| Title: | BadRegionFinder: an R/Bioconductor package for identifying regions with bad coverage |
|---|---|
| Description: | BadRegionFinder is a package for identifying regions with a bad, acceptable and good coverage in sequence alignment data available as bam files. The whole genome may be considered as well as a set of target regions. Various visual and textual types of output are available. |
| Authors: | Sarah Sandmann |
| Maintainer: | Sarah Sandmann <[email protected]> |
| License: | LGPL-3 |
| Version: | 1.35.0 |
| Built: | 2024-12-19 03:09:35 UTC |
| Source: | https://github.com/bioc/BadRegionFinder |
BadRegionFinder is a package for identifying regions with a bad, acceptable and good coverage in sequence alignment data available as bam files. The whole genome may be considered as well as a set of target regions. Various visual and textual types of output are available.
| Package: | BadRegionFinder |
| Type: | Package |
| Title: | BadRegionFinder: an R/Bioconductor package for identifying regions with bad coverage |
| Version: | 1.35.0 |
| Date: | 2016-03-07 |
| Author: | Sarah Sandmann |
| Maintainer: | Sarah Sandmann <[email protected]> |
| Description: | BadRegionFinder is a package for identifying regions with a bad, acceptable and good coverage in sequence alignment data available as bam files. The whole genome may be considered as well as a set of target regions. Various visual and textual types of output are available. |
| License: | LGPL-3 |
| Imports: | VariantAnnotation, Rsamtools, biomaRt, GenomicRanges, S4Vectors, utils, stats, grDevices, graphics |
| Suggests: | BSgenome.Hsapiens.UCSC.hg19 |
| biocViews: | Coverage, Sequencing, Alignment, WholeGenome, Classification |
| NeedsCompilation: | no |
| Config/pak/sysreqs: | make libicu-dev libpng-dev libxml2-dev libssl-dev |
| Repository: | https://bioc.r-universe.dev |
| RemoteUrl: | https://github.com/bioc/BadRegionFinder |
| RemoteRef: | HEAD |
| RemoteSha: | 7025a464be72b032f7e4eeda7acee690bb574b0e |
In the use case of targeted sequencing it is most important to design the set of used primers in a way that the targeted regions are sequenced with a sufficient coverage. Yet, due to e.g. high GC-content the aimed at coverage may not always be obtained. Thus, a tool performing a detailed coverage analysis comparing many samples at a time – and not considering all available samples individually – appears to be most useful. Furthermore, with regards to reads mapping off target, it seems helpful to have a tool for investigating those regions, which show a relatively high coverage, but which were not originally targeted.
BadRegionFinder is a package for classifying a selection of regions or the whole genome into the user-definable categories of bad, acceptable and good coverage in any sequence alignment data available as bam files. Various visual and textual types of output are available including detailed output files considering every base that is or should be covered and an overview file considering the coverage of the different genes that were targeted.
Index of help topics:
BadRegionFinder-package
BadRegionFinder: an R/Bioconductor package for
identifying regions with bad coverage
determineCoverage Determines the coverage (recommended for
whole-genome analyses)
determineCoverageQuality
Classifies the determined coverage
determineQuantiles Determines basewise user-defined quantiles
determineRegionsOfInterest
Determines the regions of interest
plotDetailed Plots a more detailed overview of the coverage
quality
plotSummary Plots a summary of the coverage quality
plotSummaryGenes Plots a summary of the coverage quality
concerning the genes only
reportBadRegionsDetailed
Gives a detailed report on the coverage quality
reportBadRegionsGenes Sums up the coverage quality on a gene basis
reportBadRegionsSummary
Sums up the coverage quality
The package contains a function performing the coverage determination - determineCoverage (switch for whole-genome- and target region analyses). The actual classification of the coverage is performed by the function determineCoverageQuality. If any subsets of regions are of interest, these may be selected by the function determineRegionsOfInterest.
There are three different forms of textual reports available: a summary variant (reportBadRegionsSummary), a detailed variant (reportBadRegionsDetailed) and a summary variant focussing on the coverage of the genes (reportBadRegionsGenes).
