials on day C. Four vials were seeded with too many larvae and excluded from analysis. In total 10448 flies successfully emerged during the observation period leaving 3552 individuals to be right censored on day 9. Censored flies were assigned sex based on the observed sex ratio of eclosees. We calculated the number of flies of each sex that emerged from each vial, and assigned the remaining censored individuals (of unknown sex) sex based on the proportion of individuals of each sex that did emerge, so that overall an equal sex ratio of females:males was assigned to each vial.
First we plot Kaplan-Meier survival curves and the median time to eclosion without considering our full experimental design.
survminer::ggsurvplot(survfit(Surv(DAY, EVENT) ~ TRT + SEX, data = ecl.dat),
conf.int = TRUE,
risk.table = FALSE,
linetype = "SEX",
palette = c("pink", "red", "skyblue", "blue"),
fun = "event",
xlim = c(12, 21),
xlab = "Days",
ylab = "Cumulative proportion eclosed",
legend = 'right',
legend.title = "",
legend.labs = c("M \u2640","M \u2642",'P \u2640','P \u2642'),
break.time.by = 2,
ggtheme = theme_bw())
Figure X: Kaplan-Meier curve for eclosion time (in days) for flies in each treatment and sex. +’s indicate censored individuals (n = 3552).
# median eclosion times
survfit(Surv(DAY, EVENT) ~ TRT + SEX, data = ecl.dat)
Call: survfit(formula = Surv(DAY, EVENT) ~ TRT + SEX, data = ecl.dat) n events median 0.95LCL 0.95UCL TRT=M, SEX=f 3637 3059 16 16 16 TRT=M, SEX=m 3363 2697 16 16 16 TRT=P, SEX=f 3568 2492 18 18 18 TRT=P, SEX=m 3432 2200 18 18 18
Next we need to check that the proportional hazards assumption is not violated before fitting the full model. Here we plot the log-log of the survival curves (take the natural logarithm of the cumulative hazard twice). Crossing hazards (lines) indicate violation of the proportional hazards assumption.
# assess proportional hazards assumption
par(mar = c(2, 2, 2, 2))
plot(survfit(Surv(DAY, EVENT) ~ TRT + SEX, data = ecl.dat),
lty = 1:2, lwd = 2,
col = c("pink", "red", "skyblue", "blue"),
fun = "cloglog")
legend("bottomright", c("M \u2640","M \u2642",'P \u2640','P \u2642'),
col = c("pink", "skyblue", "red", "blue"),
lty = 1:2,
lwd = 2,
bty = 'n'
)
Figure X: ln(-ln(survival))
Looks good so we continue to fit the model.
coxme
modelWe fit a mixed effects Cox Proportional hazards model using the coxme
package, with time (days) to event (eclosion) as the response and TRT
(Monogamy or Polyandry), SEX
(female or male) and their interaction as predictors. We also need to account for the experimental design by including the random effects of LINE
, SEED
day and vial ID
. We calculated p-values using likelihood ratio tests comparing the full model to a model where the fixed effect of interest was removed.
# coxmod <- coxme(Surv(DAY, EVENT) ~ TRT * SEX + (1|LINE) + (1|SEED) + (1|ID),
# data = ecl.dat)
# load in the model instead of running
coxmod <- readRDS('output/coxmod.rds')
# null models to generate P-values using likelihood ratio tests
# coxmod_dropTRT <- coxme(Surv(DAY, EVENT) ~ 1 + SEX + (1|LINE) + (1|SEED) + (1|ID), data = ecl.dat)
# coxmod_dropSEX <- coxme(Surv(DAY, EVENT) ~ TRT + 1 + (1|LINE) + (1|SEED) + (1|ID), data = ecl.dat)
# coxmod_dropINT <- coxme(Surv(DAY, EVENT) ~ TRT + SEX + (1|LINE) + (1|SEED) + (1|ID), data = ecl.dat)
coxmod_dropTRT <- readRDS('output/coxmod_dropTRT.rds')
coxmod_dropSEX <- readRDS('output/coxmod_dropSEX.rds')
coxmod_dropINT <- readRDS('output/coxmod_dropINT.rds')
# make a table of the results
bind_rows(
broom::tidy(anova(coxmod, coxmod_dropTRT)),
broom::tidy(anova(coxmod, coxmod_dropSEX)),
broom::tidy(anova(coxmod, coxmod_dropINT))) %>%
cbind(Parameter = rep(c('Treatment', 'Sex', 'Treatment x Sex'), each = 2)) %>%
na.omit() %>%
mutate(across(1:4, round, 3)) %>%
as_tibble %>%
relocate(Parameter, loglik, statistic, df, p.value) %>%
kable() %>%
kable_styling() %>%
kable_styling(full_width = FALSE)
Parameter | loglik | statistic | df | p.value |
---|---|---|---|---|
Treatment | -92191.30 | 7.765 | 2 | 0.021 |
Sex | -92235.26 | 95.679 | 2 | 0.000 |
Treatment x Sex | -92187.43 | 0.011 | 1 | 0.916 |
We see there is an effect of treatment and sex but not their interaction. We can calculate estimates and confidence intervals for the hazard ratio by taking the exponent of the coefficients ± 1.96 * sqrt(vcov(???)). Polyandrous flies have a reduced hazard compared to Monogamy flies, i.e. take longer to eclose (hazard ratio = 0.377, CI = 0.227, 0.627). Males have a reduced hazard compared to females (hazard ratio = 0.823, CI = 0.782, 0.867).
