Workflow example
30 April 2018
Abstract
This workflow vignette shows how to use the SNPhood package in a real-world example. For this purpose, you will use the SNPhoodData package for a more complex analysis to illustrate most of the features from SNPhood. Importantly, you will also learn in detail how to work with a SNPhood object and what its main functions and properties are. The vignette will be continuously updated whenever new functionality becomes available or when we receive user feedback.Package
SNPhood 1.10.0
Contents
- 1 Example Workflow
- 1.1 Data check and collection
- 1.2 Setting up SNPhood
- 1.3 Quality control
- 1.4 Executing the main function
- 1.5 Working with a SNPhood object: Extracting and manipulating information and metadata
- 1.6 Visualizing counts and enrichment
- 1.7 Testing for and visualizing allelic biases
- 1.8 Cluster analyses
- 1.9 Genotype analyses
- 1.10 Combined cluster and genotype analyses
- 1.11 How to continue?
- 2 Bug Reports, Feature Requests and Contact Information
- 3 References
1 Example Workflow
In the following example, you will use data from the SNPhoodData package to address the question how many of the previously identified H3K27ac quantitative trait loci (QTLs) for individuals from the Yoruban (YRI) population [1] show allele-specific signal in individuals of European origin (CEU).
First, let’s load the required libraries SNPhood and SNPhoodData.
library(SNPhoodData)
library(SNPhood)1.1 Data check and collection
Now let’s check the data you will use for the following analysis:
(files = list.files(pattern = "*", system.file("extdata", package = "SNPhoodData"),
full.names = TRUE))## [1] "/home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM10847_H3K27AC_1_reconcile.dedup.chr21.bam"
## [2] "/home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM10847_H3K27AC_1_reconcile.dedup.chr21.bam.bai"
## [3] "/home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM10847_H3K27AC_2_reconcile.dedup.chr21.bam"
## [4] "/home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM10847_H3K27AC_2_reconcile.dedup.chr21.bam.bai"
## [5] "/home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM12890_H3K27AC_1_reconcile.dedup.chr21.bam"
## [6] "/home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM12890_H3K27AC_1_reconcile.dedup.chr21.bam.bai"
## [7] "/home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM12890_H3K27AC_2_reconcile.dedup.chr21.bam"
## [8] "/home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM12890_H3K27AC_2_reconcile.dedup.chr21.bam.bai"
## [9] "/home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/cisQ.H3K27AC.chr21.txt"
## [10] "/home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/genotypes.vcf.gz"fileUserRegions = files[grep(".txt", files)]
fileGenotypes = files[grep("genotypes", files)]The data comprises:
- a file with the user-defined regions (in this case, H3K27ac hQTLs from chromosome 21: cisQ.H3K27AC.chr21.txt)
- H3K27ac ChIP-Seq files in BAM format for two individuals (two replicates each) and corresponding index files for chr21 (SNYDER_HG19_*H3K27AC*_reconcile.dedup.chr21.bam)
- corresponding genotypes for the hQTLs in a gzipped VCF file (genotypes.vcf.gz)
The first two are required to run a SNPhood analysis, genotype files are optional.
1.2 Setting up SNPhood
To set up a SNPhood analysis, you first need to create a named list that contains all parameters needed in a SNPhood analysis. This can be done by calling the function getDefaultParameterList, which generates a default list of parameters. It takes up to two (optional) arguments, which is 1) the path to the user-defined regions file and 2) whether or not the data are paired-end:
(par.l = getDefaultParameterList(path_userRegions = fileUserRegions, isPairedEndData = TRUE))## $readFlag_isPaired
## [1] TRUE
##
## $readFlag_isProperPair
## [1] TRUE
##
## $readFlag_isUnmappedQuery
## [1] FALSE
##
## $readFlag_hasUnmappedMate
## [1] FALSE
##
## $readFlag_isMinusStrand
## [1] NA
##
## $readFlag_isMateMinusStrand
## [1] NA
##
## $readFlag_isFirstMateRead
## [1] NA
##
## $readFlag_isSecondMateRead
## [1] NA
##
## $readFlag_isNotPrimaryRead
## [1] FALSE
##
## $readFlag_isNotPassingQualityControls
## [1] FALSE
##
## $readFlag_isDuplicate
## [1] FALSE
##
## $readFlag_reverseComplement
## [1] FALSE
##
## $readFlag_simpleCigar
## [1] TRUE
##
## $readFlag_minMapQ
## [1] 0
##
## $path_userRegions
## [1] "/home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/cisQ.H3K27AC.chr21.txt"
##
## $zeroBasedCoordinates
## [1] FALSE
##
## $regionSize
## [1] 500
##
## $binSize
## [1] 50
##
## $readGroupSpecific
## [1] TRUE
##
## $strand
## [1] "both"
##
## $startOpen
## [1] FALSE
##
## $endOpen
## [1] FALSE
##
## $headerLine
## [1] FALSE
##
## $linesToParse
## [1] -1
##
## $lastBinTreatment
## [1] "delete"
##
## $assemblyVersion
## [1] "hg19"
##
## $effectiveGenomeSizePercentage
## [1] -1
##
## $nCores
## [1] 1
##
## $keepAllReadCounts
## [1] FALSE
##
## $normByInput
## [1] FALSE
##
## $normAmongEachOther
## [1] TRUE
##
## $poolDatasets
## [1] FALSEIn many cases the default returned parameter values are a reasonable choice. However, always check the validity and usefulness of the parameters before starting an analysis to avoid unreasonable results.