Furthermore, there exist three different forms of visual reports: a summary variant (plotSummary), a detailed variant (plotDetailed) and a summary variant visualizing the coverage of the genes as a barplot (plotSummaryGenes).
Additionally, BadRegionFinder may be used to determine user-definable, basewise quantiles over all samples at any position (determineQuantiles).
Sarah Sandmann
Maintainer: Sarah Sandmann <[email protected]>
More information on the bam format can be found at: http://samtools.github.io/hts-specs/SAMv1.pdf
determineCoverage, determineCoverageQuality, determineRegionsOfInterest, reportBadRegionsSummary, reportBadRegionsDetailed, reportBadRegionsGenes, plotSummary, plotDetailed, plotSummaryGenes, determineQuantiles
library("BSgenome.Hsapiens.UCSC.hg19") threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = FALSE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) badCoverageSummary <- reportBadRegionsSummary(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "", output) coverage_indicators_temp <- reportBadRegionsDetailed(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "", samples, output) badCoverageOverview <- reportBadRegionsGenes(threshold1, threshold2, percentage1, percentage2, badCoverageSummary, output) plotSummary(threshold1, threshold2, percentage1, percentage2, badCoverageSummary, output) plotDetailed(threshold1, threshold2, percentage1, percentage2, coverage_indicators_temp, output) plotSummaryGenes(threshold1, threshold2, percentage1, percentage2, badCoverageOverview, output) quantiles <- c(0.5) coverage_summary2 <- determineQuantiles(coverage_summary, quantiles, output)library("BSgenome.Hsapiens.UCSC.hg19") threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = FALSE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) badCoverageSummary <- reportBadRegionsSummary(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "", output) coverage_indicators_temp <- reportBadRegionsDetailed(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "", samples, output) badCoverageOverview <- reportBadRegionsGenes(threshold1, threshold2, percentage1, percentage2, badCoverageSummary, output) plotSummary(threshold1, threshold2, percentage1, percentage2, badCoverageSummary, output) plotDetailed(threshold1, threshold2, percentage1, percentage2, coverage_indicators_temp, output) plotSummaryGenes(threshold1, threshold2, percentage1, percentage2, badCoverageOverview, output) quantiles <- c(0.5) coverage_summary2 <- determineQuantiles(coverage_summary, quantiles, output)
BadRegionFinder performs a coverage analysis of various samples at a time. The first, essential step of the analysis pipeline – the coverage determination – is performed by the function determineCoverage. Thereby, the whole genome is scanned and wherever a covered base is registered or an originally targeted base is detected, detailed information concerning this position is written out.
determineCoverage(samples, bam_input, targetRegions, output, TRonly)determineCoverage(samples, bam_input, targetRegions, output, TRonly)
samples |
Data frame object containing the names of the samples to be analyzed (in one column). |
bam_input |
Folder containing the alignment data in bam- and bai format or BamFileList. |
targetRegions |
Data frame- or GRanges object containing the target regions to be analyzed (chromosome: first column, start position: second column and end position: third column). |
output |
The folder to write the output files into. If an empty string is provided, no files are written out. |
TRonly |
Boolean, indicating whether the coverage of the whole genome should be analyzed and reported ( |
The coverage which is determined by the function determineCoverage contains different steps:
For every sample that is defined in samples, the coverage is determined using the function coverage ("Determine Coverage"). To combine information on the coverage with information on whether a set of bases were originally targeted by some sequencing experiment, the targetRegions get processed ("Determine target bases"). Finally, the information gets combined ("Combine information"): Those positions where no sample shows any coverage and no target base is registered, are summed up. All other positions are reported basewise.
Files get written out in the form: "Summary_chr<chromosomename>.txt".
As sequencing does often not mean whole-genome- or whole-exome sequencing, but targeted sequencing, the function determienCoverage contains a switch: TRonly. In case misaligned reads in a targeted sequencing experiment shall be analyzed, it is advisable to set TRonly to FALSE. Yet, if only the coverage of the targeted regions are of interest, it is advisable to set TRonly to TRUE.
