library(tidyverse)
library(magrittr)
library(rtracklayer)
library(glue)
library(pander)
library(scales)
library(plyranges)
library(yaml)
library(ngsReports)
library(ComplexUpset)
library(extraChIPs)
panderOptions("big.mark", ",")
panderOptions("missing", "")
panderOptions("table.split.table", Inf)
theme_set(
  theme_bw() +
    theme(
      text = element_text(size = 13),
      plot.title = element_text(hjust = 0.5)
    )
)
config <- read_yaml(here::here("config", "config.yml"))
macs2_path <- here::here(config$paths$macs2)
annotation_path <- here::here("output", "annotations")
macs2_cutoff <- config$params$macs2$callpeak %>% 
  str_extract("-[pq] [0-9\\.]+") %>% 
  str_remove_all("-") %>% 
  str_replace_all(" ", " < ")
if (is.na(macs2_cutoff)) macs2_cutoff <- "q < 0.05"
target <- params$target
samples <- read_tsv(here::here(config$samples))
samples <- samples[samples$target == target,]
stopifnot(nrow(samples) > 0)
samples$treatment <- as.factor(samples$treatment)
treat_levels <- levels(samples$treatment)
treat_colours <- hcl.colors(max(3, length(treat_levels)), "Zissou 1")[seq_along(treat_levels)]
names(treat_colours) <- treat_levels
accessions <- samples$accession
sq <- file.path(annotation_path, "chrom.sizes") %>% 
  read_tsv(col_names = c("seqnames", "seqlengths")) %>% 
  mutate(isCircular = FALSE, genome = config$reference$name) %>% 
  as.data.frame() %>% 
  as("Seqinfo") %>% 
  sortSeqlevels()
individual_peaks <- file.path(
  macs2_path, accessions, glue("{accessions}_peaks.narrowPeak")
) %>%
  importPeaks(seqinfo = sq) %>%
  endoapply(sort) %>% 
  endoapply(names_to_column, var = "accession") %>% 
  endoapply(mutate, accession = str_remove(accession, "_peak_[0-9]+$")) %>% 
  endoapply(
    mutate, 
    treatment = left_join(tibble(accession = accession), samples, by = "accession")$treatment
  ) %>% 
  setNames(accessions)
macs2_logs <- file.path(macs2_path, accessions, glue("{accessions}_callpeak.log")) %>%
  importNgsLogs() %>%
  dplyr::select(
    -contains("file"), -outputs, -n_reads, -alt_fragment_length
  ) %>%
  left_join(samples, by = c("name" = "accession")) %>%
  mutate(
    total_peaks = map_int(
      name,
      function(x) {
        length(individual_peaks[[x]])
      }
    )
  )

QC Visualisations

This section provides a simple series of visualisations to aid in the detection of any problematic samples.

  • Library Sizes: These are the total number of alignments contained in each bam file, as passed to macs2 callpeak (Zhang et al. 2008)
  • Peaks Detected: The number of peaks detected within each individual replicate are shown here, and provide clear guidance towards any samples where the IP may have been less successful, or there may be possible sample mis-labelling.
  • Peak Widths: The peak widths for each sample are shown as a percentile to capture the overall distribution of widths
  • FRIP: The Fraction of Reads in Peaks is a well-known ChIP-Seq metric for determining the success of the immuno-precipitation step (Landt et al. 2012)
  • Cross Correlations: This provides an estimate of average fragment length and an additional guide as to the relative success of the IP step (Landt et al. 2012)
macs2_logs %>%
  mutate(target = target) %>% 
  dplyr::select(
    sample = name, target, treatment,
    total_peaks, 
    reads = n_tags_treatment, read_length = tag_length,
    fragment_length
  ) %>%
  setNames(
    names(.) %>% str_replace_all("_", " ") %>% str_to_title()
  ) %>%
  pander(
    justify = "lllrrrr",
    caption = glue(
      "*Summary of results for `macs2 callpeak` on individual {target} samples.",
      "Total peaks indicates the number retained after applying the cutoff ", 
      "{macs2_cutoff} during the peak calling process.",
      "The fragment length as estimated by `macs2 predictd` is given in the final column.*",
      .sep = " "
    )
  )
Summary of results for macs2 callpeak on individual H3K27ac samples. Total peaks indicates the number retained after applying the cutoff q < 0.05 during the peak calling process. The fragment length as estimated by macs2 predictd is given in the final column.
Sample Target Treatment Total Peaks Reads Read Length Fragment Length
SRR8315186 H3K27ac E2 58,789 14,881,059 73 236
SRR8315187 H3K27ac E2 61,151 17,185,932 70 235
SRR8315188 H3K27ac E2 57,098 16,246,941 71 237
SRR8315189 H3K27ac E2DHT 58,802 16,401,078 68 242
SRR8315190 H3K27ac E2DHT 58,732 17,955,212 69 243
SRR8315191 H3K27ac E2DHT 58,227 15,842,936 67 236

