%\VignetteKeywords{runAbsoluteCN}
%\VignetteEngine{knitr::knitr}
%\VignetteDepends{PureCN}
%\VignettePackage{PureCN}
%\VignetteIndexEntry{PureCN overview}

\documentclass{article}

<<style-knitr, eval=TRUE, echo=FALSE, results="asis">>=
BiocStyle::latex()
@ 

\usepackage{booktabs} % book-quality tables

\title{Copy number calling and SNV classification using 
targeted short read sequencing}
\author[1]{Markus Riester}
\affil[1]{Novartis Institutes for BioMedical Research, Cambridge, MA}

\begin{document}

\maketitle

\begin{abstract}
\Biocpkg{PureCN} \cite{Riester2016} is a purity and ploidy aware copy number
caller for cancer samples inspired by the \software{ABSOLUTE} algorithm
\cite{Carter2012}.  It was designed for hybrid capture sequencing data,
especially with medium-sized targeted gene panels without matching normal
samples in mind (matched whole-exome data is of course supported). 

It can be used to supplement existing normalization and segmentation algorithms,
i.e. the software can start from BAM files, from target-level coverage data,
from copy number log-ratios or from already segmented data. After the correct
purity and ploidy solution was identified, \Biocpkg{PureCN} will accurately
classify variants as germline vs. somatic or clonal vs. sub-clonal.

\Biocpkg{PureCN} was further designed to integrate well with industry standard
pipelines \cite{McKenna2010}, but it is straightforward to generate input data
from other pipelines.
\end{abstract}

\packageVersion{\Sexpr{BiocStyle::pkg_ver("PureCN")}}

\tableofcontents
\newpage

<<load-purecn, echo=FALSE, message=FALSE>>=
library(PureCN)
set.seed(1234)
@

\section{Introduction}

This tutorial will demonstrate on a toy example how we recommend running
\Biocpkg{PureCN} on targeted sequencing data. To estimate tumor purity, we
jointly utilize both target-level\footnote{The captured genomic regions, e.g.
exons.} coverage data and allelic fractions of single nucleotide
variants (SNVs), inside - and optionally outside - the targeted regions.
Knowledge of purity will in turn allow us to accurately (i) infer integer copy
number and (ii) classify variants (somatic vs. germline, mono-clonal vs.
sub-clonal, heterozygous vs. homozygous etc.).

This requires 3 basic input files:

\begin{enumerate}
    \item A VCF file containing germline SNPs and somatic mutations. Somatic
status is not required in case the variant caller was run without matching
normal sample.
    \item The tumor BAM file.
    \item At least one BAM file from a normal control sample, either matched or
        process-matched.
\end{enumerate}

In addition, we need to know a little bit more about the assay.  This is the
annoying step since here the user needs to provide some information.  Most
importantly, we need to know the positions of all targets.  Then we need to
correct for GC-bias, for which we need GC-content for each target.  Optionally,
if gene-level calls are wanted, we also need for each target a gene symbol. We
may also observe subtile differences in coverage of tumor compared to
normal due to varying proliferation rates and we can provide replication
timing data to check and correct for such a bias. To obtain best results,
we can finally use a pool of normal samples to automatically learn more
about the assay and its biases and common artifacts.

The next sections will show how to do all this with \Biocpkg{PureCN} alone or
with the help of \software{GATK} and/or existing copy number pipelines. 

\textbf{All the steps described in the following are available in easy to use
command line scripts described in a separate vignette.}

\section{Basic input files}

\subsection{VCF}
\label{secinputvcf}
Germline SNPs and somatic mutations are expected in a single VCF file. At the
bare minimum, this VCF should contain read depths of reference and alt alleles
in an AD format field and a DB info flag for membership in germline
databases\footnote{The name of the flag is customizable in
\Rfunction{runAbsoluteCN}}. 

Without DB flag, variant ids starting with \Rcode{rs} are assumed to be in
dbSNP. Population allele frequencies are expected in a POP\_AF info field. These
frequencies are currently only used to infer membership in germline databases
when the DB flag is missing; in future versions they will be used calculate
somatic prior probabilities more accurately.

If a matched normal is available, then somatic status information is currently
expected in a SOMATIC info flag in the VCF. The \Biocpkg{VariantAnnotation}
package provides examples how to add info fields to a VCF in case the used
variant caller does not add this flag. If the VCF contains a BQ format field
containing base quality scores, \Biocpkg{PureCN} can remove low quality calls.

VCF files generated by \software{MuTect} \cite{Cibulskis2013} should work well
and in general require no post-processing. \Biocpkg{PureCN} can handle
\software{MuTect} VCF files generated in both tumor-only and matched normal
mode. Experimental support for \software{MuTect 2} and \software{FreeBayes}
VCFs generated in tumor-only mode is available.  

\subsection{Target information}
\label{sectargetinfo}
For the default segmentation function provided by \Biocpkg{PureCN}, the
algorithm first needs to calculate log-ratios of tumor vs. normal control
coverage. To do this, we need to know the locations of the captured genomic
regions (targets). These are provided by the manufacturer of your capture
kit\footnote{While \Biocpkg{PureCN} can use a pool of normal samples to learn
which intervals are reliable and which not, it is highly recommended to provide
the correct intervals. Garbage in, garbage out.}.  Please double check that the
genome version of the target file matches the reference. Usually the
manufacturer provides two files: the baits file containing the coordinates of
the actual capture baits, and the target file containing the coordinates of the
actual regions we want to capture. We recommend to use the baits file (and
recognize the confusing nomenclature that we follow due to convention in
established tools).

Default parameters assume that these targets do NOT include a "padding" to
include flanking regions.  \Biocpkg{PureCN} will automatically include variants
in the 50bp flanking regions if the variant caller was either run without
interval file or with interval padding (See section~\ref{faqinput}). 

\Biocpkg{PureCN} will attempt to optimize the targets for copy number calling
(similar to \cite{Talevich2016}):

\begin{itemize} 
\item Large targets are split to obtain a higher resolution
\item Targets in regions of low mappability are dropped
\item Optionally, accessible regions in-between the target (off-target) regions
    are included so that available coverage information in on- and off-target
    reads can be used by the segmentation function. In the following, we
    will use \textit{intervals} when something applies to both on-target and
    off-target regions and \textit{targets} when it only applies to
    on-target.
\end{itemize}

It further annotates intervals by GC-content (how coverage is normalized is
described later in Section~\ref{secgcbias}). 

\Biocpkg{PureCN} provides the \Rfunction{preprocessIntervals} 
function:

<<examplegc>>=
reference.file <- system.file("extdata", "ex2_reference.fa", 
    package = "PureCN", mustWork = TRUE)
bed.file <- system.file("extdata", "ex2_intervals.bed", 
        package = "PureCN", mustWork = TRUE)
mappability.file <- system.file("extdata", "ex2_mappability.bigWig", 
        package = "PureCN", mustWork = TRUE)

intervals <- import(bed.file)
mappability <- import(mappability.file)

preprocessIntervals(intervals, reference.file, 
    mappability=mappability, output.file = "ex2_gc_file.txt")
@

A command line script described in a separate vignette provides convenient
access to this function and also attempts to annotate the targets with gene
symbols using the \Rfunction{annotateTargets} function.

\subsection{Coverage data}
\label{purecncoverage}

The \Rfunction{calculateBamCoverageByInterval} function can be used to generate
the required coverage data from BAM files. All we need to do is providing the
desired intervals (as generated by \Rfunction{preprocessIntervals}):

<<examplecoverage>>=
bam.file <- system.file("extdata", "ex1.bam", package="PureCN", 
    mustWork = TRUE)
interval.file <- system.file("extdata", "ex1_intervals.txt", 
    package = "PureCN", mustWork = TRUE)

calculateBamCoverageByInterval(bam.file = bam.file, 
 interval.file = interval.file, output.file = "ex1_coverage.txt")
@

\subsection{Third-party coverage tools}
\label{gatkcoverage}

Calculating coverage from BAM files is a common task and your pipeline might
already provide this information. As alternative to
\Rfunction{calculateBamCoverageByInterval}, \Biocpkg{PureCN} currently supports
coverage files generated by \software{GATK3 DepthOfCoverage}, \software{GATK4
CollectFragmentCounts} and by \software{CNVkit}. By providing files with
standard file extension, \Biocpkg{PureCN} will automatically detect the correct
format and all following steps are the same for all tools. You will, however,
still need the interval file generated in Section~\ref{sectargetinfo} and the
third-party tool must use the exact same intervals. See also FAQ
Section~\ref{faqinput} for recommended settings for \software{GATK3
DepthOfCoverage}. 

\subsection{Third-party segmentation tools}

\Biocpkg{PureCN} integrates well with existing copy number pipelines. Instead of
coverage data, the user then needs to provide either already segmented data or a
wrapper function. This is described in Section~\ref{customseg}.