brms
I found this link useful for fitting survival models in brms
.
if(!file.exists("output/cox_brms.rds")){
cox_brm <- brm(DAY | cens(1 - EVENT) ~ TRT * SEX + (1|LINE) + (1|SEED) + (1|ID),
iter = 5000, chains = 4, cores = 4,
control = list(max_treedepth = 20,
adapt_delta = 0.999),
data = ecl.dat, family = cox())
saveRDS(cox_brm, "output/cox_brms.rds")
} else {
cox_brm <- readRDS('output/cox_brms.rds')
}
summary(cox_brm)
Family: cox Links: mu = log Formula: DAY | cens(1 - EVENT) ~ TRT * SEX + (1 | LINE) + (1 | SEED) + (1 | ID) Data: ecl.dat (Number of observations: 14000) Samples: 4 chains, each with iter = 5000; warmup = 2500; thin = 1; total post-warmup samples = 10000 Group-Level Effects: ~ID (Number of levels: 140) Estimate Est.Error l-95% CI u-95% CI Rhat Bulk_ESS Tail_ESS sd(Intercept) 0.44 0.03 0.38 0.50 1.00 1751 3037 ~LINE (Number of levels: 8) Estimate Est.Error l-95% CI u-95% CI Rhat Bulk_ESS Tail_ESS sd(Intercept) 0.47 0.19 0.24 0.97 1.00 2189 3550 ~SEED (Number of levels: 3) Estimate Est.Error l-95% CI u-95% CI Rhat Bulk_ESS Tail_ESS sd(Intercept) 0.51 0.61 0.08 2.25 1.00 1961 2566 Population-Level Effects: Estimate Est.Error l-95% CI u-95% CI Rhat Bulk_ESS Tail_ESS Intercept 0.89 0.49 0.00 1.98 1.00 2196 2184 TRTP -0.89 0.36 -1.64 -0.16 1.00 2504 3307 SEXm -0.18 0.03 -0.23 -0.12 1.00 7347 6755 TRTP:SEXm 0.01 0.04 -0.07 0.09 1.00 7296 7851 Samples were drawn using sampling(NUTS). For each parameter, Bulk_ESS and Tail_ESS are effective sample size measures, and Rhat is the potential scale reduction factor on split chains (at convergence, Rhat = 1).