Library Sizes

macs2_logs %>%
  ggplot(
    aes(name, n_tags_treatment)
  ) +
  geom_col(position = "dodge", fill = "royalblue") +
  geom_hline(
    aes(yintercept = mn),
    data = . %>%
      group_by(treatment) %>%
      summarise(mn = mean(n_tags_treatment)),
    linetype = 2,
    col = "grey"
  ) +
  facet_grid(~treatment, scales = "free_x", space = "free_x") +
  scale_y_continuous(expand = expansion(c(0, 0.05)), labels = comma) +
  labs(
    x = "Sample", y = "Library Size"
  ) +
  ggtitle(
    glue("{target}: Library Sizes")
  )
*Library sizes for each H3K27ac sample. The horizontal line indicates the mean library size for each treatment group.*

Library sizes for each H3K27ac sample. The horizontal line indicates the mean library size for each treatment group.

Peaks Detected

macs2_logs %>%
  ggplot(
    aes(name, total_peaks)
  ) +
  geom_col(fill = "royalblue") +
  geom_label(
    aes(x = name, y = total_peaks, label = lab),
    data = . %>%
      dplyr::filter(total_peaks > 0) %>% 
      mutate(
        lab = comma(total_peaks, accuracy = 1),
        total = total_peaks
      ),
    inherit.aes = FALSE, nudge_y = max(macs2_logs$total_peaks)/30,
    show.legend = FALSE
  ) +
  facet_grid(~treatment, scales = "free_x", space = "free_x") +
  scale_y_continuous(expand = expansion(c(0, 0.05)), labels = comma) +
  labs(
    x = "Sample",
    y = "Total Peaks",
    fill = "QC"
  ) +
  ggtitle(
    glue("{target}: Number of Peaks")
  )
*Peaks identified for each H3K27ac sample. The number of peaks passing the inclusion criteria (q < 0.05) are shown as labels.*

Peaks identified for each H3K27ac sample. The number of peaks passing the inclusion criteria (q < 0.05) are shown as labels.

Peak Widths

individual_peaks %>% 
  lapply(as_tibble, rangeAsChar = FALSE) %>% 
  bind_rows() %>% 
  arrange(accession, width) %>% 
  summarise(
    n = dplyr::n(), .by = c(accession, treatment, width)
  ) %>% 
  mutate(
    p = cumsum(n) / sum(n), .by = c(accession, treatment)
  ) %>% 
  ggplot(aes(width, p, colour = accession, group = accession)) +
  geom_line() +
  coord_cartesian(
    xlim = quantile(width(unlist(individual_peaks)), c(0.005, 0.995))
  ) +
  scale_y_continuous(labels = percent, expand = expansion(c(0, 0.05)))  +
  labs(x = "Peak Width (bp)", y = "Percentile", colour = "Sample")
*Peak widths for each sample shown as a cumulative percentile. The x-axis is restricted to the middle 99% of observed widths.*

Peak widths for each sample shown as a cumulative percentile. The x-axis is restricted to the middle 99% of observed widths.

FRIP

here::here(macs2_path, accessions, paste0(accessions, "_frip.tsv")) %>%
  setNames(accessions) %>%
  lapply(read_tsv) %>%
  bind_rows(.id = "accession") %>%
  left_join(samples, by = "accession") %>%
  ggplot(aes(accession, frip)) +
  geom_col(fill = "royalblue") +
  geom_hline(yintercept = 0.01, colour = "grey", linetype = 2) +
  facet_wrap(~treatment, scales = "free_x") +
  labs(x = "Sample", y = "FRIP") +
  scale_y_continuous(expand = expansion(c(0, 0.05)), labels = percent) +
  ggtitle(
    glue("{target}: Fraction of Reads In Peaks")
  )
*Fraction of Reads in Peaks. The common-use threshold of 1% is shown as the dashed line.*

Fraction of Reads in Peaks. The common-use threshold of 1% is shown as the dashed line.