\subsection{Example data}

We now load a few example files that we will use throughout this tutorial:

<<example_files, message=FALSE, warning=FALSE, results='hide'>>=
library(PureCN)

normal.coverage.file <- system.file("extdata", "example_normal.txt",
    package="PureCN") 
normal2.coverage.file <- system.file("extdata", "example_normal2.txt", 
    package="PureCN") 
normal.coverage.files <- c(normal.coverage.file, normal2.coverage.file) 
tumor.coverage.file <- system.file("extdata", "example_tumor.txt", 
    package="PureCN") 
seg.file <- system.file("extdata", "example_seg.txt",
    package = "PureCN")
vcf.file <- system.file("extdata", "example.vcf.gz", package="PureCN")
interval.file <- system.file("extdata", "example_intervals.txt", 
    package="PureCN")
@

\section{Library-specific coverage bias}
\label{secgcbias}

In coverage normalization, we distinguish between assay- and library-specific
biases.  Assay-specific biases, for example due to probe density, probe capture
efficiency and read mappability, are best removed with a pool of normal samples
(Section~\ref{bestnormal}, \cite{Talevich2016}). In other words, by examining
the coverage of particular intervals in a pool of normals, we can estimate how well
this assay captures these intervals and will then adjust the tumor coverage
accordingly.

Other biases are library-specific, meaning a patient sample captured in
different libraries may display dramatically different coverage profiles across
libraries. Data from great sequencing centers usually show relatively small
technical variance nowadays, but some biases are not completely avoidable. The
most important library-specific bias is due to GC-content, i.e. regions of high
AT- or GC-content are not always captured with exactly the same efficiency in
tumor and normals.

We usually also observe that early replicating regions have a slightly higher
coverage than late replicating regions \cite{Koren2014, Kleinheinz210807}.
Since there is often a significant difference in proliferation rates of tumor
and normal, the pool of normals might also not completely adjust for this small
bias.

As first step, we thus correct the raw coverage of all samples, tumor and
normal, for these two major sources of library-specific coverage biases
(Figure~\ref{figure/figuregccorrect-1}). For GC-normalization, we use a 2-step
loess normalization \cite{Ha2014}. For the replication timing bias, a linear
model of log-transformed coverage and provided replication timing score is
used.

<<figuregccorrect, fig.show='hide', fig.width=7, fig.height=7, warning=FALSE>>=
correctCoverageBias(normal.coverage.file, interval.file, 
    output.file="example_normal_loess.txt", plot.bias=TRUE)
@
\incfig{figure/figuregccorrect-1}{0.95\textwidth}{Coverage before and after
normalization for GC-content and replication timing.} 
{This plot shows coverage as a function of on- and off-target GC-content and
replication timing before and after normalization. Each dot is an
interval. The example files are already GC-normalized; real data will show more
dramatic differences.}

\textbf{All the following steps in this vignette assume that the coverage data
are normalized.} The example coverage files are already GC-normalized.  We
provide a convenient command line script for generating normalized coverage
data from BAM files or from \software{GATK} coverage files (see Quick
vignette).


\section{Pool of normals}

\subsection{Selection of normals for log-ratio calculation}
\label{bestnormal}

For calculating copy number log-ratios of tumor vs. normal, \Biocpkg{PureCN}
requires coverage from a process-matched normal sample.  Using a normal that
was sequenced using a similar, but not identical assay, rarely works.
Differently covered genomic regions simply result in too many log-ratio
outliers. This section describes how to identify good process-matched normals
in case no matched normal is available or in case the matched normal has low or
uneven coverage. 

The \Rfunction{createNormalDatabase} function builds a database of coverage
files (a command line script providing this functionality is described in
a separate vignette): 

<<normaldb>>=
normalDB <- createNormalDatabase(normal.coverage.files)
# serialize, so that we need to do this only once for each assay
saveRDS(normalDB, file="normalDB.rds")
@

Again, please make sure that all coverage files were GC-normalized prior to
building the database (Section~\ref{secgcbias}).  Internally,
\Rfunction{createNormalDatabase} determines the sex of the samples and trains a
PCA that is later used for denoising tumor coverage:

<<normaldbpca>>=
normalDB <- readRDS("normalDB.rds")
pool <- calculateTangentNormal(tumor.coverage.file, normalDB)
@

\subsection{Artifact filtering}
\label{artifactfiltering}

It is important to remove as many artifacts as possible, because low ploidy
solutions are typically punished more by artifacts than high ploidy solutions.
High ploidy solutions are complex and usually find ways of explaining artifacts
reasonably well. The following steps in this section are optional, but
recommended since they will reduce the number of samples requiring manual
curation, especially when matching normal samples are not available. 

\subsubsection{VCF}
\label{artifactfilteringvcf}

We recommend running \software{MuTect} with a pool of normal samples to filter
common sequencing errors and alignment artifacts from the VCF.
\software{MuTect} requires a single VCF containing all normal samples, for
example generated by the \software{GATK3 CombineVariants} tool (see
Section~\ref{faqinput}).

It is highly recommended to provide \Biocpkg{PureCN} this combined VCF as well;
it will help the software correcting non-reference read mapping biases. This
is described in the \Rfunction{setMappingBiasVcf} documentation.  To reduce
memory usuage, the normal panel VCF can be reduced to contain only variants
present in 4 or more samples (the VCF for \software{MuTect} should however
contain variants present in 2-3 samples). 

Because these VCFs can become huge with large pools of normals, we can
optionally pre-compute the mapping bias, thus avoiding parsing these VCFs for
every sample:

<<calculatemappingbias>>=
# speed-up future runtimes by pre-calculating variant mapping biases
normal.panel.vcf.file <- system.file("extdata", "normalpanel.vcf.gz", 
                                     package="PureCN")

bias <- calculateMappingBiasVcf(normal.panel.vcf.file, genome = "h19")
saveRDS(bias, "mapping_bias.rds")
normal.panel.vcf.file <- "mapping_bias.rds"
@

\subsubsection{Coverage data}
We next use coverage data of normal samples to estimate the expected variance
in coverage per interval:

<<targetweightfile1, message=FALSE>>=
interval.weight.file <- "interval_weights.txt"
calculateIntervalWeights(normalDB$normal.coverage.files, interval.weight.file)
@

This function calculates copy number log-ratios using all normal
samples provided in the \Rcode{normal.coverage.files} argument.  Assuming that
all normal samples are in general diploid, a high variance in log-ratio
is indicative of an interval with either common germline alterations or frequent
artifacts; high or low copy number log-ratios in these intervals are unlikely
measuring somatic copy number events.  

This \Rcode{interval.weight.file} is automatically generated by the NormalDB.R
script described in the Quick vignette. 

\subsection{Artifact filtering without a pool of normals}
\label{artifactfiltering2}

By default, \Biocpkg{PureCN} will exclude targets with coverage below 15X from
segmentation (with a pool of normals, targets are filtered based on the
coverage and variance in normal database only). For variants in the provided
VCF, the same 15X cutoff is applied.  \software{MuTect} applies more
sophisticated artifact tests and flags suspicious variants. If
\software{MuTect} was run in matched normal mode, then both potential artifacts
and germline variants are rejected, that means we cannot just filter by the
PASS/REJECT \software{MuTect} flags. The \Rfunction{filterVcfMuTect} function
optionally reads the \software{MuTect 1.1.7} stats file and will keep germline
variants, while removing potential artifacts.  Without the stats file,
\Biocpkg{PureCN} will use only the filters based on read depths as defined in
\Rfunction{filterVcfBasic}.  Both functions are automatically called by
\Biocpkg{PureCN}, but can be easily modified and replaced if necessary.

We can also use a BED file to blacklist regions expected to be problematic, for
example the simple repeats track from the UCSC:

<<ucsc_segmental>>=
# Instead of using a pool of normals to find low quality regions,
# we use suitable BED files, for example from the UCSC genome browser.

# We do not download these in this vignette to avoid build failures 
# due to internet connectivity problems.
downloadFromUCSC <- FALSE
if (downloadFromUCSC) {
    library(rtracklayer)
    mySession <- browserSession("UCSC")
    genome(mySession) <- "hg19"
    simpleRepeats <- track( ucscTableQuery(mySession, 
        track="Simple Repeats", table="simpleRepeat"))
    export(simpleRepeats, "hg19_simpleRepeats.bed")
}

snp.blacklist <- "hg19_simpleRepeats.bed"
@

\section{Recommended run}
\label{secrecommended}
Finally, we can run \Biocpkg{PureCN} with all that information:

<<runpurecn>>=
ret <-runAbsoluteCN(normal.coverage.file=pool,
# normal.coverage.file=normal.coverage.file, 
    tumor.coverage.file=tumor.coverage.file, vcf.file=vcf.file, 
    genome="hg19", sampleid="Sample1", 
    interval.file=interval.file, normalDB=normalDB,
# args.setMappingBiasVcf=list(normal.panel.vcf.file=normal.panel.vcf.file),
# args.filterVcf=list(snp.blacklist=snp.blacklist, 
# stats.file=mutect.stats.file), 
    args.segmentation=list(interval.weight.file=interval.weight.file), 
    post.optimize=FALSE, plot.cnv=FALSE, verbose=FALSE)
@

The \Rcode{normal.coverage.file} argument points to a coverage file obtained from
either a matched or a process-matched normal sample, but can be also a small
pool of best normals (Section~\ref{bestnormal}).  