In this example the default value for most parameters is a reasonable choice: the size of the regions (regionSize = 500) resulting in an analysis window of 2 * 500 (5’ and 3’ of the hQTL) + 1 (for the hQTL) = 1001 bp. Often it is useful to do a pilot analysis with only a few regions to explore the data fast. This can be done by setting the parameter linesToParse to a value > -1 to indicate the number of lines that should be parsed. Since here we work with only 178 hQTLs on chr21 we keep all of them for the analysis.
There are a few parameters that we have to adjust: First, we want to pool datasets because there are two replicates for each individual, and combining the datasets will give us more power, for example to detect allelic biases (parameter poolDatasets). You also have to carefully check if the start positions in the user regions file are 0-based or 1-based because a shift of one base pair will result in non-sensical results for the genotype distribution of the hQTLs that are determined automatically during the analysis. In this case, start coordinates are 1-based, which is also the default for the parameter zeroBasedCoordinates. Our the data are mapped allele-specifically, so perform allele-specific analysis you have to ensure to set the parameter readGroupSpecific to TRUE.
SNPhood offers a powerful and intuitive way of controlling which reads are considered valid when importing BAM files by means of the various flags that exist (see https://samtools.github.io/hts-specs/SAMv1.pdf), in complete analogy to the Rsamtools package. In essence, for each flag, a corresponding parameter (readFlag_) exists that specifies if the flag has to be set (TRUE), not set (FALSE) or if is irrelevant (NA).
We thus have to check the default values for the various readFlag_ parameters. The default values are for paired-end data, and all of our data here are also paired-end, so we can leave them at their default values. Note that the default values disregard any reads that could be problematic. For example, reads that are marked as duplicates (readFlag_isDuplicate), unmapped (either the read itself or its mate - readFlag_isUnmappedQuery and readFlag_hasUnmappedMate), not a primary read (readFlag_isNotPrimaryRead), not (properly) paired (readFlag_isPaired and readFlag_isProperPair) or fail quality controls (readFlag_isNotPassingQualityControls) are discarded. Note that all flags (as specified by the readFlag parameters) that are set to NA will be ignored when importing the reads.
Lastly, we adjust the size of each bin within the region and select a smaller value than the default one (binSize = 25 instead of 50).
# Verify that you do not have zero-based coordinates
par.l$zeroBasedCoordinates## [1] FALSEpar.l$readGroupSpecific## [1] TRUEpar.l$poolDatasets = TRUE
par.l$binSize = 25You are almost done with the preparation, all that is left is to create a data frame to tell SNPhood which data to use for the analysis and some additional meta information. For this, you can use another helper function: collectFiles. The argument patternFiles specifies the folder and file name of input files; wildcards are allowed.