Cross Correlations

here::here(
  macs2_path, accessions, glue("{accessions}_cross_correlations.tsv")
  ) %>%
  lapply(read_tsv) %>%
  setNames(accessions) %>%
  bind_rows(.id = "accession") %>%
  left_join(samples, by = "accession") %>%
  ggplot(aes(fl, correlation, colour = treatment)) +
  geom_point(alpha = 0.1) +
  geom_smooth(se = FALSE, method = 'gam', formula = y ~ s(x, bs = "cs")) +
  geom_label(
    aes(label = fl),
    data = . %>% 
      mutate(correlation = correlation + 0.025 * max(correlation)) %>% 
      dplyr::filter(correlation == max(correlation), .by = accession),
    show.legend = FALSE, alpha = 0.7
  ) +
  facet_wrap(~accession) +
  scale_colour_manual(values = treat_colours) +
  labs(
    x = "Distance (bp)",
    y = "Cross Correlation",
    colour = "Treatment"
  ) +
  ggtitle(
    glue("{target}: Cross Correlations")
  )
*Cross correlations between reads. The distance corresponding to the maximum height provides a good estimate of the most common fragment length, with labels indicating these lengths. Relative heights between replicate samples also indicate the success of the immuno-precipitation. It is important to note that no black or grey lists were applied during data preparation, and a more rigorous analysis using the outputs generated here should do so.*

Cross correlations between reads. The distance corresponding to the maximum height provides a good estimate of the most common fragment length, with labels indicating these lengths. Relative heights between replicate samples also indicate the success of the immuno-precipitation. It is important to note that no black or grey lists were applied during data preparation, and a more rigorous analysis using the outputs generated here should do so.

Individual Replicates

A brief summary of all detected peaks for the given target within each replicate is shown using an UpSet plot below.

