r-bioc-savr 1.16.0-1 / usr / lib / R / site-library / savR / doc / savR.Rnw

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\title{Using savR}
\author{R. Brent Calder}

\maketitle

<<prepare>>=
library(savR)
@

<<intro,cache=T>>=
fc <- savR(system.file("extdata", "MiSeq", package="savR"))
@

<<show>>=
fc
@

<<doPfPlot,eval=F>>=
pfBoxplot(fc)
@
<<pfPlot,echo=F>>=
png(filename="pf.png", width=400, height=300, res=72)
pfBoxplot(fc)
invisible(dev.off())
@

\begin{figure}[htb]
\begin{center}
\includegraphics[width=3in]{pf.png}
\end{center}
\caption{Boxplot of total vs. PF clusters}
\label{fig:fp}
\end{figure}

\section*{Introduction}

The Illumina Sequence Analysis Viewer (SAV) is a Windows application provided by Illumina that 
presents graphs made in real time from data collected over the course of basecalling.  This data was 
previously also made available in HTML format for inspection after the run; however, it is now
preserved in binary format and not simply parsed by users who wish to perform automated quality
assessment.  Here is presented \textit{savR}, an R package to parse the binary output, generate 
QC assessment plots and make the data available to users of Illumina sequencing instruments.
For more information about Illumina SAV, please consult the Illumina iCom website and the
Sequencing Analysis Viewer User's Guide, available
\href{https://duckduckgo.com/?q=sequencing%20analysis%20viewer%20user%27s%20guide}{\textit{online}}.

\section*{Description}

The \texttt{savR} function is passed a path to an Illumina HiSeq or MiSeq run, and returns a
\texttt{savProject} object, containing the parsed data.  Accessor methods are available for
information in the \texttt{RunInfo.xml} file as well as the parsed SAV Metrics files.  These include
corrected intensities, quality metrics, tile metrics, and extraction metrics.  The \textit{savR}
package comes with an example MiSeq data set which can be loaded thusly:

<<intro2,eval=F>>=
fc <- savR(system.file("extdata", "MiSeq", package="savR"))
@

\subsection*{RunInfo.xml}

The \texttt{RunInfo.xml} file is parsed and stored in the slots of the \texttt{savProject} object.
There are accessor methods for the project's \texttt{location}, \texttt{reads}, number of ``ends'' 
or \texttt{directions}, the \texttt{run} ID, the number of \texttt{cycles}, and a description of
the \texttt{flowcellLayout}.

<<ri>>=
directions(fc)
reads(fc)
cycles(fc)
flowcellLayout(fc)
@

\subsection*{Corrected intensitites}

Corrected intensity metrics (obtained from \texttt{CorrectedIntMetricsOut.bin}) can be inspected
by the \texttt{correctedIntestites} accessor method:

<<ciex>>=
head(correctedIntensities(fc), n=1)
@

This is a \texttt{data.frame} of intensity metrics; one line for each set of lane, tile and cycle 
measurements. Reported statistics include average intensity, corrected intensity (for cross-talk between 
bases and phasing/pre-phasing), called corrected intensities, number of called bases and signal to
noise ratio.  There are methods which act upon \texttt{savProject} objects to produce QC plots, for example 
plotIntensity to assess signal intensity for each channel as in figure \ref{fig:ci}.

<<doCiPlot,eval=F>>=
plotIntensity(fc)
@

<<ciPlot,echo=F>>=
png(filename="ci.png", width=250, height=400, res=72)
plotIntensity(fc)
invisible(dev.off())
@

\begin{figure}[htb]
\begin{center}
\includegraphics[width=2.25in]{ci.png}
\end{center}
\caption{Corrected intensity plot: cycle 1, base ``A''.}
\label{fig:ci}
\end{figure}

\subsection*{Quality Metrics}

The quality metrics (\texttt{QMetricsOut.bin}) file contains per-lane/tile/cycle metrics for the number
of clusters with quality at each PHRED value from 1-50.


<<qmex>>=
head(qualityMetrics(fc), n=1)
@

<<doQhPlot,eval=F>>=
qualityHeatmap(fc,1,1)
@

<<qhPlot,echo=F>>=
png(filename="qh.png", width=400, height=300, res=72)
qualityHeatmap(fc,1,1)
invisible(dev.off())
@

\begin{figure}[h]
\begin{center}
\includegraphics[width=3.5in]{qh.png}
\end{center}
\caption{Quality heatmap: lane 1, read 1.}
\label{fig:qh}
\end{figure}

\subsection*{Tile Metrics}

The tile metrics (\texttt{TileMetricsOut.bin}) file contains coded information about per-lane/cycle/tile cluster density,
pass-filter clusters, phasing and pre-phasing data.  Consult the \texttt{tileMetrics} help page for more information.

<<tmex>>=
head(tileMetrics(fc), n=4)
@

\subsection*{Extraction Metrics}

The extraction metrics (\texttt{ExtractionMetricsOut.bin}) file contains per-lane/cycle/tile information about per-base FWHM
(full width pixel size of clusters at half maximum) and 90th \%-ile intensity of signal intensity.

<<example2>>=
head(extractionMetrics(fc), n=1)
@

\section*{Coda}

There is a convenience function (\texttt{buildReports}), which partially reconstructs the Illumina reports folder
that was previously generated by the Illumina instrument software and which was superseded by SAV and InterOp files.





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