patternBAMFiles = paste0(dirname(files[3]), "/*.bam")
(files.df = collectFiles(patternFiles = patternBAMFiles, verbose = TRUE))## signal
## 1 /home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM10847_H3K27AC_1_reconcile.dedup.chr21.bam
## 2 /home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM10847_H3K27AC_2_reconcile.dedup.chr21.bam
## 3 /home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM12890_H3K27AC_1_reconcile.dedup.chr21.bam
## 4 /home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM12890_H3K27AC_2_reconcile.dedup.chr21.bam
## input individual genotype
## 1 NA NA NA
## 2 NA NA NA
## 3 NA NA NA
## 4 NA NA NAFinally, you assign the names of the individuals in the column “inidivual” to make pooling of the datasets possible. The column input can be ignored because there is no negative control in this analysis due to the fact that the analysis is done allele-specifically and currently, input normalization does not work in this setting (see the main vignette for details). The column genotype can also be ignored for now because (will be integrated later):
files.df$individual = c("GM10847", "GM10847", "GM12890", "GM12890")
files.df## signal
## 1 /home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM10847_H3K27AC_1_reconcile.dedup.chr21.bam
## 2 /home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM10847_H3K27AC_2_reconcile.dedup.chr21.bam
## 3 /home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM12890_H3K27AC_1_reconcile.dedup.chr21.bam
## 4 /home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/SNYDER_HG19_GM12890_H3K27AC_2_reconcile.dedup.chr21.bam
## input individual genotype
## 1 NA GM10847 NA
## 2 NA GM10847 NA
## 3 NA GM12890 NA
## 4 NA GM12890 NA1.3 Quality control
** As stated explicitly in the main vignette, SNPhood is not a designated and sufficient tool for ChIP-Seq QC and has never been designed as such. It is important to assess potential biases such as GC, mapping, contamination or other biases beforehand using dedicated tools both within and outside the Bioconductor framework (see the main vignette for designated QC tools).**
However, SNPhood does offer some rudimentary QC controls. Before executing the full pipeline, you will therefore first do a quick QC step to make sure that the datasets do not have any artefacts. For this, you will investigate the correlation of the raw read counts for our regions among the different datasets. The correlation coefficients should be very high among replicate samples and relatively high among different samples.
For this, you run the main function analyzeSNPhood with a special argument and afterwards employ the function plotCorrelationDatasets. Note that you temporarily reset the value of the parameter poolDatasets to also check the correlation among the replicates samples.
par.l$poolDatasets = FALSE
SNPhood.o = analyzeSNPhood(par.l, files.df, onlyPrepareForDatasetCorrelation = TRUE,
verbose = TRUE)## Warning in analyzeSNPhood(par.l, files.df, onlyPrepareForDatasetCorrelation
## = TRUE, : Forcing parameter normAmongEachOther to FALSE because either input
## normalization is turned on, only one files is going to be processed, or
## because allele-specific reads are requested## Warning in .parseAndProcessUserRegions(par.l, chrSizes.df, verbose =
## verbose): 4 duplicate region removed in file /home/biocbuild/bbs-3.7-bioc/
## R/library/SNPhoodData/extdata/cisQ.H3K27AC.chr21.txt out of 178 regions. New
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## cannot be changed## Warning in analyzeSNPhood(par.l, files.df, onlyPrepareForDatasetCorrelation
## = TRUE, : Note that you set the parameter "onlyPrepareForDatasetCorrelation"
## to TRUE. You will not be able to use any functionality except the sample
## correlation plots. For a full analysis, run the function again and set the
## parameter to FALSE.You can now run the correlation analysis on the SNPhood object and plot it directly (we could also plot it to a PDF file):
SNPhood.o = plotAndCalculateCorrelationDatasets(SNPhood.o, fileToPlot = NULL)
corrResults = results(SNPhood.o, type = "samplesCorrelation")
mean(corrResults$corTable[lower.tri(corrResults$corTable)])## [1] 0.9177585The correlation values are indeed very high among the replicate samples and also quite high among the individuals. The mean correlation coefficient among the datasets is 0.91, which is very high (note that only the lower triangle matrix of the correlation matrix is used for this, to not bias the analysis). There does not seem to be a problem with the datasets. However, as mentioned repeatedly, this is not sufficient for QC and should only be another verification that data quality is high and that biases have been controlled for.
1.4 Executing the main function
Now you can execute the full pipeline by setting the parameter onlyPrepareForDatasetCorrelation to FALSE again (the default). The execution of the function may take a few minutes and may issue some warnings e.g. indicating missing data and “correcting” inconsistent parameter settings:
par.l$poolDatasets = TRUE
SNPhood.o = analyzeSNPhood(par.l, files.df, onlyPrepareForDatasetCorrelation = FALSE,
verbose = TRUE)## Warning in analyzeSNPhood(par.l, files.df, onlyPrepareForDatasetCorrelation
## = FALSE, : Forcing parameter normAmongEachOther to FALSE because either
## input normalization is turned on, only one files is going to be processed, or
## because allele-specific reads are requested## Warning in .parseAndProcessUserRegions(par.l, chrSizes.df, verbose =
## verbose): 4 duplicate region removed in file /home/biocbuild/bbs-3.7-bioc/
## R/library/SNPhoodData/extdata/cisQ.H3K27AC.chr21.txt out of 178 regions. New
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## cannot be changedThe warnings indicate that 4 duplicate regions have been removed as well as that the parameter normAmongEachOther has been set to FALSE because, in this case, reads are read group(allele)-specific (parameter readGroupSpecific is set to TRUE).