The \Rcode{normalDB} argument (Section~\ref{bestnormal}) provides a pool of
normal samples and for example allows the segmentation function to skip targets
with low coverage or common germline deletions in the pool of normals.  If
available, a VCF containing all variants from the normal samples should be
provided via \Rcode{args.setMappingBiasVcf} to correct read mapping biases.  The
files specified in \Rcode{args.filterVcf} help \Biocpkg{PureCN} filtering SNVs
more efficiently for artifacts as described in Sections~\ref{artifactfiltering}
and \ref{artifactfiltering2}.  The \Rcode{snp.blacklist} is only necessary if
neither a matched normal nor a large pool of normals is available. 

The \Rcode{post.optimize} flag will increase the runtime by about a factor of
2-5, but might return slightly more accurate purity estimates. For high quality
whole-exome data, this is typically not necessary for copy number calling (but
might be for variant classification, see Section~\ref{predictsomatic}). For
smaller targeted panels, the runtime increase is typically marginal and
\Rcode{post.optimize} should be always set to \Rcode{TRUE}.

The \Rcode{plot.cnv} argument allows the segmentation function to generate
additional plots if set to \Rcode{TRUE}.  Finally, \Rcode{verbose} outputs
important and helpful information about all the steps performed and is therefore
set to \Rcode{TRUE} by default.

\section{Output}

\subsection{Plots}

We now create a few output files:

<<createoutput>>=
file.rds <- "Sample1_PureCN.rds"
saveRDS(ret, file=file.rds)
pdf("Sample1_PureCN.pdf", width=10, height=11)
plotAbs(ret, type="all")
dev.off()
@

The RDS file now contains the serialized return object of the
\Rfunction{runAbsoluteCN} call. The PDF contains helpful plots for all local
minima, sorted by likelihood.  The first plot in the generated PDF is displayed
in Figure~\ref{figure/figureexample1-1} and shows the purity and ploidy local
optima, sorted by final likelihood score after fitting both copy number and
allelic fractions.

<<figureexample1, fig.show='hide', fig.width=6, fig.height=6>>=
plotAbs(ret, type="overview")
@
\incfig{figure/figureexample1-1}{0.75\textwidth}{Overview.}
{The colors visualize the copy number fitting score from low (blue) to high
(red). The numbers indicate the ranks of the local optima. Yellow fonts
indicate that the corresponding solutions were flagged, which does not
necessarily mean the solutions are wrong. The correct solution (number 1) of
this toy example was flagged due to large amount of LOH.} 

We now look at the main plots of the maximum likelihood solution in more detail.

<<figureexample2, fig.show='hide', fig.width=6, fig.height=6>>=
plotAbs(ret, 1, type="hist")
@
\incfig{figure/figureexample2-1}{0.75\textwidth}{Log-ratio histogram.}

Figure~\ref{figure/figureexample2-1} displays a histogram of tumor vs. normal
copy number log-ratios for the maximum likelihood solution (number 1 in
Figure~\ref{figure/figureexample1-1}). The height of a bar in this plot is
proportional to the fraction of the genome falling into the particular
log-ratio copy number range. The vertical dotted lines and numbers visualize
the, for the given purity/ploidy combination, expected log-ratios for all
integer copy numbers from 0 to 7. It can be seen that most of the log-ratios of
the maximum likelihood solution align well to expected values for copy numbers
of 0, 1, 2 and 4. 

<<figureexample3, fig.show='hide', fig.width=8, fig.height=8>>=
plotAbs(ret, 1, type="BAF")
@
\incfig{figure/figureexample3-1}{0.95\textwidth}{B-allele frequency plot.}{Each
dot is a (predicted) germline SNP. The first panel shows the allelic fractions
as provided in the VCF file.  The alternating grey and white background colors
visualize odd and even chromosome numbers, respectively. The black lines
visualize the expected (not the average!) allelic fractions in the segment.
These are calculated using the estimated purity and the total and minor segment
copy numbers. These are visualized in black and grey, respectively, in the
second and third panel.  The second panel shows the copy number log-ratios, the
third panel the integer copy numbers.}

Germline variant data are informative for calculating integer copy number
because unbalanced maternal and paternal chromosome numbers in the tumor
portion of the sample lead to unbalanced germline allelic fractions.
Figure~\ref{figure/figureexample3-1} shows the allelic fractions of predicted
germline SNPs. The goodness of fit (GoF) is provided on an arbitrary scale in
which 100\% corresponds to a perfect fit and 0\% to the worst possible fit.
The latter is defined as a fit in which allelic fractions on average differ by
0.2 from their expected fractions. Note that this does not take purity into
account and low purity samples are expected to have a better fit. In the
middle panel, the corresponding copy number log-ratios are shown. The lower
panel displays the calculated integer copy numbers, corrected for purity and
ploidy. We can zoom into particular chromosomes 
(Figure~\ref{figure/figureexample3b-1}).

<<figureexample3b, fig.show='hide', fig.width=9, fig.height=8>>=
plotAbs(ret, 1, type="BAF", chr="chr19")
@
\incfig{figure/figureexample3b-1}{0.95\textwidth}{Chromosome plot.}{Similar
    to Figure~\ref{figure/figureexample3-1}, but zoomed into a particular 
    chromosome. The grey dots in the middle panel visualize copy number 
    log-ratios of targets without heterozygous SNPs, which are omitted from the 
    previous genome-wide plot. The x-axis now indicates genomic coordinates in
    kbps.}

<<figureexample4, fig.show='hide', fig.width=8, fig.height=8>>=
plotAbs(ret, 1, type="AF")
@
\incfig{figure/figureexample4-1}{0.95\textwidth}{Allele fraction plots.}{Each
dot is again a (predicted) germline SNP. The size of dots indicate quality,
defined here as the product of mapping bias and coverage. The shapes visualize
the different SNV groups based on prior and posterior probabilities.  The labels
show the expected values for all called states; 2m1 would be diploid,
heterozygous, 2m2 diploid, homozygous.  The relationship of allelic fraction and
coverage is shown in the top right panel.  This plot normally also shows somatic
mutations in two additional panels, with the left panel showing the same plot as
for germline SNPs and the bottom right panel a histogram of cellular fraction of
predicted somatic mutations. This toy example contains only germline SNPs
however.}

Finally, Figure~\ref{figure/figureexample4-1} provides more insight into how
well the variants fit the expected values.
\clearpage

\subsection{Data structures}

The R data file (\Rcode{file.rds}) contains gene-level copy number calls,
SNV status and LOH calls.  The purity/ploidy combinations are sorted by
likelihood and stored in \Rcode{ret\$results}.

<<output1>>=
names(ret)
@

We provide convenient functions to extract information from this data structure
and show their usage in the next sections. We recommend using these functions
instead of accessing the data directly since data structures might change in
future versions. 

\subsubsection{Prediction of somatic status and cellular fraction}
\label{predictsomatic}
To understand allelic fractions of particular SNVs, we must know the (i)
somatic status, the (ii) tumor purity, the (iii) local copy number, as well as
the (iv) number of chromosomes harboring the mutations or SNPs. One of
\Biocpkg{PureCN} main functions is to find the most likely combination of these
four values. We further assign posterior probabilities to all possible
combinations or states. Availability of matched normals reduces the search space
by already providing somatic status. 

The \Rfunction{predictSomatic} function provides access to these probabilities.
For predicted somatic mutations, this function also provides cellular fraction
estimates, i.e. the fraction of tumor cells with mutation. Fractions
significantly below 1 indicate sub-clonality\footnote{This number can be above 1
when the observed allelic fraction is higher than expected for a clonal
mutation. This may be due to random sampling, wrong copy number, sub-clonal copy
number events, or wrong purity/ploidy estimates.}:

<<output3>>=
head(predictSomatic(ret), 3)
@

The output columns are explained in Table~\ref{tbl:predictSomatic}.