A GRangesList is returned. Every GRanges object contains the coverage information of one chromosome. The metadata columns contain information on the concrete coverage of each sample at a specific position. Furthermore, the column 'TargetBases' contains information on whether the considered region or position contains target bases (value 1) or not (value 0). A region cannot contain both as two regions would be defined in this case.
If a chromosome is not covered and was not targeted as well, the GRanges object solely contains a single line, considering a whole chromosome if TRonly=FALSE. If TRonly=TRUE the starting and end position of the corresponding chromosome is set to zero.
Sarah Sandmann <[email protected]>
BadRegionFinder, determineCoverageQuality, determineRegionsOfInterest, reportBadRegionsSummary, reportBadRegionsDetailed, reportBadRegionsGenes, plotSummary, plotDetailed, plotSummaryGenes, determineQuantiles
sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = FALSE)sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = FALSE)
The previously determined coverage (using determineCoverage with TRonly = TRUE or TRonly = FALSE) for all samples gets combined to be classified into six categories: bad coverage off target, bad coverage on target, acceptable coverage off target, acceptable coverage on target, good coverage off target, good coverage on target. These categories are user-defined.
determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary)determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary)
threshold1 |
Integer, threshold defining the number of reads that have to be registered for a sample that its coverage is classified as acceptable. |
threshold2 |
Integer, threshold defining the number of reads that have to be registered for a sample that its coverage is classified as good. To obtain useful results, |
percentage1 |
Float, defining the percentage of samples that have to feature a coverage of at least |
percentage2 |
Float, defining the percentage of samples that have to feature a coverage of at least |
coverage_summary |
GRangesList object, return value of function |
Every chromosome is analyzed individually. First, the coverage of each sample is categorized according to threshold1 and threshold2 into three different categories:
bad coverage: less than threshold1 reads
acceptable coverage: at least threshold1, but less than threshold2 reads
good coverage: at least threshold2 reads
Subsequently this information gets combined with the defined precentages to obtain a numerically coded quality value for each region saved in the previously created list object coverage_summary:
0: off target; not even percentage1 percent of all samples have a good or acceptable coverage (bad region)
1: on target; not even percentage1 percent of all samples have a good or acceptable coverage (bad region)
2: off target; at least percentage1 percent of all samples have a good or acceptable coverage, but less than percentage2 percent of all samples have a good coverage (acceptable region)
3: on target; at least percentage1 percent of all samples have a good or acceptable coverage, but less than percentage2 percent of all samples have a good coverage (acceptable region)
4: off target; at least percentage2 percent of all samples have a good coverage (good region)
5: on target; at least percentage2 percent of all samples have a good coverage (good region)
A list is returned. Every component contains the coverage information of one chromosome as a GRanges object. The metadata columns contain information on the concrete coverage of each sample at a specific position. Furthermore, the column 'TargetBases' contains information on whether the considered region or position contains target bases (value 1) or not (value 0). The column 'indicator' contains information on the coverage quality of the corresponding region/position.
If a chromosome is not covered and was not targeted as well, the GRanges object solely contains a single line, considering a whole chromosome if TRonly=FALSE. If TRonly=TRUE the corresponding component is "NA".
Sarah Sandmann <[email protected]>
BadRegionFinder, determineCoverage, determineRegionsOfInterest, reportBadRegionsSummary, reportBadRegionsDetailed, reportBadRegionsGenes, plotSummary, plotDetailed, plotSummaryGenes, determineQuantiles
threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = FALSE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary)threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = FALSE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary)
The function determineQuantiles provides a possibility to determine user-definable quantiles for every base previously analyzed. Thereby, the quantiles are determined over all samples.
determineQuantiles(coverage_summary, quantiles, output)determineQuantiles(coverage_summary, quantiles, output)
coverage_summary |
List object, return value of function |
quantiles |
Vector determining the quantiles to be calculated. |
output |
The folder to write the output files into. |
The function determineQuantiles serves to determine a set of user-defined quantiles at each position over all samples. Every single base is analyzed, except for the case when the bases were not originally targeted and if no coverage is detected by any of the samples. In this case the corresponding region is summed up.
Files get written out in the form: "Quantiles_chr<chromosomename>.txt".