1.5 Working with a SNPhood object: Extracting and manipulating information and metadata
Now you can start analyzing the results already, as the main pipeline finished. To familiarize ourselves with the SNPhood object let’s first take a look at some of its properties and helper functions. Generally, there are four “entities” stored in a SNPhood object: regions, datasets, bins, and read groups. For all of them, methods to extract and alter the stored information exist (see below).
SNPhood.o
## Retrieve number of regions, datasets, bins, and read groups
nRegions(SNPhood.o)
nDatasets(SNPhood.o)
nBins(SNPhood.o)
nReadGroups(SNPhood.o)
## Retrieve general annotation of SNPhood object with all its different
## elements
names(annotation(SNPhood.o))
annotation(SNPhood.o)$regions
annotation(SNPhood.o)$files
## Retrieve the parameters that were used for the analysis
head(parameters(SNPhood.o))
names(parameters(SNPhood.o))
## Retrieve annotation of regions
head(annotationRegions(SNPhood.o))
## Retrieve annotation of bins
annotationBins(SNPhood.o)
head(annotationBins2(SNPhood.o, fullAnnotation = TRUE))
SNP_names = c("rs7275860", "rs76473124")
head(annotationBins2(SNPhood.o, regions = 1:10, fullAnnotation = FALSE))
annotationBins2(SNPhood.o, regions = SNP_names, fullAnnotation = TRUE)
## Retrieve annotation of datasets
annotationDatasets(SNPhood.o)
annotationReadGroups(SNPhood.o)
## Extract counts after the binning
# Extract one count matrix from the paternal read group from the first dataset
head(counts(SNPhood.o, type = "binned", readGroup = "paternal", dataset = 1))
# Extract count matrices from all read groups from the first dataset
str(counts(SNPhood.o, type = "binned", readGroup = NULL, dataset = 1))
# Extract count matrices from all read groups from the first dataset (using
# its name)
DataSetName <- annotationDatasets(SNPhood.o)[1]
str(counts(SNPhood.o, type = "binned", readGroup = NULL, dataset = DataSetName))
# Extract count matrices from all read groups from the all dataset
str(counts(SNPhood.o, type = "binned", dataset = NULL))
## Similarly, you can also extract counts before the binning
head(counts(SNPhood.o, type = "unbinned", readGroup = "paternal", dataset = 1))
## If you had enrichments instead of counts, you would employ the enrichments
## method in analogy to counts
enrichment(SNPhood.o, readGroup = "paternal")## Warning in .getCounts(SNPhood.o, type = "enrichmentBinned", readGroup,
## dataset): Returning an empty list, could not find the requested data in the
## object. Did you ask for the correct type of data? See also the help pages.You can modify the information stored in a SNPhood object:
SNPhood.o
## Rename regions, datasets, bins, and read groups
mapping = as.list(paste0(annotationRegions(SNPhood.o), ".newName"))
names(mapping) = annotationRegions(SNPhood.o)
SNPhood_mod.o = renameRegions(SNPhood.o, mapping)
mapping = list("Individual1", "Individual2")
names(mapping) = annotationDatasets(SNPhood.o)
SNPhood_mod.o = renameDatasets(SNPhood.o, mapping)
mapping = list("Bin1_NEW")
names(mapping) = annotationBins(SNPhood.o)[1]
SNPhood_mod.o = renameBins(SNPhood.o, mapping)
mapping = list("a", "b", "c")
names(mapping) = annotationReadGroups(SNPhood.o)
SNPhood_mod.o = renameReadGroups(SNPhood.o, mapping)
## Delete regions, datasets, and read groups (deleting bins is still in
## development)
SNPhood_mod.o = deleteRegions(SNPhood.o, regions = 1:5)
SNPhood_mod.o = deleteRegions(SNPhood.o, regions = c("rs9984805", "rs59121565"))
SNPhood_mod.o = deleteDatasets(SNPhood.o, datasets = 1)
SNPhood_mod.o = deleteDatasets(SNPhood.o, datasets = "GM12890")
# For read groups, we currently support only a name referral
SNPhood_mod.o = deleteReadGroups(SNPhood.o, readGroups = "paternal")
## Merge read groups
SNPhood_merged.o = mergeReadGroups(SNPhood.o)
nReadGroups(SNPhood_merged.o)
annotationReadGroups(SNPhood_merged.o)1.6 Visualizing counts and enrichment
Let’s first visualize the number of overlapping reads for the regions before (plotRegionCounts) and after binning (plotBinCounts) datasets and read groups. The two functions have a number of arguments to make the visualization as flexible as possible. See the help pages for details, we here only touch upon a few parameters:
plotBinCounts(SNPhood.o, regions = 2)
plotBinCounts(SNPhood.o, regions = 2, plotGenotypeRatio = TRUE, readGroups = c("paternal",
"maternal"))## Warning: Removed 14 rows containing missing values (geom_path).