fixef(cox_brm) %>%
data.frame() %>%
rownames_to_column() %>%
rename(Parameter = rowname) %>%
kable() %>%
kable_styling() %>%
kable_styling(full_width = FALSE)
Parameter | Estimate | Est.Error | Q2.5 | Q97.5 |
---|---|---|---|---|
Intercept | 0.8907941 | 0.4862808 | 0.0025523 | 1.9775770 |
TRTP | -0.8934343 | 0.3635685 | -1.6424421 | -0.1644978 |
SEXm | -0.1750960 | 0.0264880 | -0.2270423 | -0.1228412 |
TRTP:SEXm | 0.0103648 | 0.0397943 | -0.0672883 | 0.0888395 |
1 / exp(fixef(cox_brm)[, -2])
Estimate Q2.5 Q97.5 Intercept 0.4103298 0.9974509 0.1384042 TRTP 2.4435070 5.1677742 1.1788010 SEXm 1.1913606 1.2548830 1.1307049 TRTP:SEXm 0.9896887 1.0696037 0.9149924
hyp_test <- bind_rows(
hypothesis(cox_brm, 'TRTP = 0')$hypothesis,
hypothesis(cox_brm, 'SEXm = 0')$hypothesis,
hypothesis(cox_brm, 'TRTP:SEXm = 0')$hypothesis
) %>%
mutate(Parameter = c('Polandry', 'Male', 'Polyandry x Male'),
across(2:5, round, 3)) %>%
#select(-Hypothesis) %>%
relocate(Parameter, Estimate, Est.Error, CI.Lower, CI.Upper, Star)
pvals <- bayestestR::p_direction(cox_brm) %>%
as.data.frame() %>%
mutate(vars = map_chr(str_split(Parameter, "_"), ~ .x[2]),
p_val = 1 - pd,
star = ifelse(p_val < 0.05, "\\*", "")) %>%
select(vars, p_val, star)
hyp_test %>%
mutate(vars = c('TRTP', 'SEXm', 'TRTP:SEXm')) %>%
left_join(pvals %>% filter(vars != 'Intercept'),
by = c("vars")) %>%
select(Parameter, Estimate, Est.Error, CI.Lower, CI.Upper, `p` = p_val, star) %>%
rename(` ` = star) %>%
kable() %>%
kable_styling(full_width = FALSE)
Parameter | Estimate | Est.Error | CI.Lower | CI.Upper | p | |
---|---|---|---|---|---|---|
Polandry | -0.893 | 0.364 | -1.642 | -0.164 | 0.0125 | * |
Male | -0.175 | 0.026 | -0.227 | -0.123 | 0.0000 | * |
Polyandry x Male | 0.010 | 0.040 | -0.067 | 0.089 | NA | NA |
Plot posteriors.
# get posterior predictions
post_cox <- posterior_samples(cox_brm) %>%
as_tibble() %>%
select(contains("b_"), -contains("Intercept")) %>%
mutate(draw = 1:n()) %>%
pivot_longer(-draw) %>%
mutate(key = str_remove_all(name, "b_"))
post_cox %>%
ggplot(aes(value, key, fill = key)) +
geom_vline(xintercept = 0, linetype = 2) +
stat_halfeye(alpha = .8) +
scale_fill_brewer(palette = "Spectral") +
ylab("Model parameter") +
xlab("Effect on Wing length") +
theme_ridges() +
theme(legend.position = "none") +
NULL
# wrangle
f <-
fixef(cox_brm) %>%
data.frame() %>%
rownames_to_column() %>%
mutate(param = str_remove(rowname, "m|P")) %>%
tidyr::expand(nesting(Estimate, Q2.5, Q97.5, param),
days = 0:24) %>%
mutate(m = 1 - pexp(days, rate = 1 / exp(Estimate)),
ll = 1 - pexp(days, rate = 1 / exp(Q2.5)),
ul = 1 - pexp(days, rate = 1 / exp(Q97.5)))
# plot!
f %>%
ggplot(aes(x = days)) +
# geom_hline(yintercept = .5, linetype = 3, aes(color = param)) +
geom_ribbon(aes(ymin = ll, ymax = ul, fill = param),
alpha = 1/2) +
# geom_line(aes(y = m, aes(color = cols))) +
# scale_fill_manual(values = wes_palette("Moonrise2")[c(4, 1)], breaks = NULL) +
# scale_color_manual(values = wes_palette("Moonrise2")[c(4, 1)], breaks = NULL) +
# scale_y_continuous("proportion remaining", , breaks = c(0, .5, 1), limits = c(0, 1)) +
xlab("days to adoption") +
NULL
f %>%
ggplot(aes(x = days, y = m, colour = param)) +
geom_line() +
geom_ribbon(aes(ymin = ll, ymax = ul, fill = param), alpha = 1/2) +
scale_colour_manual(values = c("pink", "red", "skyblue", "blue")) +
scale_fill_manual(values = c("pink", "red", "skyblue", "blue")) +
theme_bw() +
theme() +
NULL
We measured the length of wing vein IV for a subset of flies as a correlate of body size, which may influence development time.
if(!file.exists("output/wing_brms.rds")){
wing_brms <- brm(Length ~ Treatment * Sex + (1|LINE) + (1|Seed),
data = wing_length,
iter = 5000, chains = 4, cores = 4,
prior = c(set_prior("normal(0,1)", class = "b"),
set_prior("cauchy(0,1)", class = "sd")),
control = list(max_treedepth = 20,
adapt_delta = 0.999)
)
saveRDS(wing_brms, "output/wing_brms.rds")
} else {
wing_brms <- readRDS('output/wing_brms.rds')
}
pp_check(wing_brms)
Plot posteriors.