To annotate the input VCF file with these values:
<<output4>>=
vcf <- predictSomatic(ret, return.vcf=TRUE)
writeVcf(vcf, file="Sample1_PureCN.vcf") 
@

For optimal classification results:
\begin{itemize}
    \item Set \Rcode{post.optimize=TRUE} since small inaccuracies in purity can
decrease the classification performance significantly
    \item Provide \Rcode{args.setMappingBias} a pool of normal VCF to
        obtain position-specific mapping bias information
    \item Exclude variants in regions of low mappability    
    \item Use a somatic posterior probability cutoff of 0.8 and 0.2 for somatic
        and germline variants, respectively.  This appears to be a good
        compromise of call rate and accuracy. If the beta-binomial model was
        selected in the \Rcode{model} argument of \Rfunction{runAbsoluteCN},
        these cutoffs might need to be relaxed to get acceptable call rates.
    \item Add a \Rcode{Cosmic.CNT} info field to the VCF or provide a COSMIC VCF
        in \Rfunction{runAbsoluteCN} (see Section~\ref{seccosmic}). 
\end{itemize}        

\textbf{Note that the posterior probabilities assume that the purity and ploidy
combination is correct. Before classifying variants, it is thus recommended to
manually curate flagged samples.}

\begin{table*}
\caption{\Rfunction{predictSomatic} output columns.}
\begin{tabular}{lp{9.5cm}}
\toprule
Column name       & Description \\
\midrule
\Rcode{chr, start, end}   & Variant coordinates \\
\Rcode{ID}   & The variant ID as provided in the VCF  \\
\Rcode{REF, ALT}   & The reference and alt alleles  \\
\Rcode{SOMATIC.M*} & Posterior probabilities for all somatic states.  M0 to M7 
are multiplicity values, i.e. the number of chromosomes harboring the 
mutation (e.g.  1 heterozygous, 2 homozygous if copy number C is 2). SOMATIC.M0
represents a sub-clonal state (somatic mutations by definition have a 
multiplicity larger than 0). \\
\Rcode{GERMLINE.M*} & Posterior probabilities for all heterozygous germline 
states \\
\Rcode{GERMLINE.CONTHIGH} & Posterior probability for contamination.  This 
state corresponds to homozygous germline SNPs that were not filtered out 
because reference alleles from another individual were sequenced, resulting in 
allelic fractions smaller than 1. \\
\Rcode{GERMLINE.CONTLOW} & Posterior probability for contamination. This state
corresponds to non-reference alleles only present in the contamination. \\
\Rcode{GERMLINE.HOMOZYGOUS} & Posterior probability that SNP is homozygous in 
normal. Requires the \Rcode{model.homozygous} option in 
\Rfunction{runAbsoluteCN}. See Section~\ref{sec:celllines}. \\
\Rcode{ML.SOMATIC} & \Rcode{TRUE} if the maximum likelihood state is a somatic
    state \\
\Rcode{POSTERIOR.SOMATIC} & The posterior probability that the variant is 
somatic (sum of all somatic state posterior probabilities) \\
\Rcode{ML.M}  & Maximum likelihood multiplicity \\
\Rcode{ML.C}  & Maximum likelihood integer copy number \\
\Rcode{ML.M.SEGMENT} & Maximum likelihood minor segment copy number \\
\Rcode{M.SEGMENT.POSTERIOR} & Posterior probability of \Rcode{ML.M.SEGMENT} \\
\Rcode{M.SEGMENT.FLAGGED} & Segment flag indicating \Rcode{ML.M.SEGMENT} is
unreliable, either due to low posterior probability (< 0.5) or few variants (<
\Rcode{min.variants.segment}). Indels are always flagged. \\
\Rcode{ML.AR}  & Expected allelic fraction of the maximum likelihood state \\
\Rcode{AR}   & Observed allelic fraction (as provided in VCF) \\
\Rcode{AR.ADJUSTED} & Observed allelic fraction adjusted for mapping bias\\
\Rcode{ML.LOH} & \Rcode{TRUE} if variant is most likely in LOH \\
\Rcode{CN.SUBCLONAL} & \Rcode{TRUE} if copy number segment is sub-clonal \\
\Rcode{CELLFRACTION} & Fraction of tumor cells harboring the somatic mutation\\
\Rcode{FLAGGED} & Flag indicating that call is unreliable 
    (currently only due to high mapping bias and high pool of normal counts)\\
\Rcode{log.ratio} & The copy number log-ratio (tumor vs. normal) for this 
    variant \\
\Rcode{depth} & The total sequencing depth at this position \\
\Rcode{prior.somatic} & Prior probability of variant being somatic \\
\Rcode{prior.contamination} & Prior probability that variant is contamination 
    from another individual \\
\Rcode{on.target} & 1 for variants within intervals, 2 for variants in flanking
 regions, 0 for off-target variants \\
\Rcode{seg.id}       & Segment id  \\
\Rcode{pon.count}  & Number of hits in the \Rcode{normal.panel.vcf.file} \\
\Rcode{gene.symbol} & Gene symbol if available \\ 
\bottomrule
\end{tabular}
\label{tbl:predictSomatic}
\end{table*}

\subsubsection{Amplifications and deletions}

To call amplifications, we recommend using a cutoff of 6 for focal
amplifications and a cutoff of 7 otherwise. For homozygous deletions, a cutoff
of 0.5 is useful to allow some heterogeneity in copy number.

For samples that failed \Biocpkg{PureCN} calling we recommended using
common log-ratio cutoffs to call amplifications, for example 0.9.

This strategy is implemented in the \Rfunction{callAlterations} function:

<<calling2>>=
gene.calls <- callAlterations(ret)
head(gene.calls)
@

It is also often useful to filter the list further by known biology, for
example to exclude non-focal amplifications of tumor suppressor genes. The
Sanger Cancer Gene Census \cite{Futreal2004} for example provides such a list.

The output columns of \Rfunction{callAlterations} are explained in
Table~\ref{tbl:callAlterations}.

\begin{table*}
\caption{\Rfunction{callAlterations} output columns.}
\begin{tabular}{lp{9.5cm}}
\toprule
Column name       & Description \\
\midrule
\Rcode{chr, start, end} & Gene coordinates \\
\Rcode{C}            & Segment integer copy number \\
\Rcode{seg.mean}     & Segment mean of copy number log-ratios 
(not adjusted for purity/ploidy)  \\
\Rcode{seg.id}       & Segment id  \\
\Rcode{number.targets} & Number of targets annotated with this symbol \\
\Rcode{gene.*}       & Gene copy number log-ratios  
(not adjusted for purity/ploidy)  \\
\Rcode{focal}       & \Rcode{TRUE} for focal amplification, as defined by the 
\Rfunction{fun.focal} argument in \Rfunction{runAbsoluteCN} \\
\Rcode{breakpoints} & Number of breakpoints between \Rcode{start} and 
\Rcode{end} \\
\Rcode{type}        & Amplification or deletion \\
\Rcode{num.snps.segment} & Number of SNPs in this segment informative for 
    LOH detection (requires VCF)\\
\Rcode{M} & minor copy number of segment (requires VCF) \\
\Rcode{M.flagged} & flag indicating that \Rcode{M} is unreliable (requires VCF) \\
\Rcode{loh}         & \Rcode{TRUE} if segment is in LOH, \Rcode{FALSE} if not 
    and \Rcode{NA} if number of SNPs is insufficient (requires VCF) \\
\bottomrule
\end{tabular}
\label{tbl:callAlterations}
\end{table*}


\subsubsection{Find genomic regions in LOH}

The \Rcode{gene.calls} data.frame described above provides gene-level LOH
information. To find the corresponding genomic regions in LOH, we can use the
\Rfunction{callLOH} function:

<<loh>>=
loh <- callLOH(ret)
head(loh)
@

The output columns are explained in Table~\ref{tbl:callLOH}.

\begin{table*}
\caption{\Rfunction{callLOH} output columns.}
\begin{tabular}{lp{9.5cm}}
\toprule
Column name & Description \\
\midrule
\Rcode{chr, start, end} & Segment coordinates \\
\Rcode{arm}         & Chromosome arm \\
\Rcode{C}           & Segment integer copy number \\
\Rcode{M}           & Minor integer copy number ($M+N=C,M\leq N$) \\
\Rcode{type}        & LOH type if $M=0$ \\
\bottomrule
\end{tabular}
\label{tbl:callLOH}
\end{table*}

\section{Curation}

For prediction of variant status (germline vs. somatic, sub-clonal vs. clonal,
homozygous vs. heterozygous), it is important that both purity and ploidy are
correct. We provide functionality for curating results:

<<curationfile>>=
createCurationFile(file.rds) 
@

This will generate a CSV file in which the correct purity and ploidy values
can be manually entered. It also contains a column "Curated", which should be
set to \Rcode{TRUE}, otherwise the file will be overwritten when re-run.

Then in R, the correct solution (closest to the combination in the CSV file)
can be loaded with the \Rfunction{readCurationFile} function:

<<readcurationfile>>=
ret <- readCurationFile(file.rds)
@

This function has various handy features, but most importantly it will re-order
the local optima so that the curated purity and ploidy combination is ranked
first. This means \Rcode{plotAbs(ret,1,type="hist")} would show the plot for
the curated purity/ploidy combination, for example.