df <- individual_peaks %>%
  makeConsensus(var = "qValue") %>% 
  as_tibble() %>% 
  mutate(qValue = vapply(qValue, min, numeric(1))) 
has_peaks <- vapply(accessions, function(x) sum(df[[x]]) > 0, logical(1)) %>%
  which() %>%
  names()
ql <- samples$accession %>% 
  intersect(has_peaks) %>%
  lapply(
    function(x) {
      col <- as.character(dplyr::filter(samples, accession == x)$treatment)
      upset_query(
        set = x, 
        fill = treat_colours[col]
      )
    }
  ) 
## Add the set of common peaks
if (nrow(dplyr::filter(df, if_all(all_of(accessions))))) {
  ql <- c(
    ql, 
    list(
      upset_query(intersect = accessions, color = "royalblue", only_components = "intersections_matrix")
    )
  ) 
}
## Add the set of common peaks
if (nrow(dplyr::filter(df, if_all(all_of(accessions))))) {
  ql <- c(
    ql, 
    list(
      upset_query(intersect = accessions, color = "royalblue", only_components = "intersections_matrix")
  )
  )
}
## Treatment specific common peaks
split_acc <- lapply(split(samples, samples$treatment), pull, accession)
if (length(split_acc) > 1) {
  treat_ql <- seq_along(split_acc) %>% 
    lapply(
      function(i) {
        ids <- split_acc[[i]]
        others <- setdiff(unlist(split_acc), ids)
        nr <- df %>% 
          dplyr::filter(!if_all(all_of(ids)), !if_any(all_of(others))) %>% 
          nrow()
        if (nr > 10) return(ids)
        NULL
      }
    ) %>% 
    .[vapply(., length, integer(1)) > 0] %>% 
    lapply(
      function(x) {
        upset_query(intersect = x, color = "red", only_components = "intersections_matrix")        
      }
    )
  if (length(treat_ql)) ql <- c(ql, treat_ql)
}
size <- get_size_mode('exclusive_intersection')
df %>% 
  upset(
    intersect = accessions,
    base_annotations = list(
      `Peaks in Intersection` = intersection_size(
        text_mapping = aes(label = comma(!!size)),
        bar_number_threshold = 1, text_colors = "black", 
        text = list(size = 3, angle = 90, vjust = 0.5, hjust = -0.1)
      ) +
        scale_y_continuous(expand = expansion(c(0, 0.2)), label = comma) +
        theme(
          panel.grid = element_blank(), 
          axis.line = element_line(colour = "grey20"),
          panel.border = element_rect(colour = "grey20", fill =  NA)
        )
    ),
    annotations = list(
      qValue = ggplot(mapping = aes(y = qValue)) +
        geom_boxplot(na.rm = TRUE, outlier.colour = rgb(0, 0, 0, 0.2)) +
        coord_cartesian(ylim = c(0, quantile(df$qValue, 0.95))) +
        scale_y_continuous(expand = expansion(c(0, 0.05))) +
        ylab(expr(paste("Macs2 ", q[min]))) +
        theme(
          panel.grid = element_blank(), 
          axis.line = element_line(colour = "grey20"),
          panel.border = element_rect(colour = "grey20", fill =  NA)
        )
    ),
    set_sizes = (
      upset_set_size() +
        geom_text(
          aes(label = comma(after_stat(count))), 
          hjust = 1.1, stat = 'count', size = 3
        ) +
        scale_y_reverse(expand = expansion(c(0.2, 0)), label = comma) +
        ylab(glue("Macs2 Peaks")) +
        theme(
          panel.grid = element_blank(), 
          axis.line = element_line(colour = "grey20"),
          panel.border = element_rect(colour = "grey20", fill =  NA)
        )
    ),
    queries = ql,
    min_size = 10,
    n_intersections = 35,
    keep_empty_groups = TRUE,
    sort_sets = FALSE
  ) +
  theme(
    panel.grid = element_blank(), 
    panel.border = element_rect(colour = "grey20", fill =  NA),
    axis.line = element_line(colour = "grey20")
  ) +
  labs(x = "Intersection") +
  patchwork::plot_layout(heights = c(2, 3, 2))
*UpSet plot showing all samples. Any potential sample/treatment mislabelling will show up clearly here as samples from each group may show a preference to overlap other samples within the same treatment group. Peaks shared between all samples, and exclusive to those within each treatment group, are highlighted if found. Intersections are only included if 10 or more sites are present. The top panel shows a boxplot of the min $q$-values produced by `macs2 callpeak` for each peak in the intersection as representative of the sample with the weakest signal for each peak. The y-axis for the top panel is truncated at the 95^th^ percentile of values.*

UpSet plot showing all samples. Any potential sample/treatment mislabelling will show up clearly here as samples from each group may show a preference to overlap other samples within the same treatment group. Peaks shared between all samples, and exclusive to those within each treatment group, are highlighted if found. Intersections are only included if 10 or more sites are present. The top panel shows a boxplot of the min \(q\)-values produced by macs2 callpeak for each peak in the intersection as representative of the sample with the weakest signal for each peak. The y-axis for the top panel is truncated at the 95th percentile of values.

Merged Replicates

In addition to the above sets of peaks, sets of peaks were called using merged samples within each treatment group.

merged_peaks <- file.path(
  macs2_path, target, glue("{treat_levels}_merged_peaks.narrowPeak")
) %>% 
  importPeaks(seqinfo = sq) %>% 
  setNames(treat_levels) %>% 
  endoapply(plyranges::remove_names) %>% 
  endoapply(sort)
merged_logs <- file.path(
  macs2_path, target, glue("{treat_levels}_merged_callpeak.log")
) %>%
  importNgsLogs() %>%
  dplyr::select(
    -contains("file"), -outputs, -n_reads, -alt_fragment_length
  ) %>% 
  mutate(
    name = str_remove_all(name, "_merged"),
    total_peaks = map_int(
      name,
      function(x) {
        length(merged_peaks[[x]])
      }
    )
  ) 
merged_logs %>%
  mutate(target = target) %>% 
  dplyr::select(
    target, treatment = name, 
    total_peaks, 
    reads = n_tags_treatment, read_length = tag_length,
    fragment_length
  ) %>%
  setNames(
    names(.) %>% str_replace_all("_", " ") %>% str_to_title()
  ) %>%
  pander(
    justify = "llrrrr",
    caption = glue(
      "*Summary of results for `macs2 callpeak` on merged {target} samples within treatment groups.",
      "Total peaks indicates the number retained after applying the specified FDR", 
      "threshold during the peak calling process.",
      "The fragment length as estimated by `macs2 predictd` is given in the final column.*",
      .sep = " "
    )
  )
Summary of results for macs2 callpeak on merged H3K27ac samples within treatment groups. Total peaks indicates the number retained after applying the specified FDR threshold during the peak calling process. The fragment length as estimated by macs2 predictd is given in the final column.
Target Treatment Total Peaks Reads Read Length Fragment Length
H3K27ac E2 51,513 48,313,932 71 226
H3K27ac E2DHT 50,912 50,199,226 68 230