plotRegionCounts(SNPhood.o, regions = 1:5, plotRegionBoundaries = TRUE, sizePoints = 2,
plotRegionLabels = TRUE, mergeReadGroupCounts = TRUE)
plotRegionCounts(SNPhood.o, regions = NULL, plotChr = "chr21", sizePoints = 2)
We can also create aggregate plots and plot the bin counts for all regions or a subset of regions:
plotBinCounts(SNPhood.o, regions = NULL, readGroups = c("paternal", "maternal"))## Warning in plotBinCounts(SNPhood.o, regions = NULL, readGroups =
## c("paternal", : Cannot add genotype as the number of regions to plot is 174
## and not 1.
1.7 Testing for and visualizing allelic biases
We now test for allelic biases. To determine the significance across regions, one can select the lowest p-value for each region. This ensures to select the bin with the most power (which often has the highest number of reads) to detect allelic bias. However, the tests for bins are not independent from one another and the p-values are not adjusted for multiple testing. We therefore implemented a permutation-based procedure to control the false discovery rate (FDR). This option is also enabled by default (parameters calcBackgroundDistr and nRepetitions).
# Run the analysis, perform no time-consuming background calculation for now
SNPhood.o = testForAllelicBiases(SNPhood.o, readGroups = c("paternal", "maternal"),
calcBackgroundDistr = TRUE, nRepetitions = 100, verbose = FALSE)
# Extract the results of the analysis, again using the results function
names(results(SNPhood.o, type = "allelicBias"))## [1] "pValue" "confIntervalMin" "confIntervalMax"
## [4] "fractionEstimate" "background" "FDR_results"
## [7] "parameters"head(results(SNPhood.o, type = "allelicBias", elements = "pValue")[[1]], 4)## bin_1 bin_2 bin_3 bin_4 bin_5 bin_6
## rs75359783 1.00000000 1.00000000 1.00000000 1.0000000 1.0000000 1.0000000
## rs8132276 0.25587508 0.34888888 0.40503225 0.6776395 1.0000000 0.8145294
## rs113783782 0.02127075 0.07681274 0.01921082 0.1184692 0.5488281 0.7539063
## rs7275860 0.50000000 0.50000000 0.62500000 0.3750000 1.0000000 1.0000000
## bin_7 bin_8 bin_9 bin_10 bin_11 bin_12
## rs75359783 1.00000000 1.0000000 1.0000000 1.00000000 1.00000000 1.0000000
## rs8132276 1.00000000 1.0000000 0.6290588 0.14346313 0.40487289 1.0000000
## rs113783782 0.03857422 0.1796875 0.1184692 0.05737305 0.03515625 0.1795654
## rs7275860 0.62500000 0.2500000 0.0312500 0.03125000 0.03125000 0.0703125
## bin_13 bin_14 bin_15 bin_16 bin_17 bin_18
## rs75359783 1.0000000 1.0000000 1.0000000 1.0000000 1.0000000 1.0000000
## rs8132276 1.0000000 0.8238029 0.1670685 0.3017578 0.8238029 0.8238029
## rs113783782 0.2668457 0.5078125 0.3876953 0.5078125 0.6875000 0.6875000
## rs7275860 0.1796875 1.0000000 0.4531250 0.4531250 0.3750000 0.2187500
## bin_19 bin_20 bin_21 bin_22 bin_23 bin_24
## rs75359783 1.0000000 1.0000000 1.0000000 1.000000 1.0000 1.0000000
## rs8132276 0.4048729 0.8318119 0.8238029 1.000000 1.0000 0.5810547
## rs113783782 0.3750000 1.0000000 1.0000000 1.000000 0.6875 1.0000000
## rs7275860 0.1250000 0.4531250 0.4531250 0.453125 