A list is returned. Every component contains the coverage information of one chromosome as a GRanges object. The metadata columns contain information on the coverage according to the previously defined quantiles. Furthermore, the column 'TargetBases' contains information on whether the considered region or position contains target bases (value 1) or not (value 0).
If a chromosome has not been targeted and/or not covered by any sample, but defined in regionsOfInterest, the component is "NA".
Sarah Sandmann <[email protected]>
BadRegionFinder, determineCoverage, determineCoverageQuality, determineRegionsOfInterest, reportBadRegionsSummary, reportBadRegionsDetailed, reportBadRegionsGenes, plotSummary, plotDetailed, plotSummaryGenes
library("BSgenome.Hsapiens.UCSC.hg19") sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = TRUE) quantiles <- c(0.25, 0.5, 0.75) coverage_summary2 <- determineQuantiles(coverage_summary, quantiles, output)library("BSgenome.Hsapiens.UCSC.hg19") sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = TRUE) quantiles <- c(0.25, 0.5, 0.75) coverage_summary2 <- determineQuantiles(coverage_summary, quantiles, output)
The function determineRegionsOfInterest serves to select the coverage information (including the coverage of all samples and - depending on the input object - their assigned quality value) of one or more subsets of regions.
determineRegionsOfInterest(regionsOfInterest, coverage_indicators)determineRegionsOfInterest(regionsOfInterest, coverage_indicators)
regionsOfInterest |
Data frame- or GRanges object containing the regions of interest (if data frame: chromosome: first column, start position: second column and end position: third column). |
coverage_indicators |
List object, return value of function |
Every chromosome is analyzed individually. For every base defined in regionsOfInterest the previously determined coverage information is written out. The function thereby serves to select special subsets of regions, e.g. targeted and untargeted regions when using determineCoverage with TRonly=FALSE in the first place or particular targeted regions when using TRonly=TRUE.
It is not recommended to use TRonly=TRUE, but to select regions off target using determineRegionsOfInterest. In this case, no coverage will be registered for all bases off target, as this information was not saved during the step of determineCoverage.
If regionsOfInterest is identical compared to targetRegions and the function determineCoverage with TRonly=TRUE has been used in the first place, the selection of regions that is returned is not changed at all.
A list is returned. Every component contains the coverage information of one chromosomeas as a GRanges object. The metadata columns contain information on the concrete coverage of each sample at a specific position. Furthermore, the column 'TargetBases' contains information on whether the considered region or position contains target bases (value 1) or not (value 0). The column 'indicator' contains information on the coverage quality of the corresponding region/position (0: bad region off target; 1: bad region on target; 2: acceptable region off target; 3: acceptable region on target; 4: good region off target; 5: good region on target).
If a chromosome has not been targeted and/or not covered by any sample, but defined in regionsOfInterest, the component is "NA".
Sarah Sandmann <[email protected]>
BadRegionFinder, determineCoverage, determineCoverageQuality, reportBadRegionsSummary, reportBadRegionsDetailed, reportBadRegionsGenes, plotSummary, plotDetailed, plotSummaryGenes, determineQuantiles
threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = FALSE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) coverage_indicators_2 <- determineRegionsOfInterest(targetRegions, coverage_indicators)threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = FALSE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) coverage_indicators_2 <- determineRegionsOfInterest(targetRegions, coverage_indicators)
The function plotDetailed provides a possibility to visualize the output of reportBadRegionsDetailed. A line graph is returned, visualizing the median coverage at each base that was chosen to be in the region of interest. For each base, the category of coverage quality is color coded. Furthermore, information on the genes that are located at the positions analyzed is included.
plotDetailed(threshold1, threshold2, percentage1, percentage2, coverage_indicators_temp, output)plotDetailed(threshold1, threshold2, percentage1, percentage2, coverage_indicators_temp, output)
threshold1 |
Integer, threshold defining the number of reads that have to be registered for a sample that its coverage is classified as acceptable. |
threshold2 |
Integer, threshold defining the number of reads that have to be registered for a sample that its coverage is classified as good. |
percentage1 |
Float, defining the percentage of samples that have to feature a coverage of at least |
percentage2 |
Float, defining the percentage of samples that have to feature a coverage of at least |
coverage_indicators_temp |
List object, return value of function |
output |
The folder to write the output file into. If this argument is an empty string, the plot is printed on the screen. |
The function plotDetailed serves to summarize the previously determined coverage quality in a visual way, including additionally information on the mean coverage over all samples at every position.