Merged Peak Overlaps

plotOverlaps(merged_peaks, set_col = treat_colours)
*Overlap between peaks in each treatment-specific set of peaks called by merging replicates.*

Overlap between peaks in each treatment-specific set of peaks called by merging replicates.

Merged Peak Widths

merged_peaks %>% 
  lapply(as_tibble, rangeAsChar = FALSE) %>% 
  bind_rows(.id = "treatment") %>% 
  ggplot(aes(width, after_stat(density), fill = treatment, colour = treatment)) +
  geom_density(alpha = 0.3) +
  scale_x_log10(expand = expansion(c(0, 0.05))) +
  scale_y_continuous(expand = expansion(c(0, 0.05))) +
  scale_fill_manual(values = treat_colours) +
  scale_colour_manual(values = treat_colours) +
  labs(x = "Peak Width (bp)", y  = "Density", fill = "Treatment", colour = "Treatment") +
  ggtitle(
    glue("{target}: Peak Width Distributions for Merged Samples")
  )
*Distributions of peak widths when using merged samples for H3K27ac within each treatment group*

Distributions of peak widths when using merged samples for H3K27ac within each treatment group

Merged Peaks By Individal Replicate

samples %>% 
  split(.$treatment) %>% 
  lapply(
    function(x) {
      trt <- unique(x$treatment)
      smp <- unique(x$accession)
      ol <- lapply(individual_peaks[smp], overlapsAny, subject = merged_peaks[[trt]])
      tbl <- lapply(ol, function(i) tibble(n = length(i), ol = sum(i), p = mean(i)))
      bind_rows(tbl, .id = "accession")
    }
  ) %>% 
  bind_rows(.id = "treatment") %>% 
  mutate(target = target) %>% 
  ggplot() +
  geom_col(aes(accession, n), fill = "grey70", alpha = 0.7) +
  geom_col(aes(accession, ol), fill = "royalblue") +
  geom_label(
    aes(accession, 0.5 * ol, label = percent(p, 0.1)),
    fill = "white", alpha = 0.7
  ) +
  facet_wrap(~treatment, nrow = 1, scales = "free_x") +
  scale_y_continuous(expand = expansion(c(0, 0.05)), labels = comma) +
  labs(x = "Sample", y = "Individual Peaks")
*Peaks indentified using merged samples and the overlap with individual samples. Bar heights indicate the total number of peaks identified in each replicate, with the blue segments indicating those also identified when merging replicates.*

Peaks indentified using merged samples and the overlap with individual samples. Bar heights indicate the total number of peaks identified in each replicate, with the blue segments indicating those also identified when merging replicates.