0.1250 0.2890625
## bin_25 bin_26 bin_27 bin_28 bin_29 bin_30 bin_31
## rs75359783 1.0000000 1.0000000 1.0000000 1.000000 1.00000 1.0000000 1.00000
## rs8132276 0.1795654 0.2668457 0.5078125 0.453125 0.37500 1.0000000 1.00000
## rs113783782 0.6875000 0.2890625 0.0390625 0.218750 0.12500 0.1250000 0.21875
## rs7275860 0.2187500 0.0703125 0.2890625 0.453125 0.21875 0.0390625 0.12500
## bin_32 bin_33 bin_34 bin_35 bin_36
## rs75359783 1.00000000 1.0000000000 1.0000000000 1.00000000 1.00000000
## rs8132276 1.00000000 0.5000000000 1.0000000000 1.00000000 1.00000000
## rs113783782 0.21875000 0.0214843750 0.0117187500 0.00390625 0.00781250
## rs7275860 0.00390625 0.0009765625 0.0004882813 0.00390625 0.00390625
## bin_37 bin_38 bin_39 bin_40
## rs75359783 1.000000 1.000 1.000 1.000
## rs8132276 1.000000 1.000 1.000 1.000
## rs113783782 0.015625 0.375 0.625 0.125
## rs7275860 0.015625 0.250 0.500 0.125# Extract the results of the FDR calculation for the first dataset
FDR_dataset1 = results(SNPhood.o, type = "allelicBias", elements = "FDR_results")[[1]]
head(FDR_dataset1, 20)## pValueThreshold FDR nReal nSim
## 1 0.0001 0.00000000 5 0.00
## 2 0.0005 0.01153213 6 0.07
## 3 0.0010 0.01800327 6 0.11
## 4 0.0050 0.06542056 7 0.49
## 5 0.0100 0.09584087 10 1.06
## 6 0.0200 0.16932907 13 2.65
## 7 0.0300 0.21700224 14 3.88
## 8 0.0400 0.28635147 16 6.42
## 9 0.0500 0.28741093 18 7.26
## 10 0.0600 0.30224018 19 8.23
## 11 0.0700 0.32033097 23 10.84
## 12 0.0800 0.34282585 24 12.52
## 13 0.0900 0.35135135 24 13.00
## 14 0.1000 0.36958235 24 14.07
## 15 0.1100 0.38728619 24 15.17
## 16 0.1200 0.39113492 25 16.06
## 17 0.1300 0.41955378 32 23.13
## 18 0.1400 0.42238267 32 23.40
## 19 0.1500 0.42317777 33 24.21
## 20 0.1600 0.42458588 33 24.35# Extract the results of the FDR calculation for the second dataset
FDR_dataset2 = results(SNPhood.o, type = "allelicBias", elements = "FDR_results")[[2]]
head(FDR_dataset2, 20)## pValueThreshold FDR nReal nSim
## 1 0.0001 0.002493766 8 0.02
## 2 0.0005 0.006323397 11 0.07
## 3 0.0010 0.013452915 11 0.15
## 4 0.0050 0.051787017 13 0.71
## 5 0.0100 0.068901304 20 1.48
## 6 0.0200 0.138943249 22 3.55
## 7 0.0300 0.200000000 22 5.50
## 8 0.0400 0.245852187 25 8.15
## 9 0.0500 0.271986022 25 9.34
## 10 0.0600 0.298147108 25 10.62
## 11 0.0700 0.317561419 30 13.96
## 12 0.0800 0.341825902 31 16.10
## 13 0.0900 0.336016097 33 16.70
## 14 0.1000 0.345272482 34 17.93
## 15 0.1100 0.360180655 34 19.14
## 16 0.1200 0.370020382 34 19.97
## 17 0.1300 0.384571347 43 26.87
## 18 0.1400 0.388770434 43 27.35
## 19 0.1500 0.391340434 44 28.29
## 20 0.1600 0.394439857 44 28.66maxFDR = 0.1
signThresholdFDR_dataset1 = FDR_dataset1$pValueThreshold[max(which(FDR_dataset1$FDR <
maxFDR))]
signThresholdFDR_dataset2 = FDR_dataset2$pValueThreshold[max(which(FDR_dataset1$FDR <
maxFDR))]From the FDR results we see that for both datasets, the FDR is below 10% for p-values < 0.01. For this analysis, 10% is an acceptable number, so we will use this as significance threshold for the upcoming visualization functions.