On the y axis the median coverage over all samples is coded. Every position is considered individually.
On the x axis the detected genes are printed. Wherever a new region covering a new gene is registered, a dashed line is drawn.
Yet, additionally to the mere median coverage, the corresponding coverage quality at each position is also included in the plot. The different categories of coverage quality are color coded in the following way: red - bad region on target; yellow - acceptable region on target; green - good region on target; black - bad region off target; dark gray - acceptable region off target; light gray - good region off target.
No value is returned.
Sarah Sandmann <[email protected]>
BadRegionFinder, determineCoverage, determineCoverageQuality, determineRegionsOfInterest, reportBadRegionsSummary, reportBadRegionsDetailed, reportBadRegionsGenes, plotSummary, plotSummaryGenes, determineQuantiles
library("BSgenome.Hsapiens.UCSC.hg19") threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = FALSE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) coverage_indicators_temp <- reportBadRegionsDetailed(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "", samples, output) plotDetailed(threshold1, threshold2, percentage1, percentage2, coverage_indicators_temp, output)library("BSgenome.Hsapiens.UCSC.hg19") threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = FALSE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) coverage_indicators_temp <- reportBadRegionsDetailed(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "", samples, output) plotDetailed(threshold1, threshold2, percentage1, percentage2, coverage_indicators_temp, output)
The function plotSummary provides a possibility to visualize the output of reportBadRegionsSummary. A line graph is returned, visualizing the number of bases that fall into each category of coverage quality. Furthermore, information on the genes located in these regions is included.
plotSummary(threshold1, threshold2, percentage1, percentage2, badCoverageSummary, output)plotSummary(threshold1, threshold2, percentage1, percentage2, badCoverageSummary, output)
threshold1 |
Integer, threshold defining the number of reads that have to be registered for a sample that its coverage is classified as acceptable. |
threshold2 |
Integer, threshold defining the number of reads that have to be registered for a sample that its coverage is classified as good. |
percentage1 |
Float, defining the percentage of samples that have to feature a coverage of at least |
percentage2 |
Float, defining the percentage of samples that have to feature a coverage of at least |
badCoverageSummary |
GRanges object, return value of function |
output |
The folder to write the output file into. If this argument is an empty string, the plot is printed on the screen. |
The function plotSummary serves to summarize the previously determined coverage quality in a visual way.
On the y axis the coverage quality is coded. The different categories are color coded as well as height coded. As numbers from 0 to 5 were previously assigned to the different categories, thick lines are now drawn at the height of the category. Furthermore, the categories are color coded in the following way: red - bad region on target; yellow - acceptable region on target; green - good region on target; black - bad region off target; dark gray - acceptable region off target; light gray - good region off target.
On the x axis the detected genes are printed. Wherever a new region covering a new gene is registered, a dashed line is drawn.
No value is returned.
Sarah Sandmann <[email protected]>
BadRegionFinder, determineCoverage, determineCoverageQuality, determineRegionsOfInterest, reportBadRegionsSummary, reportBadRegionsDetailed, reportBadRegionsGenes, plotDetailed, plotSummaryGenes, determineQuantiles
library("BSgenome.Hsapiens.UCSC.hg19") threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = TRUE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) badCoverageSummary <- reportBadRegionsSummary(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "", output) plotSummary(threshold1, threshold2, percentage1, percentage2, badCoverageSummary, output)library("BSgenome.Hsapiens.UCSC.hg19") threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = TRUE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) badCoverageSummary <- reportBadRegionsSummary(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "", output) plotSummary(threshold1, threshold2, percentage1, percentage2, badCoverageSummary, output)
The function plotSummaryGenes provides a possibility to visualize the output of reportBadRegionsGenes. A barplot is returned, visualizing the percent of each gene that falls into each category of coverage quality. The plot thereby serves to quickly distinguish well from bad covered genes.
