---
title: "Using oligonucleotide microarray reporter sequence information
  for preprocessing and quality assessment"
author:
- name: Wolfgang Huber
- name: Robert Gentleman
date: "`r format(Sys.time(), '%d %B, %Y')`"
package: Biostrings
vignette: >
  %\VignetteIndexEntry{Handling probe sequence information}
  %\VignetteEngine{knitr::rmarkdown}
  %\VignetteEncoding{UTF-8}
  %\VignetteDepends{Biostrings, hgu95av2probe, hgu95av2cdf, affy, affydata}
  %\VignetteKeywords{Expression Analysis}
  %\VignettePackage{Biostrings}
output:
  BiocStyle::html_document
fig_caption: true
---

# Overview

This document presents some basic and simple tools for dealing with the
oligonucleotide microarray reporter sequence information in the Bioconductor
*probe* packages. This information is used, for example, in the
`r Biocpkg("gcrma")` package.

*Probe* packages are a convenient way for distributing and storing the probe
sequences (and related information) of a given chip.

As an example, the package `r Biocpkg("hgu95av2probe")` provides microarray
reporter sequences for Affymetrix' *HgU95a version 2* genechip, and for almost
all major Affymetrix genechips, the corresponding packages can be downloaded
from the Bioconductor website. If you have the reporter sequence information of
a particular chip, you can also create such a package yourself. This is
described in the makeProbePackage vignette of the `r Biocpkg("AnnotationForge")`
package.

This document assumes some basic familiarity with R and with the design of the
*AffyBatch* class in the `r Biocpkg("affy")` package, Bioconductor's basic
container for Affymetrix genechip data.

First, let us load the `r Biocpkg("Biostrings")` package and some other packages
we will use.

```{r loadPackages,message=FALSE,warning=FALSE}
library(Biostrings) 
library(hgu95av2probe) 
library(hgu95av2cdf) 
```

# Using probe packages

Help for the probe sequence data packages can be accessed through

```{r hgu95av2probe,eval=FALSE}
?hgu95av2probe
```

One of the issues that you have to deal with is that the *probe* packages do not
provide the reporter sequences of all the features present in an *AffyBatch*.
Some sequences are missing, some are implied; in particular, the data structure
in the *probe* packages does not explicitly contain the sequences of the
mismatch probes, since they are implied by the perfect match probes. Also, some
other features, typically harboring control probes or empty, do not have
sequences. This is the choice that Affymetrix made when they made files with
probe sequences available, and we followed it.

Practically, this means that the vector of probe sequences in a *probe* package
does not align 1:1 with the rows of the corresponding *AffyBatch*; you need to
keep track of this mapping, and some tools for this are provided and explained
below (see Section [2.2](#subsec.relating)). It also means that some functions from the
`r Biocpkg("affy")` package, such as `pm`, cannot be used when the sequences of
the probes corresponding to their result are needed; since `pm` reports the
intensities, but not the identity of the probes it has selected, yet the latter
would be needed to retrieve their sequences.

## Basic functions

Let us look at some basic operations on the sequences.

### Reverse and complementary sequence

DNA sequences can be reversed and/or complemented with the `reverse`,
`complement` and `reverseComplement` functions.

```{r reverseComplement,eval=FALSE}
?reverseComplement
```

### Matching sets of probes against each other

```{r MatchingSetsofProbesAgasintEachOther}
pm <- DNAStringSet(hgu95av2probe) 
dict <- pm[3801:4000]
pdict <- PDict(dict)
m <- vcountPDict(pdict, pm)
dim(m) 
table(rowSums(m))
which(rowSums(m) == 3)
ii <- which(m[77, ] != 0)
pm[ii]
```

### Base content

The base content (number of occurrence of each character) of the sequences can
be computed with the function `alphabetFrequency`.

```{r alphabetFrequency}
bcpm <- alphabetFrequency(pm, baseOnly=TRUE)
head(bcpm) 
alphabetFrequency(pm, baseOnly=TRUE, collapse=TRUE)
```

## Relating to the features of an *AffyBatch* {#subsec.relating}

```{r hgu95av2dimncol}
nc = hgu95av2dim$NCOL
nc
```

```{r hgu95av2dimnrow}
nr = hgu95av2dim$NROW
nr
```