You can now visualize the results to get an overview of the allelic bias in our dataset. You may start by visualizing the results of the allelic bias analysis across regions or a user-defined genomic range such as the full chromosome 21. For this, the function plotAllelicBiasResultsOverview is employed, which either plots the minimum or median p-value for each region in the selected genomic region.
plotAllelicBiasResultsOverview(SNPhood.o, regions = NULL, plotChr = "chr21", signThreshold = 0.01,
pValueSummary = "min")
plotAllelicBiasResultsOverview(SNPhood.o, regions = 3:5, plotRegionBoundaries = TRUE,
plotRegionLabels = TRUE, signThreshold = 0.01, pValueSummary = "min")
The first plot indicates that a considerable amount of regions seems to show an allelic bias. You can then look at the results for the allelic bias analysis in detail for a specific dataset and region with the function plotAllelicBiasResults:
plots = plotAllelicBiasResults(SNPhood.o, region = 2, signThreshold = 0.01, readGroupColors = c("blue",
"red", "gray"))## Warning: Ignoring unknown aesthetics: shape
## Warning: Ignoring unknown aesthetics: shape## Warning: Removed 6 rows containing missing values (geom_point).plots = plotAllelicBiasResults(SNPhood.o, region = 7, signThreshold = 0.01, readGroupColors = c("blue",
"red", "gray"))## Warning: Ignoring unknown aesthetics: shape## Warning: Ignoring unknown aesthetics: shape## Warning: Removed 5 rows containing missing values (geom_point).
While the first plot shows an example of a region for which no allelic bias can be found, the second one shows significant allelic bias across many bins. In this case, the allelic bias might be caused by the genotype, as paternal and maternal reads have different genotypes (see the legend, A versus C).
1.8 Cluster analyses
SNPhood provides some clustering functionalities to cluster SNPs based on their local neighbourhood. Let’s try them out. First, you will cluster the counts for the paternal read group (allele) from the first dataset using 2 and 5 clusters, respectively. The function plotAndClusterMatrix returns an object of class SNPhood so that the clustering results can be accessed directly for subsequent visualization.
SNPhood.o = plotAndClusterMatrix(SNPhood.o, readGroup = "paternal", nClustersVec = 2,
dataset = 1, verbose = FALSE)
SNPhood.o = plotAndClusterMatrix(SNPhood.o, readGroup = "paternal", nClustersVec = 5,
dataset = 1, verbose = FALSE)
str(results(SNPhood.o, type = "clustering", elements = "paternal"), list.len = 3)## List of 1