The default curation file will list the maximum likelihood solution:

<<curationfileshow>>=
read.csv("Sample1_PureCN.csv")
@

\Biocpkg{PureCN} currently only flags samples with warnings, it does not mark
any samples as failed. The \Rcode{Failed} column in the curation file can be
used to manually flag samples for exclusion in downstream analyses. See
Table~\ref{tbl:flags} for an explanation of all flags.

\begin{table*}
\caption{\Rfunction{createCurationFile} flags.}
\begin{tabular}{lp{6cm}}
\toprule
Flag & Description \\
\midrule
EXCESSIVE LOH & $> 50$\% of genome in LOH and ploidy $< 2.6$ \\
EXCESSIVE LOSSES & $\geq 1$\% of genome deleted \\
HIGH AT- OR GC-DROPOUT & High GC-bias exceeding cutoff in \Rcode{max.dropout} \\
HIGH PURITY & (when \Rcode{model.homozygous=FALSE}). For very high purity
samples, it is recommended to set \Rcode{model.homozygous=TRUE}. See
Section~\ref{sec:celllines}. \\ 
LOW PURITY & Purity $< 30$\% \\
LOW BOOTSTRAP VALUE & \Rfunction{bootstrapResults} identified multiple plausible solutions \\
NOISY LOG-RATIO & Log-ratio standard deviation $>$ \Rcode{max.logr.sdev} \\ 
NOISY SEGMENTATION & More than \Rcode{max.segments} \\ 
NON-ABERRANT & $\geq 99 $\% of genome has identical copy number and $\geq 0.5$\% has second most common state \\
POLYGENOMIC & $\geq 0.75 \times$ \Rcode{max.non.clonal} fraction of the genome in sub-clonal state \\
POOR GOF & GoF $<$ \Rcode{min.gof} \\
POTENTIAL SAMPLE CONTAMINATION & Significant portion of dbSNP variants potentially cross-contaminated \\
RARE KARYOTYPE & Ploidy $< 1.5$ or  $> 4.5$  \\
\bottomrule
\end{tabular}
\label{tbl:flags}
\end{table*}

\section{Cell lines}
\label{sec:celllines}

Default parameters assume some normal contamination. In 100\% pure samples
without matching normal samples such as cell lines, we cannot distinguish
homozygous SNPs from LOH by looking at single allelic fractions alone. It is
thus necessary to keep homozygous variants and include this uncertainty in the
likelihood model. This is done by setting the \Rfunction{runAbsoluteCN} argument
\Rcode{model.homozygous=TRUE}. If matched normals are available, then variants
homozygous in normal are automatically removed since they are uninformative.

For technical reasons, the maximum purity \Biocpkg{PureCN} currently models is
0.99. We recommend setting \Rcode{test.purity=seq(0.9,0.99,by=0.01)} in
\Rfunction{runAbsoluteCN} for cell lines. 

Please note that in order to detect homozygous deletions in 100\% pure samples,
you will need to provide a \Rcode{normalDB} in \Rfunction{runAbsoluteCN} to
filter low quality targets efficiently (Section~\ref{secrecommended}).

\section{Maximizing the number of heterozygous SNPs}
\label{secmaxsnps}

It is possible to use SNPs in off-target reads in the variant fitting step by
running \software{MuTect} without interval file and then setting the
\Rfunction{filterVcfBasic} argument \Rcode{remove.off.target.snvs} to
\Rcode{FALSE}. We recommend a large pool of normals in this case and then
generating SNP blacklists as described in Sections~\ref{artifactfiltering} and
\ref{artifactfiltering2}.  Remember to also run all the normals in
\software{MuTect} without interval file. 

An often better alternative to including all off-target reads is only including
variants in the flanking regions of targets (between 50-100bp). This will usually
significantly increase the number of heterozygous SNPs (see
Section~\ref{faqinput}). These SNPs are automatically added if the variant
caller was run without interval file or with interval padding.

\section{Advanced usage}
\subsection{Custom normalization and segmentation}
\label{customseg}
Copy number normalization and segmentation are crucial for obtaining good
purity and ploidy estimates. If you have a well-tested pipeline that produces
clean results for your data, you might want to use \Biocpkg{PureCN} as add-on
to your pipeline. By default, we will use \CRANpkg{DNAcopy}
\cite{Venkatraman2007} to segment normalized target-level coverage log-ratios.
It is straightforward to replace the default with other methods and the
\Rfunction{segmentationCBS} function can serve as an example.

The next section describes how to replace the default segmentation. For the
probably more uncommon case that only the coverage normalization is performed by
third-party tools, see Section~\ref{customnorm}.

\subsubsection{Custom segmentation}
It is possible to provide already segmented data, which is especially
recommended when matched SNP6 data are available or when third-party
segmentation tools are not written in R. Otherwise it is usually however better
to customize the default segmentation function, since the algorithm then has
access to the raw log-ratio distribution\footnote{If the third-party tool
provides target-level log-ratios, then these can be provided via the
\Rcode{log.ratio} argument in addition to \Rcode{seg.file} though. See also
Section~\ref{customnorm}.}. The expected file format for already segmented copy
number data is\footnote{This segmentation file can contain multiple samples, in
which case the provided \Rcode{sampleid} must match a sample in the column
\Rcode{ID}}:

\begin{verbatim}
    ID  chrom   loc.start   loc.end num.mark    seg.mean
    Sample1   1   61723   5773942 2681    0.125406444072723
    Sample1   1   5774674 5785170 10  -0.756511807441712
\end{verbatim}

Since its likelihood model is exon-based, \Biocpkg{PureCN} currently still
requires an interval file to generate simulated target-level log-ratios from a
segmentation file.  For simplicity, this interval file is expected either via
the \Rcode{tumor.coverage.file} or via the \Rcode{interval.file} argument (see
Figure~\ref{figure/figurecustombaf-1}). Note that \Biocpkg{PureCN} will
re-segment the simulated log-ratios using the default
\Rfunction{segmentationCBS} function, in particular to identify regions of
copy-number neutral LOH and to cluster segments with similar allelic imbalance
and log-ratio. The provided interval file should therefore cover all
significant copy number alterations\footnote{If this behaviour is not wanted,
because maybe the custom function already identifies CNNLOH reliably,
\Rfunction{segmentationCBS} can be replaced with a minimal version.}. Please
check that the log-ratios are similar to the ones obtained by the default
\Biocpkg{PureCN} segmentation and normalization.

<<customseg>>=
retSegmented <- runAbsoluteCN(seg.file=seg.file, 
    interval.file=interval.file, vcf.file=vcf.file, 
    max.candidate.solutions=1, genome="hg19",
    test.purity=seq(0.3,0.7,by=0.05), verbose=FALSE, 
    plot.cnv=FALSE)
@

The \Rcode{max.candidate.solutions} and \Rcode{test.purity} arguments are set
to non-default values to reduce the runtime of this vignette. 

<<figurecustombaf, fig.show='hide', fig.width=8, fig.height=8>>=
plotAbs(retSegmented, 1, type="BAF")
@
\incfig{figure/figurecustombaf-1}{0.95\textwidth}{B-allele frequency plot for
segmented data.}{This plot shows the maximum likelihood solution for an example
where segmented data are provided instead of coverage data. Note that the middle
panel shows no variance in log-ratios, since only segment-level log-ratios are
available.}
  
\subsubsection{Custom normalization}
\label{customnorm}
If third-party tools such as \software{GATK4} are used to calculate
target-level copy number log-ratios, and \Biocpkg{PureCN} should be used for
segmentation and purity/ploidy inference only, it is possible to provide these
log-ratios:

<<customlogratio, message=FALSE>>=
# We still use the log-ratio exactly as normalized by PureCN for this
# example
log.ratio <- calculateLogRatio(readCoverageFile(normal.coverage.file),
    readCoverageFile(tumor.coverage.file))

retLogRatio <- runAbsoluteCN(log.ratio=log.ratio,
    interval.file=interval.file, vcf.file=vcf.file, 
    max.candidate.solutions=1, genome="hg19",
    test.purity=seq(0.3,0.7,by=0.05), verbose=FALSE, 
    normalDB=normalDB, plot.cnv=FALSE)
@

Again, the \Rcode{max.candidate.solutions} and \Rcode{test.purity} arguments
are set to non-default values to reduce the runtime of this vignette. It is
highly recommended to compare the log-ratios obtained by \Biocpkg{PureCN} and
the third-party tool, since some pipelines automatically adjust log-ratios for
a default purity value. Note that this example uses a pool of normals to filter
low quality targets. Interval coordinates are again expected in either a
\Rcode{interval.file} or a \Rcode{tumor.coverage.file}. If a tumor coverage file is
provided, then all targets below the coverage minimum are further excluded.