plotSummaryGenes(threshold1, threshold2, percentage1, percentage2, badCoverageGenes, output)plotSummaryGenes(threshold1, threshold2, percentage1, percentage2, badCoverageGenes, output)
threshold1 |
Integer, threshold defining the number of reads that have to be registered for a sample that its coverage is classified as acceptable. |
threshold2 |
Integer, threshold defining the number of reads that have to be registered for a sample that its coverage is classified as good. |
percentage1 |
Float, defining the percentage of samples that have to feature a coverage of at least |
percentage2 |
Float, defining the percentage of samples that have to feature a coverage of at least |
badCoverageGenes |
Data frame object, return value of function |
output |
The folder to write the output file into. If this argument is an empty string, the plot is printed on the screen. |
The function plotSummaryGenes serves to summarize the previously determined coverage quality in a visual way concerning the genes only.
For every gene either one or two stacked bars are plotted. If a gene is covered, but it was not originally targeted, a bar is plotted containing the following color code: black - bad region off target; dark gray - acceptable region off target; light gray - good region off target. If a gene was originally targeted, a bar is plotted containing the following color code: red - bad region on target; yellow - acceptable region on target; green - good region on target.
No value is returned.
Sarah Sandmann <[email protected]>
BadRegionFinder, determineCoverage, determineCoverageQuality, determineRegionsOfInterest, reportBadRegionsSummary, reportBadRegionsDetailed, reportBadRegionsGenes, plotSummary, plotDetailed, determineQuantiles
library("BSgenome.Hsapiens.UCSC.hg19") threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = TRUE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) badCoverageSummary <- reportBadRegionsSummary(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "", output) badCoverageGenes <- reportBadRegionsGenes(threshold1, threshold2, percentage1, percentage2, badCoverageSummary, output) plotSummaryGenes(threshold1, threshold2, percentage1, percentage2, badCoverageGenes, output)library("BSgenome.Hsapiens.UCSC.hg19") threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = TRUE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) badCoverageSummary <- reportBadRegionsSummary(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "", output) badCoverageGenes <- reportBadRegionsGenes(threshold1, threshold2, percentage1, percentage2, badCoverageSummary, output) plotSummaryGenes(threshold1, threshold2, percentage1, percentage2, badCoverageGenes, output)
The function reportBadRegionsDetailed creates a detailed report containing all regions of interest (basewise), the coverage of each sample at the corresponding positions, the indicator whether the bases were originally targeted, their coverage quality and the corresponding gene (name and geneID).
reportBadRegionsDetailed(threshold1, threshold2, percentage1, percentage2, coverage_indicators, mart, samples, output)reportBadRegionsDetailed(threshold1, threshold2, percentage1, percentage2, coverage_indicators, mart, samples, output)
threshold1 |
Integer, threshold defining the number of reads that have to be registered for a sample that its coverage is classified as acceptable. |
threshold2 |
Integer, threshold defining the number of reads that have to be registered for a sample that its coverage is classified as good. |
percentage1 |
Float, defining the percentage of samples that have to feature a coverage of at least |
percentage2 |
Float, defining the percentage of samples that have to feature a coverage of at least |
coverage_indicators |
List object, return value of function |
mart |
mart as defined in the manual for package 'biomaRt'. If the human genome (hg19) shall be used, an empty string may be provided and the mart is automatically generated. |
samples |
Data frame object containing the names of the samples to be analyzed (in one column). |
output |
The folder to write the output files into. If |
To gain more detailed information of the coverage quality, a file for every chromosome to be analyzed may be created by the function reportBadRegionsDetailed. The function may either take information on the whole genome (output from determineCoverage with TRonly=FALSE, processed using determineCoverageQuality) as an input, or information on the target regions (output from determineCoverage with TRonly=TRUE, processed using determineCoverageQuality), or information on a selection of regions of interest (output from determineRegionsOfInterest).
