--- title: 'Report breakdown by amplicon sequence' author: 'ampliCan' date: '`r format(Sys.time(), "%d %B %Y")`' output: html_document: toc: true theme: paper toc_float: true number_sections: true params: alignments: !r system.file('extdata', 'results', 'alignments', 'events_filtered_shifted_normalized.csv', package = 'amplican') config_summary: !r system.file('extdata', 'results', 'config_summary.csv', package = 'amplican') cut_buffer: 5 xlab_spacing: 4 vignette: > %\VignetteIndexEntry{example amplicon_report report} %\VignetteEngine{knitr::rmarkdown} %\VignetteEncoding{UTF-8} --- ```{r load data, message=F, warning=FALSE, include=FALSE} library(amplican) library(ggplot2) alignments <- data.table::fread(params$alignments) data.table::setDF(alignments) config <- data.frame(data.table::fread(params$config_summary)) config$AmpliconUpper <- toupper(config$Amplicon) uniqueAmlicons <- unique(config$AmpliconUpper) labels <- sapply(uniqueAmlicons, function(x) { toString(config$ID[config$AmpliconUpper == x]) }) height <- amplican::plot_height(length(uniqueAmlicons)) ``` *** # Description *** **Read distribution plot** - plot shows number of reads assigned during read grouping **Filtered Reads** - plot shows percentage of assigned reads that have been recognized as PRIMER DIMERS or filtered based on low alignment score **Cutting rates** - plot gives overview of percentage of reads (not filtered as PRIMER DIMER) that have cut **Frameshift** - plot shows what percentage of reads that have frameshift **Frameshift overlapping** - shows what percentage of reads have frameshift counting only deletions and insertions that overlap expected cut site (should be more accurate when controls are not available) **Read heterogeneity plot** - shows what is the share of each of the unique reads in total count of all reads. The more yellow each row, the less heterogeneity in the reads, more black means reads don't repeat often and are unique **Deletions plot** - shows summary of deletions detected after alignments with distinction for forward (top plot) and reverse (bottom) reads, blue dotted lines represent primers as black dotted line represents cut site box, for deletions overlapping with cut site box there is distinction in color **Mismatches plot** - shows summary of mismatches detected after alignments split by forward (top plot) and reverse (bottom) reads, mismatches are colored in the same manner as amplicon **Insertions plot** - shows summary of insertions detected after alignments split by forward (top plot) and reverse (bottom) reads, insertion is shown as right-angled triangle where size of the insertion corresponds to the width of the triangle, size and transparency of triangle reflect on the frequency of the insertion **Cuts plot** - shows summary of cuts in the amplicon (insertions that are overlapping with specified box of uppercase letters in the amplicon, and are supported by both forward and reverse reads) *** # Amplicon Summary *** ## Amplicon groups ```{r amplicon groups, echo=FALSE, fig.height=height, fig.width=14, message=F, warning=FALSE} library(knitr) ampliconDF <- data.frame(group = 1:length(uniqueAmlicons), IDs = unname(labels)) kable(ampliconDF) ampliconDF$Amplicon <- uniqueAmlicons #for sorting config$group <- match(config$AmpliconUpper, ampliconDF$Amplicon) ``` ## Read distribution ```{r plot_total_reads, echo=FALSE, fig.height=height, fig.width=14, message=F, warning=FALSE} ampliconTable <- aggregate(cbind(Reads, Reads_Filtered, PRIMER_DIMER, Low_Score, Reads_Indel, Reads_Frameshifted) ~ group, data = config, sum) ggplot(data = ampliconTable, aes(x = as.factor(group), y = log10(Reads + 1)), order = group) + geom_bar(stat = 'identity') + ylab('Number of reads + 1, log10 scaled') + xlab('Amplicon Group') + theme(legend.position = 'none', axis.text = element_text(size = 12), axis.title = element_text(size = 14, face = 'bold')) + coord_flip() + geom_text(aes(x = as.factor(group), y = log10(Reads + 1), label = Reads), hjust = -1) ``` ## Filtered Reads ```{r plot_F_per, echo=FALSE, fig.height=height, fig.width=14, message=F, warning=FALSE} ampliconTable$F_percentage <- (ampliconTable$PRIMER_DIMER + ampliconTable$Low_Score) * 100/ampliconTable$Reads ampliconTable$F_percentage[is.nan(ampliconTable$PD_percentage)] <- 0 ggplot(data = ampliconTable, aes(x = as.factor(group), y = F_percentage, order = group)) + geom_bar(stat='identity') + ylab('Percentage of filtered reads') + xlab('Amplicon Group') + theme(axis.text = element_text(size=12), axis.title = element_text(size=14, face = 'bold')) + ylim(0, 100) + coord_flip() + geom_text(aes(x = as.factor(group), y = F_percentage, label = PRIMER_DIMER), hjust = -1) ``` ## Indel rates ```{r plot_indel_rate, echo=FALSE, fig.height=height, fig.width=14, message=F, warning=FALSE} ampliconTable$indel_percentage <- ampliconTable$Reads_Indel * 100/ampliconTable$Reads_Filtered ampliconTable$indel_percentage[is.nan(ampliconTable$indel_percentage)] <- 0 ggplot(data = ampliconTable, aes(x = as.factor(group), y = indel_percentage, order = group)) + geom_bar(stat = 'identity') + ylab('Percentage of reads (not filtered) that have indel') + xlab('Amplicon Group') + theme(axis.text = element_text(size=12), axis.title = element_text(size=14, face = 'bold')) + ylim(0,100) + coord_flip() + geom_text(aes(x = as.factor(group), y = indel_percentage, label = Reads_Indel), hjust = -1) ``` ## Frameshift ```{r plot_frameshift_per, echo=FALSE, fig.height=height, fig.width=14, message=F, warning=FALSE} ampliconTable$frameshift_percentage <- ampliconTable$Reads_Frameshifted * 100/ampliconTable$Reads_Filtered ampliconTable$frameshift_percentage[is.nan(ampliconTable$frameshift_percentage)] <- 0 ggplot(data = ampliconTable, aes(x = as.factor(group), y = frameshift_percentage, order = group)) + geom_bar(position = 'stack', stat = 'identity') + ylab('Percentage of reads (not filtered) that have frameshift') + xlab('Amplicon Group') + theme(axis.text = element_text(size=12), axis.title = element_text(size=14,face = 'bold')) + ylim(0, 100) + coord_flip() + geom_text(aes(x = as.factor(group), y = frameshift_percentage, label = Reads_Frameshifted), hjust = -1) ``` ## Heterogeneity of reads ```{r plot read heterogeneity, echo=FALSE, fig.height=height + 1, fig.width=14, message=F, warning=FALSE} plot_heterogeneity(alignments, config, level = 'group') ``` *** # Alignments plots *** ```{r plot_alignments, results='asis', echo=F, message=F, warning=F} alignments <- alignments[alignments$consensus & alignments$overlaps, ] alignments$strand <- "+" # strand does not matter after consensus filtering src = sapply(seq_along(uniqueAmlicons), function(i) { ID = config$ID[config$AmpliconUpper == uniqueAmlicons[i]] ID = paste0("c('", paste0(ID, collapse = "','"), "')", collapse = "") knitr::knit_expand(text = c( "## Group {{i}} \n", "### Deletions \n", paste('```{r del-{{i}}, echo = F, results = "asis", ', 'fig.width=25, message=F, warning=F}', collapse = ''), paste('amplican::plot_deletions(alignments, config, {{ID}},', ' params$cut_buffer, params$xlab_spacing)', collapse = ''), '```\n', "### Insertions \n", paste('```{r ins-{{i}}, echo = F, results = "asis", ', 'fig.width=25, message=F, warning=F}', collapse = ''), paste('amplican::plot_insertions(alignments, config, {{ID}},', ' params$cut_buffer, params$xlab_spacing)', collapse = ''), '```\n', "### Mismatches \n", paste('```{r mis-{{i}}, echo = F, results = "asis", ', 'fig.width=25, message=F, warning=F}', collapse = ''), paste('amplican::plot_mismatches(alignments, config, {{ID}},', ' params$cut_buffer, params$xlab_spacing)', collapse = ''), '```\n', "### Cuts \n", paste('```{r cuts-{{i}}, echo = F, results = "asis", ', 'fig.width=25, message=F, warning=F}', collapse = ''), paste('amplican::plot_cuts(alignments, config, {{ID}},', ' params$cut_buffer, params$xlab_spacing)', collapse = ''), '```\n', "### Variants \n", paste('```{r var-{{i}}, echo = F, message=F, results = "asis", ', 'message=F, warning=F}', collapse = ''), paste('p <- amplican::plot_variants(alignments, config, {{ID}}, ', ' params$cut_buffer)', collapse = ''), '```\n')) }) # knit the source res = knitr::knit_child(text = src, quiet = TRUE) cat(res, sep = '\n') ```