\subsection{COSMIC annotation}
\label{seccosmic}
If a matched normal is not available, it is also helpful to provide
\Rfunction{runAbsoluteCN} the COSMIC database \cite{Forbes2015} via
\Rcode{cosmic.vcf.file} (or via a \Rcode{Cosmic.CNT} INFO field in the VCF).
While this has limited effect on purity and ploidy estimation due the sparsity
of hotspot mutations, it often helps in the manual curation to compare how well
high confidence germline (dbSNP) vs. somatic (COSMIC) variants fit a particular
purity/ploidy combination. 

For variant classification (Section~\ref{predictsomatic}), providing COSMIC
annotation also avoids that hotspot mutations with dbSNP id get assigned a very
low prior probability of being somatic.

\subsection{ExAC and gnomAD annotation}

\Biocpkg{PureCN} is not automatically annotating input VCFs with data from
common germline databases such as ExAC. See Section~\ref{secinputvcf} for ways
to tell \Biocpkg{PureCN} where to find either a summary binary flag (i.e. likely
germline yes/no) or population allele frequencies.

\subsection{Mutation burden}

The \Rfunction{predictSomatic} function described in
Section~\ref{predictsomatic} can be used to efficiently remove private germline
mutations. This in turn allows the calculation of mutation burden for unmatched
tumor samples. A wrapper function for this specific task is included as
\Rfunction{callMutationBurden}:

<<callmutationburden>>=
callableBed <- import(system.file("extdata", "example_callable.bed.gz", 
    package = "PureCN"))

callMutationBurden(ret, callable=callableBed)
@

The \Rcode{callableBed} file should be a file parsable by
\Biocpkg{rtracklayer}. This file can specify genomic regions that are callable,
for example as obtained by \software{GATK3 CallableLoci}. This is optional, but
if provided can be used to accurately calculate mutation rates per
megabase. Variants outside the callable regions are not counted. Private
germline rates should be fairly constant across samples; outliers here should be
manually inspected.

The output columns are explained in Table~\ref{tbl:callMutationBurden}.

\begin{table*}
\caption{\Rfunction{callMutationBurden} output columns.}
\begin{tabular}{lp{6cm}}
\toprule
Column name & Description \\
\midrule
\Rcode{somatic.ontarget} & Number of mutations in target regions \\
\Rcode{somatic.all}      & Total number of mutations. Depending on input
VCF and \Rfunction{runAbsoluteCN} arguments, this might include calls in 
flanking regions and off-targets reads.  \\
\Rcode{private.germline.ontarget} & Number of private germline SNPs in 
targets. For matched tumor/normal samples, this is the number of variants
annotated as somatic, but classified as germline.  \\
\Rcode{private.germline.all} & Total number of private germline SNPs \\
\Rcode{callable.bases.ontarget} & Number of callable on-target bases \\
\Rcode{callable.bases.flanking} & Number of callable on-target and 
flanking bases \\
\Rcode{callable.bases.all} & Total number of callable bases. With
default parameters includes off-target regions that were ignored by 
\Rfunction{runAbsoluteCN}. \\
\Rcode{somatic.rate.ontarget} & Somatic mutations per megabase in target regions \\
\Rcode{somatic.rate.ontarget.95.lower} & Lower 95\% of confidence interval \\
\Rcode{somatic.rate.ontarget.95.upper} & Upper 95\% of confidence interval \\
\Rcode{private.germline.rate.ontarget} & Private germline mutations per megabase in target regions \\
\Rcode{private.germline.rate.ontarget.95.lower} & Lower 95\% of confidence interval \\
\Rcode{private.germline.rate.ontarget.95.upper} & Upper 95\% of confidence interval \\
\bottomrule
\end{tabular}
\label{tbl:callMutationBurden}
\end{table*}

\subsection{Detect cross-sample contamination}
\label{seccontamination}
It is important to correctly handle heterozygous common SNPs that do not have an
expected allelic fraction of 0.5 in normal samples. These can be SNPs in poor
quality regions (as already described, see
Section~\ref{artifactfilteringvcf}), but also SNPs from cross-sample
contaminated DNA. Without matched normals, detection of those problematic SNPs
is not trivial. 

For cross-sample contamination, \Biocpkg{PureCN} by default always tests for a
1\% contamination and assigns common SNPs to a contamination state when allelic
fractions are either close to 0 or close to 1 and when this cannot be explained
by CNAs. The main purpose of these states is to provide a bin for common SNPs
that for artifactual reasons do not fit any other state.

This tool applies a simple heuristic to flag samples for cross-contamination:
Given the coverage and putative contamination rate based on allelic fractions of
potentially contaminated SNPs, how many SNPs do we expect to detect based on our
power to detect variants at that contamination rate? If the expected number is
much higher than observed, then significant contamination is unlikely; 
observed SNPs close to 0 or 1 are more likely artifacts or the contamination
rate is much lower than the minimum tested. Otherwise \Biocpkg{PureCN} will
perform a post-optimization in which contamination rate is optimized in
additional variant fitting steps.

Cross-sample contamination can also result in increased observed heterozygosity
on chrX for males, which in turn often results in a \Biocpkg{PureCN} warning
that sex inferred from coverage and VCF are in conflict.

By default, cross-contamination is tested in the range from 1 to 7.5\%.
Catastrophic failures with higher contamination might not get flagged.

\subsection{Power to detect somatic mutations}

As final quality control step, we can test if coverage and tumor purity are
sufficent to detect mono-clonal or even sub-clonal somatic mutations. We
strictly follow the power calculation by Carter et al. \cite{Carter2012}. 

The following Figure~\ref{figure/power1-1} shows the power to detect mono-clonal
somatic mutations as a function of tumor purity and sequencing coverage
(reproduced from \cite{Carter2012}):

<<power1, fig.show='hide', fig.width=6, fig.height=6>>=
purity <- c(0.1,0.15,0.2,0.25,0.4,0.6,1)
coverage <- seq(5,35,1)
power <- lapply(purity, function(p) sapply(coverage, function(cv) 
    calculatePowerDetectSomatic(coverage=cv, purity=p, ploidy=2, 
    verbose=FALSE)$power))

# Figure S7b in Carter et al.
plot(coverage, power[[1]], col=1, xlab="Sequence coverage", 
    ylab="Detection power", ylim=c(0,1), type="l")

for (i in 2:length(power)) lines(coverage, power[[i]], col=i)
abline(h=0.8, lty=2, col="grey")     
legend("bottomright", legend=paste("Purity", purity),
    fill=seq_along(purity))
@
\incfig{figure/power1-1}{0.95\textwidth}{Power to detect mono-clonal somatic
mutations.}{Reproduced from \cite{Carter2012}.}

Figure~\ref{figure/power2-1} then shows the same plot for sub-clonal mutations
present in 20\% of all tumor cells: 

<<power2, fig.show='hide', fig.width=6, fig.height=6>>=
coverage <- seq(5,350,1)
power <- lapply(purity, function(p) sapply(coverage, function(cv) 
    calculatePowerDetectSomatic(coverage=cv, purity=p, ploidy=2, 
    cell.fraction=0.2, verbose=FALSE)$power))
plot(coverage, power[[1]], col=1, xlab="Sequence coverage", 
    ylab="Detection power", ylim=c(0,1), type="l")

for (i in 2:length(power)) lines(coverage, power[[i]], col=i)
abline(h=0.8, lty=2, col="grey")     
legend("bottomright", legend=paste("Purity", purity),
    fill=seq_along(purity))
@
\incfig{figure/power2-1}{0.95\textwidth}{Power to detect sub-clonal somatic
mutations present in 20\% of all tumor cells.}{Reproduced from \cite{Carter2012}.}

\clearpage

\section{Limitations}
\label{sec:limits}
\Biocpkg{PureCN} currently assumes a completely diploid normal genome. For
human samples, it tries to detect sex by calculating the coverage ratio of
chromosomes X and Y and will then remove sex chromosomes in male 
samples\footnote{Loss of Y chromosome (LOY) can result in wrong female calls,
especially in high purity samples or if LOY is in both tumor and contaminating
normal cells. The software throws a warning in this case when germline SNPs
are provided.}. For non-human samples, the user needs to manually remove all
non-diploid chromosomes from the coverage data and specify
\Rcode{sex="diploid"} in the \Biocpkg{PureCN} call.

While \Biocpkg{PureCN} supports and models sub-clonal somatic copy number
alterations, it currently assumes that the majority of alterations are
mono-clonal. For most clinical samples, this is reasonable, but very
heterogeneous samples are likely not possible to call without manual curation.
Poly-genomic tumors are often called as high ploidy or low purity. The former
usually happens when sub-clonal losses are called as 2 copies and mono-clonal
losses correctly as 1 copy. The latter when sub-clonal losses are called
mono-clonal, which only happens when there are far more sub-clonal than
mono-clonal losses. Please note however that unless purities are very high,
algorithms that model poly-genomic tumors do not necessarily have a higher call
rate, since they tend to overfit noisy samples or similarly confuse true
high-ploidy with poly-genomic tumors.  Due to the lack of signal, manual
curation is also recommended in low purity samples or very quiet genomes.