Each column of an *AffyBatch* corresponds to an array, each row to a certain
probe on the arrays. The probes are stored in a way that is related to their
geometrical position on the array. For example, the *hgu95av2* array is
geometrically arranged into `r nc` columns and `r nr` rows. We call the column
and row indices the `x`- and `y`-coordinates, respectively. This results in
`r nc` ⨉ `r nr` = `r format(nc*nr, scientific=FALSE)` probes of the *AffyBatch*;
we also call them indices. To convert between `x`-and `y`-coordinates and
indices, you can use the functions `xy2indices` and `indices2xy` from the
`r Biocpkg("affy")` package.

The sequence data in the *probe* packages is addressed by their `x` and
`y`-coordinates. Let us construct a vector `abseq` that aligns with the indices
of an *hgu95av2* *AffyBatch* and fill in the sequences:

```{r abseq}
library(affy) 
abseq = rep(as.character(NA), nc*nr) 
ipm = with(hgu95av2probe, xy2indices(x, y, nc=nc)) 
any(duplicated(ipm)) # just a sanity check 
abseq[ipm] = hgu95av2probe$sequence
table(is.na(abseq))
```

The mismatch sequences are not explicitly stored in the probe packages. They are
implied by the match sequences, by flipping the middle base. This can be done
with the `pm2mm` function defined below. For Affymetrix GeneChips the length of
the probe sequences is 25, and since we start counting at 1, the middle position
is 13.

```{r pm2mm}
mm <- pm
subseq(mm, start=13, width=1) <- complement(subseq(mm, start=13, width=1))
cat(as.character(pm[[1]]), as.character(mm[[1]]), sep="\n")
```

We compute `imm`, the indices of the mismatch probes, by noting that each
mismatch has the same `x`-coordinate as its associated perfect match, while its
`y`-coordinate is increased by 1.

```{r imm}
imm = with(hgu95av2probe, xy2indices(x, y+1, nc=nc))
intersect(ipm, imm) # just a sanity check
abseq[imm] = as.character(mm)
table(is.na(abseq))
```

See Figures \@ref(fig:bap)-\@ref(fig:p2p) for some applications of
the probe sequence information to preprocessing and data quality related plots.

# Some sequence related "preprocessing and quality" plots

The function `alphabetFrequency` counts the number of occurrences of each of the
four bases A, C, G, T in each probe sequence.

```{r alphabetFrequency2}
freqs <- alphabetFrequency(DNAStringSet(abseq[!is.na(abseq)]), baseOnly=TRUE)
bc <- matrix(nrow=length(abseq), ncol=5)
colnames(bc) <- colnames(freqs)
bc[!is.na(abseq), ] <- freqs
head(na.omit(bc))
```

Let us define an ordered factor variable for GC content:

```{r gc}
GC = ordered(bc[,"G"] + bc[,"C"])
colores = rainbow(nlevels(GC))
```

And let us create an *AffyBatch* object.

```{r bap, fig.cap="Distribution of probe GC content. The height of each bar corresponds to the number of probes with the corresponding GC content."}
library(affydata)
f <- system.file("extracelfiles", "CL2001032020AA.cel", package="affydata")
pd <- new("AnnotatedDataFrame", data=data.frame(fromFile=I(f), row.names="f"))
abatch <- read.affybatch(filenames=f, compress=TRUE, phenoData=pd)
barplot(table(GC), col=colores, xlab="GC", ylab="number")
```

```{r bxp, fig.cap="Boxplots of log~2~ intensity stratifed by probe GC content."}
boxplot(log2(exprs(abatch)[,1]) ~ GC, outline=FALSE,
        col=colores, , xlab="GC", ylab=expression(log[2]~intensity))
```

```{r p2p, fig.cap="Scatterplot of PM vs MM intensities, colored by probe GC content."}
plot(exprs(abatch)[ipm,1], exprs(abatch)[imm,1], asp=1, pch=".", log="xy",
     xlab="PM", ylab="MM", col=colores[GC[ipm]])
abline(a=0, b=1, col="#404040", lty=3)
```

```{r devoff,include=FALSE}
dev.off()
```