# get posterior predictions
post_dat <- posterior_samples(wing_brms) %>%
as_tibble() %>%
select(contains("b_"), -contains("Intercept")) %>%
mutate(draw = 1:n()) %>%
pivot_longer(-draw) %>%
mutate(key = str_remove_all(name, "b_"))
post_dat %>%
ggplot(aes(value, key, fill = key)) +
geom_vline(xintercept = 0, linetype = 2) +
stat_halfeye(alpha = .8) +
scale_fill_brewer(palette = "Spectral") +
ylab("Model parameter") +
xlab("Effect on Wing length") +
theme_ridges() +
theme(legend.position = "none") +
NULL
wing_test <- bind_rows(
hypothesis(wing_brms, 'TreatmentP = 0')$hypothesis,
hypothesis(wing_brms, 'SexM = 0')$hypothesis,
hypothesis(wing_brms, 'TreatmentP:SexM = 0')$hypothesis
) %>%
mutate(Parameter = c('Polandry', 'Male', 'Polyandry x Male'),
across(2:5, round, 3)) %>%
#select(-Hypothesis) %>%
relocate(Parameter, Estimate, Est.Error, CI.Lower, CI.Upper, Star)
pvals <- bayestestR::p_direction(wing_brms) %>%
as.data.frame() %>%
mutate(vars = map_chr(str_split(Parameter, "_"), ~ .x[2]),
p_val = 1 - pd,
star = ifelse(p_val < 0.05, "\\*", "")) %>%
select(vars, p_val, star)
wing_test %>% mutate(vars = c('TreatmentP', 'SexM', 'TreatmentP.SexM')) %>%
left_join(pvals %>% filter(vars != 'Intercept'),
by = c("vars")) %>%
select(Parameter, Estimate, Est.Error, CI.Lower, CI.Upper, `p` = p_val, star) %>%
mutate(p = ifelse(p > 0.001, round(p, 3), '< 0.001')) %>%
rename(` ` = star) %>%
kable() %>%
kable_styling(full_width = FALSE)
Parameter | Estimate | Est.Error | CI.Lower | CI.Upper | p | |
---|---|---|---|---|---|---|
Polandry | 0.125 | 0.289 | -0.457 | 0.701 | 0.316 | |
Male | -1.764 | 0.042 | -1.847 | -1.681 | < 0.001 | * |
Polyandry x Male | 0.040 | 0.060 | -0.077 | 0.159 | 0.256 |
fitbrms <- wing_length %>%
modelr::data_grid(Treatment, Sex, LINE, Seed) %>%
add_fitted_draws(wing_brms) %>%
sample_frac(size = .5)
fitbrms %>%
mutate(Treatment = ifelse(Treatment == "M", "Monogamy", "Polyandry"),
Sex = ifelse(Sex == "F", "Female", "Male"),
var = paste(Treatment, Sex)) %>%
ggplot(aes(x = Sex, y = .value, colour = var)) +
geom_hline(yintercept = 0, linetype = 2) +
stat_pointinterval(position = position_dodge(0.4),
fill = NA, .width = c(0.5, 0.95), alpha = 0.7) +
scale_colour_manual(values = c("pink", "skyblue", "red", "blue"), name = "") +
labs(y = 'Wing vein IV') +
theme_bw() +
theme(legend.position = 'top',
axis.title.x = element_blank()) +
NULL
Males are smaller than females and there is no effect of selection treatment on wing length.