Merged Peaks By Number Of Overlaps

samples %>% 
  split(.$treatment) %>% 
  lapply(
    function(x) {
      trt <- unique(x$treatment)
      acc <- unique(x$accession)
      ol <- lapply(acc, function(i) overlapsAny(merged_peaks[[trt]], individual_peaks[i]))
      names(ol) <- acc
      tbl <- as.matrix(as.data.frame(ol))
      counts <- table(rowSums(tbl))
      tibble(reps = names(counts), total = as.integer(counts))
    }
  ) %>% 
  bind_rows(.id = "treatment") %>% 
  mutate(reps = as.factor(reps)) %>% 
  arrange(desc(reps)) %>% 
  mutate(cumsum = cumsum(total), p = total / sum(total), .by = treatment) %>% 
  mutate(y = cumsum - 0.5 * total) %>% 
  ggplot(aes(treatment, total, fill = reps)) +
  geom_col() +
  geom_label(
    aes(treatment, y, label = percent(p, 0.1)),
    data = . %>% dplyr::filter(p > 0.05),
    fill = "white", alpha = 0.7
  ) +
  scale_y_continuous(expand = expansion(c(0, 0.05)), labels = comma) +
  scale_fill_viridis_d(direction = -1) +
  labs(x = "Treatment", y = "Merged Peaks", fill = "Overlapping\nReplicates")
*Treatment-specific peaks called by merging individual replcates are shown by the number of individual replicates they overlap. The percentages of peaks with each overlapping number are shown within each bar.*

Treatment-specific peaks called by merging individual replcates are shown by the number of individual replicates they overlap. The percentages of peaks with each overlapping number are shown within each bar.

References

Landt, Stephen G, Georgi K Marinov, Anshul Kundaje, Pouya Kheradpour, Florencia Pauli, Serafim Batzoglou, Bradley E Bernstein, et al. 2012. “ChIP-seq Guidelines and Practices of the ENCODE and modENCODE Consortia.” Genome Res. 22 (9): 1813–31.
Zhang, Yong, Tao Liu, Clifford A Meyer, Jérôme Eeckhoute, David S Johnson, Bradley E Bernstein, Chad Nusbaum, et al. 2008. “Model-Based Analysis of ChIP-Seq (MACS).” Genome Biol. 9 (9): R137.

R version 4.2.3 (2023-03-15)

Platform: x86_64-conda-linux-gnu (64-bit)

locale: LC_CTYPE=en_AU.UTF-8, LC_NUMERIC=C, LC_TIME=en_AU.UTF-8, LC_COLLATE=en_AU.UTF-8, LC_MONETARY=en_AU.UTF-8, LC_MESSAGES=en_AU.UTF-8, LC_PAPER=en_AU.UTF-8, LC_NAME=C, LC_ADDRESS=C, LC_TELEPHONE=C, LC_MEASUREMENT=en_AU.UTF-8 and LC_IDENTIFICATION=C

attached base packages: stats4, stats, graphics, grDevices, utils, datasets, methods and base

other attached packages: extraChIPs(v.1.2.0), SummarizedExperiment(v.1.28.0), Biobase(v.2.58.0), MatrixGenerics(v.1.10.0), matrixStats(v.1.0.0), BiocParallel(v.1.32.5), ComplexUpset(v.1.3.3), ngsReports(v.2.0.0), patchwork(v.1.1.2), yaml(v.2.3.7), plyranges(v.1.18.0), scales(v.1.2.1), pander(v.0.6.5), glue(v.1.6.2), rtracklayer(v.1.58.0), GenomicRanges(v.1.50.0), GenomeInfoDb(v.1.34.9), IRanges(v.2.32.0), S4Vectors(v.0.36.0), BiocGenerics(v.0.44.0), magrittr(v.2.0.3), lubridate(v.1.9.2), forcats(v.1.0.0), stringr(v.1.5.0), dplyr(v.1.1.2), purrr(v.1.0.1), readr(v.2.1.4), tidyr(v.1.3.0), tibble(v.3.2.1), ggplot2(v.3.4.2) and tidyverse(v.2.0.0)