\section{Support}

If you encounter bugs or have problems running \Biocpkg{PureCN},
please post them at

\begin{itemize}
    \item \url{https://support.bioconductor.org/p/new/post/?tag\_val=PureCN} or
    \item \url{https://github.com/lima1/PureCN/issues}.
\end{itemize}

If \Biocpkg{PureCN} throws user errors, then there is likely a problem with the
input files.  If the error message is not self-explanatory, feel free to seek
help at the support site. 

In your report, please add the outputs of the \Rfunction{runAbsoluteCN} call
(with \Rcode{verbose=TRUE}, or the \Rcode{*.log} file in PureCN.R) and
\Rfunction{sessionInfo()}. Please also check that your problem is not already
covered in the following sections.

For general feedback such as suggestions for improvements, feature requests,
complaints, etc. please do not hesitate to send us an email.

\subsection{Checklist}

\begin{itemize}
    \item Used the correct interval files provided by the manufacturer of the
    capture kit and the genome version of the interval file matches the
    reference. Ideally used the baits file, not the targets file (in Agilent
    data, the baits files are called "covered" and the targets are "regions").
    \item For hybrid capture data, included off-target reads in the coverage
        calculation 
    \item BAM files were generated following established best practices and
    tools finished successfully.
    \item Checked standard QC metrics such as AT/GC dropout and duplication 
    rates.
    \item Tumor and normal data were obtained using the same capture kit and
    pipeline.
    \item Coverage data of tumor and normal were GC-normalized.
    \item The VCF file contains germline variants (i.e. not only somatic calls).
    \item Maximized the number of high coverage heterozygous SNPs, for example
    by running \software{MuTect} with a 50-75bp interval padding
    (Section~\ref{secmaxsnps}).  The \Rfunction{runAbsoluteCN} output lists the
    percentage of targets with variants and this should be around 10-15\%.
    Ultra-deep sequencing data can provide good SNP allelic fractions in the
    100-200bp flanking regions.
    \item If a pool of normal samples is available, followed the steps in
    Section~\ref{artifactfiltering}.
    \item Read the output of \Rfunction{runAbsoluteCN} with
        \Rcode{verbose=TRUE}, fixed all warnings.
    \item If third-party segmentation tools are used, checked that normalized
    log-ratios are not biased, i.e. very similar compared to \Biocpkg{PureCN}
    log-ratios (some pipelines already adjust for a default normal 
    contamination).
\end{itemize}

\subsection{FAQ}

\paragraph{If the ploidy is frequently too high, please check:}

\begin{itemize}
    \item Does the log-ratio histogram (Figure~\ref{figure/figureexample2-1})
look noisy? If yes, then
    \begin{itemize}
    \item Is the coverage sufficient? Tumor coverages below 80X can be
difficult, especially in low purity samples. Normal coverages below 50X might
result in high variance of log-ratios. See Section~\ref{bestnormal} for finding
a good normal sample for log-ratio calculation.
    \item Is the coverage data of both tumor and normal GC-normalized? If not, 
    see \Rfunction{correctCoverageBias}.
    \item Is the quality of both tumor and normal sufficient? A high AT or
GC-dropout might result in high variance of log-ratios. Challenging FFPE samples
also might need parameter tuning of the segmentation function. See 
\Rfunction{segmentationCBS}. A high expected tumor purity allows more aggressive
segmentation parameters, such as \Rcode{prune.hclust.h=0.2} or higher.
    \item Was the correct target interval file used (genome version and capture
    kit, see Section~\ref{gatkcoverage})? If unsure, ask the help desk of your
    sequencing center.
    \item Were the normal samples run with the same assay and pipeline? 
    \item Did you provide \Rfunction{runAbsoluteCN} all the recommended files
    as described in Section~\ref{secrecommended}?  
    \item For whole-genome data, you will get better results using a
    specialized third-party segmentation method as described in
    section~\ref{customseg}, since our default is optimized for targeted
    sequencing.
    \end{itemize}
    \item Otherwise, if log-ratio peaks are clean as in 
Figure~\ref{figure/figureexample2-1}:
    \begin{itemize}
        \item Was MuTect run without a matched normal? If yes, then make sure
        to provide either a pool of normal VCF or a SNP blacklist (if no pool
        of normal samples is available) as described in
        Sections~\ref{artifactfiltering} and \ref{artifactfiltering2}.
        \item A high fraction of sub-clonal copy-number alterations might also
    result in a low ranking of correct low ploidy solutions (see
    Section~\ref{sec:limits}). 
    \end{itemize}
\end{itemize} 

\paragraph{If the ploidy is frequently too low:}
\begin{itemize}
    \item \Biocpkg{PureCN} with default parameters is conservative in calling
    genome duplications.
    \item This should only affect low purity samples (< 35\%), since in higher
    purity samples the duplication signal is usually strong enough to
    reliably detect it.  
    \item In whole-exome data, it is usually safe to decrease the
    \Rcode{max.homozygous.loss} default, since such large losses are rare.
\end{itemize}

\paragraph{Will PureCN work with my data?}

\begin{itemize}
    \item \Biocpkg{PureCN} was designed for medium-sized (>2-3Mb) targeted
        panels.  The more data, the better, best results are typically achieved
        in whole-exome data.

    \item The number of heterozygous SNPs is also important (>1000 per sample).
        Copy number baits enriched in SNPs are therefore very helpful (see
        Section~\ref{secmaxsnps}).

    \item Some users got acceptable results with small (<1Mb) panels.
        Try to find a perfect off-target bin width
        (\Rcode{average.off.target.width} in \Rfunction{preprocessIntervals})
        and maximize the number of heterozygous SNPs by including as much
        padding as possible. Keep in mind that without tiling baits, you will
        only have a poor resolution to detect LOH. 

    \item Coverages below 80X are difficult unless purities are high and
        coverages are even.

    \item \Biocpkg{PureCN} also needs process-matched normal samples, again, the
        more the better. 

    \item Samples with tumor purities below 15-20\% usually cannot be analyzed
        with this algorithm and \Biocpkg{PureCN} might return very wrong purity
        estimates. In high coverage samples with low duplication rates, this
        limit can be close to 10\%.

    \item Whole-genome data is not officially supported and specialized tools
        will likely provide better results.  Third-party segmentation tools
        designed for this data type would be again required.

    \item Amplicon sequencing data is also not officially supported. If the
        assay contains tiling probes (at least with 1Mb spacing) and uses a
        barcode protocol that reduces PCR bias of measured allelic fractions,
        then this method might produce acceptable results. Setting the
        \Rcode{model} argument of \Rfunction{runAbsoluteCN} to \Rcode{betabin}
        is recommended. Specialized segmentation tools might be again better
        than our default.

\end{itemize}

\paragraph{If you have trouble generating input data PureCN accepts, please check:}
\label{faqinput}
\begin{itemize}
    \item For problems related to generating valid coverage data, either consult
        the \software{GATK} manual for the \software{DepthOfCoverage} or
        \software{CollectFragmentCounts} tools or Section~\ref{purecncoverage}
        for the equivalent function in \Biocpkg{PureCN}. If you use
        \software{DepthOfCoverage} and off-target intervals as generated by
        IntervalFile.R (See Quick vignette), make sure to run it with parameters
        \Rcode{ --omitDepthOutputAtEachBase} and \Rcode{--interval\_merging
        OVERLAPPING\_ONLY}.

    \item Currently only VCF files generated by \software{MuTect 1} are
        officially supported and well tested. A minimal example 
        \software{MuTect} call would be:
    \begin{verbatim}
    $JAVA7 -Xmx6g -jar $MUTECT \
    --analysis_type MuTect -R $REFERENCE \
    --dbsnp $DBSNP_VCF \
    --cosmic $COSMIC_VCF \
    -I:normal $BAM_NORMAL \
    -I:tumor $BAM_TUMOR  \
    -o $OUT/${ID}_bwa_mutect_stats.txt \
    -vcf $OUT/${ID}_bwa_mutect.vcf
    \end{verbatim}
    The normal needs to be matched; without matched normal, only provide the
    tumor BAM file (do NOT provide a process-matched normal here). The default
    output file is the stats or call-stats file; this can be provided in
    addition to the required VCF file via \Rcode{args.filterVcf} in
    \Rfunction{runAbsoluteCN}. If provided, it may help \Biocpkg{PureCN} filter
    artifacts. This requires \software{MuTect} in version 1.1.7.  This version
    is currently available at
    \url{https://software.broadinstitute.org/gatk/download/mutect} and requires
    Java 1.7 (it does not work with Java 1.8).
    