sessionInfo()
R version 4.0.3 (2020-10-10) Platform: x86_64-apple-darwin17.0 (64-bit) Running under: macOS Catalina 10.15.4 Matrix products: default BLAS: /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRblas.dylib LAPACK: /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRlapack.dylib locale: [1] en_AU.UTF-8/en_AU.UTF-8/en_AU.UTF-8/C/en_AU.UTF-8/en_AU.UTF-8 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] knitrhooks_0.0.4 knitr_1.30 kableExtra_1.1.0 tidybayes_2.0.3 [5] brms_2.14.4 Rcpp_1.0.4.6 nlme_3.1-149 lme4_1.1-23 [9] Matrix_1.2-18 coxme_2.2-16 bdsmatrix_1.3-4 survival_3.2-7 [13] ggridges_0.5.2 forcats_0.5.0 stringr_1.4.0 dplyr_1.0.0 [17] purrr_0.3.4 readr_1.3.1 tidyr_1.1.0 tibble_3.0.1 [21] ggplot2_3.3.2 tidyverse_1.3.0 workflowr_1.6.2 loaded via a namespace (and not attached): [1] readxl_1.3.1 backports_1.1.7 plyr_1.8.6 [4] igraph_1.2.5 svUnit_1.0.3 splines_4.0.3 [7] crosstalk_1.1.0.1 TH.data_1.0-10 rstantools_2.1.1 [10] inline_0.3.15 digest_0.6.25 htmltools_0.5.0 [13] rsconnect_0.8.16 fansi_0.4.1 magrittr_2.0.1 [16] openxlsx_4.1.5 modelr_0.1.8 RcppParallel_5.0.1 [19] matrixStats_0.56.0 xts_0.12-0 sandwich_2.5-1 [22] prettyunits_1.1.1 colorspace_1.4-1 blob_1.2.1 [25] rvest_0.3.5 haven_2.3.1 xfun_0.19 [28] callr_3.4.3 crayon_1.3.4 jsonlite_1.7.0 [31] zoo_1.8-8 glue_1.4.2 survminer_0.4.8 [34] gtable_0.3.0 emmeans_1.4.7 webshot_0.5.2 [37] V8_3.4.0 car_3.0-8 pkgbuild_1.0.8 [40] rstan_2.21.2 abind_1.4-5 scales_1.1.1 [43] mvtnorm_1.1-0 DBI_1.1.0 rstatix_0.5.0 [46] miniUI_0.1.1.1 viridisLite_0.3.0 xtable_1.8-4 [49] foreign_0.8-80 km.ci_0.5-2 stats4_4.0.3 [52] StanHeaders_2.21.0-3 DT_0.13 htmlwidgets_1.5.1 [55] httr_1.4.1 threejs_0.3.3 arrayhelpers_1.1-0 [58] ellipsis_0.3.1 farver_2.0.3 reshape_0.8.8 [61] pkgconfig_2.0.3 loo_2.3.1 dbplyr_1.4.4 [64] labeling_0.3 tidyselect_1.1.0 rlang_0.4.6 [67] reshape2_1.4.4 later_1.0.0 munsell_0.5.0 [70] cellranger_1.1.0 tools_4.0.3 cli_2.0.2 [73] generics_0.0.2 broom_0.5.6 evaluate_0.14 [76] fastmap_1.0.1 yaml_2.2.1 processx_3.4.2 [79] fs_1.4.1 zip_2.0.4 survMisc_0.5.5 [82] whisker_0.4 mime_0.9 projpred_2.0.2 [85] xml2_1.3.2 compiler_4.0.3 bayesplot_1.7.2 [88] shinythemes_1.1.2 rstudioapi_0.11 gamm4_0.2-6 [91] curl_4.3 ggsignif_0.6.0 reprex_0.3.0 [94] statmod_1.4.34 stringi_1.5.3 highr_0.8 [97] ps_1.3.3 Brobdingnag_1.2-6 lattice_0.20-41 [100] nloptr_1.2.2.1 markdown_1.1 KMsurv_0.1-5 [103] shinyjs_1.1 vctrs_0.3.0 pillar_1.4.4 [106] lifecycle_0.2.0 bridgesampling_1.0-0 estimability_1.3 [109] insight_0.8.4 data.table_1.12.8 httpuv_1.5.3.1 [112] R6_2.4.1 promises_1.1.0 rio_0.5.16 [115] gridExtra_2.3 codetools_0.2-16 boot_1.3-25 [118] colourpicker_1.0 MASS_7.3-53 gtools_3.8.2 [121] assertthat_0.2.1 rprojroot_1.3-2 withr_2.2.0 [124] shinystan_2.5.0 multcomp_1.4-13 bayestestR_0.6.0 [127] mgcv_1.8-33 parallel_4.0.3 hms_0.5.3 [130] grid_4.0.3 coda_0.19-3 minqa_1.2.4 [133] rmarkdown_2.5 carData_3.0-4 ggpubr_0.3.0 [136] git2r_0.27.1 shiny_1.4.0.2 lubridate_1.7.8 [139] base64enc_0.1-3 dygraphs_1.1.1.6