loaded via a namespace (and not attached): utf8(v.1.2.3), tidyselect(v.1.2.0), RSQLite(v.2.3.1), AnnotationDbi(v.1.60.0), htmlwidgets(v.1.6.2), grid(v.4.2.3), munsell(v.0.5.0), codetools(v.0.2-19), interp(v.1.1-4), DT(v.0.28), withr(v.2.5.0), colorspace(v.2.1-0), filelock(v.1.0.2), highr(v.0.10), knitr(v.1.43), rstudioapi(v.0.14), ggside(v.0.2.2), labeling(v.0.4.2), GenomeInfoDbData(v.1.2.9), polyclip(v.1.10-4), bit64(v.4.0.5), farver(v.2.1.1), rprojroot(v.2.0.3), vctrs(v.0.6.3), generics(v.0.1.3), lambda.r(v.1.2.4), xfun(v.0.39), biovizBase(v.1.46.0), timechange(v.0.2.0), csaw(v.1.32.0), BiocFileCache(v.2.6.0), R6(v.2.5.1), doParallel(v.1.0.17), clue(v.0.3-64), locfit(v.1.5-9.8), AnnotationFilter(v.1.22.0), bitops(v.1.0-7), cachem(v.1.0.8), DelayedArray(v.0.24.0), vroom(v.1.6.3), BiocIO(v.1.8.0), nnet(v.7.3-19), gtable(v.0.3.3), ensembldb(v.2.22.0), rlang(v.1.1.1), splines(v.4.2.3), GlobalOptions(v.0.1.2), lazyeval(v.0.2.2), dichromat(v.2.0-0.1), broom(v.1.0.5), checkmate(v.2.2.0), GenomicFeatures(v.1.50.2), backports(v.1.4.1), Hmisc(v.5.1-0), EnrichedHeatmap(v.1.27.2), tools(v.4.2.3), jquerylib(v.0.1.4), RColorBrewer(v.1.1-3), ggdendro(v.0.1.23), Rcpp(v.1.0.10), base64enc(v.0.1-3), progress(v.1.2.2), zlibbioc(v.1.44.0), RCurl(v.1.98-1.12), prettyunits(v.1.1.1), rpart(v.4.1.19), deldir(v.1.0-9), GetoptLong(v.1.0.5), zoo(v.1.8-12), ggrepel(v.0.9.3), cluster(v.2.1.4), here(v.1.0.1), data.table(v.1.14.8), futile.options(v.1.0.1), circlize(v.0.4.15), ProtGenerics(v.1.30.0), hms(v.1.1.3), evaluate(v.0.21), XML(v.3.99-0.14), VennDiagram(v.1.7.3), jpeg(v.0.1-10), gridExtra(v.2.3), shape(v.1.4.6), compiler(v.4.2.3), biomaRt(v.2.54.0), crayon(v.1.5.2), htmltools(v.0.5.5), mgcv(v.1.8-42), tzdb(v.0.4.0), Formula(v.1.2-5), DBI(v.1.1.3), tweenr(v.2.0.2), formatR(v.1.14), dbplyr(v.2.3.2), ComplexHeatmap(v.2.14.0), MASS(v.7.3-60), GenomicInteractions(v.1.32.0), rappdirs(v.0.3.3), Matrix(v.1.5-4.1), cli(v.3.6.1), parallel(v.4.2.3), Gviz(v.1.42.0), metapod(v.1.6.0), igraph(v.1.4.3), pkgconfig(v.2.0.3), GenomicAlignments(v.1.34.0), foreign(v.0.8-84), plotly(v.4.10.2), xml2(v.1.3.4), InteractionSet(v.1.26.0), foreach(v.1.5.2), bslib(v.0.5.0), XVector(v.0.38.0), VariantAnnotation(v.1.44.0), digest(v.0.6.31), Biostrings(v.2.66.0), rmarkdown(v.2.21), htmlTable(v.2.4.1), edgeR(v.3.40.0), restfulr(v.0.0.15), curl(v.5.0.1), Rsamtools(v.2.14.0), rjson(v.0.2.21), nlme(v.3.1-162), lifecycle(v.1.0.3), jsonlite(v.1.8.5), futile.logger(v.1.4.3), viridisLite(v.0.4.2), limma(v.3.54.0), BSgenome(v.1.66.3), fansi(v.1.0.4), pillar(v.1.9.0), lattice(v.0.21-8), KEGGREST(v.1.38.0), fastmap(v.1.1.1), httr(v.1.4.6), png(v.0.1-8), iterators(v.1.0.14), bit(v.4.0.5), ggforce(v.0.4.1), stringi(v.1.7.12), sass(v.0.4.6), blob(v.1.2.4), latticeExtra(v.0.6-30) and memoise(v.2.0.1)


Workflow developed by Dr Stevie Pederson1,2,3

1.Black Ochre Data Laboratories, Telethon Kids Institute, Adelaide, SA, Australia
2.John Curtin School of Medical Resarch, Australian National University, Canberra, ACT, Australia
3.Dame Roma Mitchell Cancer Research Laboratories, Adelaide Medical School, University of Adelaide, SA, Australia