    Note that this \software {MuTect} VCF will contain variants in off-target
    reads.  By default, \Biocpkg{PureCN} will remove variants outside the
    provided targets and their 50bp flanking regions. We highly recommend
    finding good values for each assay. A good cutoff will maximize the number
    of heterozygous SNPs and keep only an acceptable number of lower quality
    calls.  This cutoff is set via \Rcode{interval.padding} in
    \Rcode{args.filterVcf}.  See Section~\ref{secmaxsnps}. 

    \item Support for GATK4 and Mutect2 is still experimental. When matched
    normals are available, this will require version 4.0.3.0 and higher and
    specifying the \Rcode{--genotype-germline-sites} flag. 

    \item For VCFs generated by other callers, the required dbSNP annotation 
    can be added for example with \software{bcftools}:
    \begin{verbatim}
    bcftools annotate --annotation $DBSNP_VCF \
        --columns ID --output $OUT/${ID}_bwa_dbsnp.vcf.gz --output-type z \
            $OUT/${ID}_bwa.vcf.gz
    \end{verbatim}
    \item To generate a mappability file with the \software{GEM} library:
         \begin{itemize}
         \item Calculate mappability, set kmer size to length of mapped reads. 
         \begin{verbatim}
    REFERENCE="hg38.fa"
    PREF=`basename $REFERENCE .fa`     
    THREADS=4
    KMER=100
    gem-indexer -T ${THREADS} -c dna -i ${REFERENCE} -o ${PREF}_index
    gem-mappability -T ${THREADS} -I ${PREF}_index.gem -l ${KMER} \
        -o ${PREF}_${KMER} -m 2 -e 2
    gem-2-wig -I ${PREF}_index.gem -i ${PREF}_${KMER}.mappability \
        -o ${PREF}_${KMER}
         \end{verbatim}
        \item Convert to bigWig format, for example using the UCSC
        \software{wigToBigWig} tool:
         \begin{verbatim}
    cut -f1,2 ${REFERENCE}.fai > ${PREF}.sizes
    wigToBigWig ${PREF}_${KMER}.wig ${PREF}.sizes ${PREF}_${KMER}.bw
         \end{verbatim}
         \end{itemize}   

    \item To generate the normal panel VCF (\Rcode{normal.panel.vcf.file})
    with \software{GATK3}:
    \begin{itemize}
        \item Run \software{MuTect} on the normal with
        \Rcode{-I:tumor \$BAM\_NORMAL} and optionally with the 
        \Rcode{--artifact\_detection\_mode} flag.
        \item Remove the empty \Rcode{none} sample from the VCF:
        \begin{verbatim}
    $JAVA -Xmx6g -jar $GATK3 \
    --analysis_type SelectVariants -R $REFERENCE \
    --exclude_sample_expressions none \
    -V $OUT/${ID}_bwa_mutect_artifact_detection_mode.vcf \
    -o $OUT/${ID}_bwa_mutect_artifact_detection_mode_no_none.vcf 
        \end{verbatim}
        \item Merge the VCFs:
        \begin{verbatim}
    CMD="java  -Xmx12g -jar $GATK3 -T CombineVariants --minimumN 3 -R $REFERENCE"
    for VCF in $OUT/*bwa_mutect_artifact_detection_mode_no_none.vcf;
    do
        CMD="$CMD --variant $VCF"
    done
    CMD="$CMD -o $OUT/normals_merged_min3.vcf"
    echo $CMD > $OUT/merge_normals_min3.sh
    bgzip $OUT/normals_merged_min3.vcf
    tabix $OUT/normals_merged_min3.vcf.gz
        \end{verbatim}
    \end{itemize}
\end{itemize}

\paragraph{Questions related to pool of normals.}

As described in detail in Sections~\ref{bestnormal} and
\ref{artifactfiltering}, a pool of normal samples is used in \Biocpkg{PureCN}
for coverage normalization (to adjust for target-specific capture biases) and
for artifact filtering. A few recommendations:

\begin{itemize}
    \item Matched normals are obviously perfect for identifying most alignment
        artifacts and mapping biases. While not critical, we still recommend
        generating a \Rcode{normal.panel.vcf.file} for \software{MuTect} and
        \Rfunction{setMappingBiasVcf} using the available normals.

    \item Without matched normals, we need process-matched normals for coverage
        normalization. We recommend more than 5 from 2-3 different library
        preparation and sequencing batches. The more, the better.

    \item These normals used for coverage normalization should be ideally
        sequenced to at least half the coverage as the tumor samples. This is
        completely different from matched normals for germline calling where
        30-40X provide sufficient power to detect heterozygosity.

    \item For artifact removal, the more normals available, the more rare
        artifacts are removed. We recommend at least 10, 50 or more are ideal.
        The more artifacts are removed, the less likely \Biocpkg{PureCN} output
        requires manual curation (Section~\ref{artifactfiltering}).

    \item For position-specific mapping bias correction, the more normals are
        available, the more rare SNPs will have reliable mapping bias estimates.
        This requires again at least about 10 normals to be useful, 100 or more
        are ideal.

    \item With smaller pool of normals, we additionally recommend filtering SNPs
        from low quality regions (Section~\ref{artifactfiltering2}).
        Additionally, it is worth trying the beta-binomial function instead of
        the default in the \Rcode{model} argument of \Rfunction{runAbsoluteCN}.
        This will incorporate uncertainty of observed variant allelic fractions
        in the variant fitting step.
    
    \item \textit{Do I really need a pool of normals? I only have tumor samples.}
        Unfortunately, yes. If you used a commercial capture kit, you might be
        able to obtain control samples from the vendor or the public domain.
        This is not optimal, but usually better than nothing. 
    
    \item It is safe to include multiple normals from the same individual.
        Fewer common germline CNVs in  \Rfunction{calculateIntervalWeights} and
        fewer SNPs in \Rfunction{calculateMappingBiasVcf} will be detected. But
        especially when the controls were sequenced in multiple batched, these
        replicates will still provide useful information for coverage
        normalization.
         
\end{itemize}

\paragraph{Questions related to manual curation.}

\Biocpkg{PureCN}, like most other related tools, essentially finds the most
simple explanation of the data. There are three major problems with this
approach:
\begin{itemize}
    \item First, hybrid capture data can be noisy and the algorithm must
        distinguish signal from noise; if the algorithm mistakes noise for
        signal, then this often results in wrong high ploidy calls (see
        Sections~\ref{artifactfiltering} and \ref{artifactfiltering2}). If all
        steps in this vignette were followed, then \Biocpkg{PureCN} should
        ignore common artifacts. Noisy samples thus often have outlier ploidy
        values and are often automatically flagged by \Biocpkg{PureCN}. The
        correct solution is in most of these cases ranked second or third.

    \item The second problem is that signal can be sparse, i.e. when the tumor
        purity is very low or when there are only few somatic events.  Manual
        curation is often easy in the latter case. For example when small losses
        are called as homozygous, but corresponding germline allele-frequencies
        are unbalanced (a complete loss would result in balanced germline allele
        frequencies, since only normal DNA is left). Future versions might
        improve calling in these cases by underweighting uninformative genomic
        regions.

    \item The third problem is that tumor evolution is fast and complex and very
        difficult to incorporate into general likelihood models. Sometimes
        multiple solutions explain the data equally well, but one solution is
        then often clearly more consistent with known biology, for example LOH
        in tumor suppressor genes such as \textit{TP53}.  A basic understanding
        of both the algorithm and the tumor biology of the particular cancer
        type are thus important for curation.  Fortunately, in most cancer
        types, such ambiguity is rather rare. See also Section~\ref{sec:limits}.

\end{itemize}

\paragraph{If all or most of the samples are flagged as:}

\begin{description}
    \item[Noisy segmentation:] The default of 300 for \Rcode{max.segments} is
    calibrated for high quality and high coverage whole-exome data. For
    whole-genome data or lower coverage data, this value needs to be
    re-calibrated.  In case the copy number data looks indeed noisy, please see
    the first FAQ. Please be aware that \Biocpkg{PureCN} will apply more
    aggressive segmentation parameters when the number of segments exceeds this
    cutoff. If the high segment count is real, this might confound downstream
    analyses.
    \item[High AT/GC dropout:] If the data is GC-normalized, then there might
    be issues with either the target intervals or the provided GC content.
    Please double check that all files are correct and that all the coverage
    files are GC-normalized (Section~\ref{secgcbias}).
    \item[Sex mismatch of coverage and VCF:] If the panel contains baits for
    chromosome Y, then the interval file was probably generated without 
    mappability file (Section~\ref{sectargetinfo}). Cross-sample contamination
    (Section~\ref{seccontamination}) can also cause sex mismatches.
\end{description}

\appendix

\bibliography{PureCN}

\section*{Session Info}
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toLatex(sessionInfo())
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\end{document}