## $ GM10847:List of 2
## ..$ nClusters2:List of 3
## .. ..$ clusteringMatrix :List of 1
## .. .. ..$ :'data.frame': 174 obs. of 41 variables:
## .. .. .. ..$ cluster: int [1:174] 2 2 2 2 2 2 2 2 2 2 ...
## .. .. .. ..$ bin_1 : num [1:174] 0.186 -0.309 -0.455 -0.582 -0.908 ...
## .. .. .. ..$ bin_2 : num [1:174] -0.0705 -0.3092 -0.4548 -0.5823 -0.9081 ...
## .. .. .. .. [list output truncated]
## .. ..$ averageSilhouette: num 0.559
## .. ..$ plots :List of 1
## .. .. ..$ :List of 45
## .. .. .. ..$ formula :Class 'formula' language z ~ row * column
## .. .. .. .. .. ..- attr(*, ".Environment")=<environment: 0x2cf74ae8>
## .. .. .. ..$ as.table : logi FALSE
## .. .. .. ..$ aspect.fill : logi TRUE
## .. .. .. .. [list output truncated]
## .. .. .. ..- attr(*, "class")= chr "trellis"
## ..$ nClusters5:List of 3
## .. ..$ clusteringMatrix :List of 1
## .. .. ..$ :'data.frame': 174 obs. of 41 variables:
## .. .. .. ..$ cluster: int [1:174] 5 5 5 5 5 5 5 4 4 4 ...
## .. .. .. ..$ bin_1 : num [1:174] 0.186 -0.309 -0.664 0.457 -0.733 ...
## .. .. .. ..$ bin_2 : num [1:174] -0.0705 -0.3092 -0.6637 -0.3209 -0.7334 ...
## .. .. .. .. [list output truncated]
## .. ..$ averageSilhouette: num 0.605
## .. ..$ plots :List of 1
## .. .. ..$ :List of 45
## .. .. .. ..$ formula :Class 'formula' language z ~ row * column
## .. .. .. .. .. ..- attr(*, ".Environment")=<environment: 0x2c7d6390>
## .. .. .. ..$ as.table : logi FALSE
## .. .. .. ..$ aspect.fill : logi TRUE
## .. .. .. .. [list output truncated]
## .. .. .. ..- attr(*, "class")= chr "trellis"You can also plot only a subset of the clusters to remove clusters with invariant regions. Here, let’s only plot clusters 2 to 5 instead of all of them (that is, 1 to 5):
SNPhood.o = plotAndClusterMatrix(SNPhood.o, readGroup = "paternal", nClustersVec = 5,
dataset = 1, clustersToPlot = 2:5, verbose = FALSE)
As you can see, most of the regions have now been removed because they belonged to cluster 1.
To summarize the cluster results and to more easily detect the patterns, you can also calculate the average enrichment across bins per cluster using the function plotClusterAverage. Note that this function returns the plots only:
p = plotClusterAverage(SNPhood.o, readGroup = "paternal", dataset = 1)## Warning in plotClusterAverage(SNPhood.o, readGroup = "paternal", dataset
## = 1): Multiple clustering results found, summarizing all of them. Multiple
## plots will be produced. Specify a filename for the parameter fileToPlot to
## see them all.
The warning can be ignored here, it simply informs us that multiple plots have been generated even though no file name has been specified. Thus, you may only seem the last plot.
1.9 Genotype analyses
Next you can integrate the genotype from external sources for our regions. For this, you first create a data frame to specify which datasets to integrate genotypes with, the path to the VCF file that provides the genotypes, and the name of the column in the VCF file that corresponds to the dataset.
(mapping = data.frame(samples = annotationDatasets(SNPhood.o), genotypeFile = rep(fileGenotypes,
2), sampleName = c("NA10847", "NA12890")))## samples
## 1 GM10847
## 2 GM12890
## genotypeFile
## 1 /home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/genotypes.vcf.gz
## 2 /home/biocbuild/bbs-3.7-bioc/R/library/SNPhoodData/extdata/genotypes.vcf.gz
## sampleName
## 1 NA10847
## 2 NA12890SNPhood.o = associateGenotypes(SNPhood.o, mapping)Let’s take a look at the imported genotypes to check the uniformity across datasets:
p = plotGenotypesPerSNP(SNPhood.o, regions = 1:20)
1.10 Combined cluster and genotype analyses
An additional feature that might be useful when performing clustering on several individuals is to group individuals for each SNP according to their genotype, which we divide into “strong” and “weak” genotypes. They are determined based on the signal obtained for the different genotypes. For this, we use the function plotAndCalculateWeakAndStrongGenotype. This function can only be executed when read groups have been merged (i.e., when only one read group is present). That’s easy to fix:
SNPhood_merged.o = mergeReadGroups(SNPhood.o)
SNPhood_merged.o = plotAndCalculateWeakAndStrongGenotype(SNPhood_merged.o, normalize = TRUE,
nClustersVec = 3)## [[1]]
##
## [[1]]
The first plot shows the results for the strong genotype, the second one for the weak genotype.
Note that you now have two SNPhood objects in your workspace: One without and one with merged read groups. Because merging read groups is irreversible (see ?mergeReadGroups), we keep both objects in the workspace.
You can now combine clustering and genotype one more time and perform the clustering only on the signal averaged across all high-genotype individuals at each SNP to increase the signal to noise ratio (analogous to the analysis in Figure 6 in Grubert et al. [1]):
p = plotGenotypesPerCluster(SNPhood_merged.o, printPlot = FALSE, printBinLabels = TRUE,
returnOnlyPlotNotObject = TRUE)
p[[1]]## [[1]]
1.11 How to continue?
From here on, possibilities are endless, and you can further investigate patterns and trends in the data! We hope that the SNPhood package is useful for your research and encourage you to contact us if you have any question or feature request!
2 Bug Reports, Feature Requests and Contact Information
We value all the feedback that we receive and will try to reply in a timely manner.
Please report any bug that you encounter as well as any feature request that you may have to SNPhood@gmail.com.