Different from the summed-up variant reportBadRegionsSummary, information on every single base of interest gets reported (except for completely uncovered and untargeted regions, which are summed up). For every base its position, the coverage of each sample, information on whether this base was originally targeted (value 1) or not (value 0), the coverage quality and the most likely gene (name and geneID) that was targeted by the original experiment get reported. Information on the gene names and the geneIDs results from biomaRt. If no gene can be found at a position, "NA" is reported for the corresponding base.
The output files are saved as: "BadCoverageChromosome<chromosomename>;threshold1;percentage1;threshold2;percentage2.txt". The output file may be visualized using plotDetailed.
A list is returned. Every component contains the coverage information of one chromosome as a GRanges object. The metadata columns contain information on the concrete coverage of each sample at a specific position. Furthermore, the column 'TargetBases' contains information on whether the considered region or position contains target bases (value 1) or not (value 0). The column 'indicator' contains information on the coverage quality of the corresponding region/position (0: bad region off target; 1: bad region on target; 2: acceptable region off target; 3: acceptable region on target; 4: good region off target; 5: good region on target). Furthermore, the name and the geneID of the gene that is located at the corresponding position is saved.
If a chromosome is not covered and was not targeted as well, the component is "NA".
Sarah Sandmann <[email protected]>
More information on the R/Bioconductor package 'biomaRt' may be found at:
http://www.bioconductor.org/packages/release/bioc/html/biomaRt.html
BadRegionFinder, determineCoverage, determineCoverageQuality, determineRegionsOfInterest, reportBadRegionsSummary, reportBadRegionsGenes, plotSummary, plotDetailed, plotSummaryGenes, determineQuantiles
library("BSgenome.Hsapiens.UCSC.hg19") threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = TRUE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) coverage_indicators_temp <- reportBadRegionsDetailed(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "", samples, output)library("BSgenome.Hsapiens.UCSC.hg19") threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = TRUE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) coverage_indicators_temp <- reportBadRegionsDetailed(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "", samples, output)
The function reportBadRegionsGenes creates a summary report considering the coverage quality on a genewise level. Taking the output of reportBadRegionsSummary as an input, the coverage quality for every previously identified gene is reported.
reportBadRegionsGenes(threshold1, threshold2, percentage1, percentage2, badCoverageSummary, output)reportBadRegionsGenes(threshold1, threshold2, percentage1, percentage2, badCoverageSummary, output)
threshold1 |
Integer, threshold defining the number of reads that have to be registered for a sample that its coverage is classified as acceptable. |
threshold2 |
Integer, threshold defining the number of reads that have to be registered for a sample that its coverage is classified as good. |
percentage1 |
Float, defining the percentage of samples that have to feature a coverage of at least |
percentage2 |
Float, defining the percentage of samples that have to feature a coverage of at least |
badCoverageSummary |
Data frame object, return value of function |
output |
The folder to write the output file into. If |
To gain an overview of the coverage quality of each targeted/covered gene, a summary file may be created by the function reportBadRegionsGenes. The function takes the output of reportBadRegionsSummary as an input.
All regions covering the same gene are summed up in the following way:
The number of bases falling into each quality category is summed up. Thereby, regions which were orignially targeted may easily be separated from those which were not, as targeted regions always feature an uneven number characterizing their coverage quality. If a region is broader than the detected gene, but the quality category is the same for the whole region, the whole region is assigned to the gene.
If no gene is reported in the input file, the coverage quality is summed up for a gene named "NA".
The output file is saved as: "BadCoverageGenesthreshold1;percentage1;threshold2;percentage2.txt". The output file may be visualized using plotSummaryGenes.
A data frame object is returned. The first column contains the name and the geneID of the gene. The following columns contain the percentage of bases falling into the following categories: bad region off traget, bad region on target, acceptable region off target, acceptable region on target, good region off target, good region on target.
Sarah Sandmann <[email protected]>
BadRegionFinder, determineCoverage, determineCoverageQuality, determineRegionsOfInterest, reportBadRegionsSummary, reportBadRegionsDetailed, plotSummary, plotDetailed, plotSummaryGenes, determineQuantiles
library("BSgenome.Hsapiens.UCSC.hg19") threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = TRUE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) badCoverageSummary <- reportBadRegionsSummary(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "",output) badCoverageGenes <- reportBadRegionsGenes(threshold1, threshold2, percentage1, percentage2, badCoverageSummary, output)library("BSgenome.Hsapiens.UCSC.hg19") threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = TRUE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) badCoverageSummary <- reportBadRegionsSummary(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "",output) badCoverageGenes <- reportBadRegionsGenes(threshold1, threshold2, percentage1, percentage2, badCoverageSummary, output)
The function reportBadRegionsSummary creates a summary report containing all regions of interest, their coverage quality and the corresponding gene (name and geneID).
reportBadRegionsSummary(threshold1, threshold2, percentage1, percentage2, coverage_indicators, mart, output)reportBadRegionsSummary(threshold1, threshold2, percentage1, percentage2, coverage_indicators, mart, output)
threshold1 |
Integer, threshold defining the number of reads that have to be registered for a sample that its coverage is classified as acceptable. |
threshold2 |
Integer, threshold defining the number of reads that have to be registered for a sample that its coverage is classified as good. |
percentage1 |
Float, defining the percentage of samples that have to feature a coverage of at least |
percentage2 |
Float, defining the percentage of samples that have to feature a coverage of at least |
coverage_indicators |
List object, return value of function |
mart |
mart as defined in the manual for package 'biomaRt'. If the human genome (hg19) shall be used, an empty string may be provided and the mart is automatically generated. |
output |
The folder to write the output file into. If |
To gain an overview of the coverage quality, a summary file may be created by the function reportBadRegionsSummary. The function may either take information on the whole genome (output from determineCoverage with TRonly=FALSE, processed using determineCoverageQuality) as an input, or information on the target regions (output from determineCoverage with TRonly=TRUE, processed using determineCoverageQuality), or information on a selection of regions of interest (output from determineRegionsOfInterest).
Wherever subsequent bases feature the same coverage quality, the region gets summed up. Although it is not directly reported whether a region contains on or off target bases, this information can be gained from the coverage quality: all bases off target feature an even number characterizing the coverage quality; all bases on target feature an uneven number characterizing the coverage quality.
For each summed up region the gene that is most likely to be targeted by the original experiment gets reported using biomaRt. If no gene can be found, "NA" is saved for the corresponding region. If not all bases in the summed up region cover a gene, the gene gets reported for the whole region nonetheless.
The output file is saved as: "BadCoverageSummarythreshold1;percentage1;threshold2;percentage2.txt". The output file may be visualized using plotSummary.
A GRanges object is returned. It represents a summary of the those adjacent regions that feature the same base quality. In the metadata columns the coverage quality of the region, the name and the geneID of the gene that is located in the corresponding region is saved.
Sarah Sandmann <[email protected]>
More information on the R/Bioconductor package 'biomaRt' may be found at:
http://www.bioconductor.org/packages/release/bioc/html/biomaRt.html
BadRegionFinder, determineCoverage, determineCoverageQuality, determineRegionsOfInterest, reportBadRegionsDetailed, reportBadRegionsGenes, plotSummary, plotDetailed, plotSummaryGenes, determineQuantiles
library("BSgenome.Hsapiens.UCSC.hg19") threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = TRUE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) badCoverageSummary <- reportBadRegionsSummary(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "", output)library("BSgenome.Hsapiens.UCSC.hg19") threshold1 <- 20 threshold2 <- 100 percentage1 <- 0.80 percentage2 <- 0.90 sample_file <- system.file("extdata", "SampleNames.txt", package = "BadRegionFinder") samples <- read.table(sample_file) bam_input <- system.file("extdata", package = "BadRegionFinder") output <- system.file("extdata", package = "BadRegionFinder") target_regions <- system.file("extdata", "targetRegions.bed", package = "BadRegionFinder") targetRegions <- read.table(target_regions, header = FALSE, stringsAsFactors = FALSE) coverage_summary <- determineCoverage(samples, bam_input, targetRegions, output, TRonly = TRUE) coverage_indicators <- determineCoverageQuality(threshold1, threshold2, percentage1, percentage2, coverage_summary) badCoverageSummary <- reportBadRegionsSummary(threshold1, threshold2, percentage1, percentage2, coverage_indicators, "", output)