Introduction: microarray quality assessment

with arrayQualityMetrics

Audrey Kauffmann, Wolfgang Huber

April 25, 2023

Contents

1 Basic use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.1 Affymetrix data - before preprocessing . . . . . . . . . . . . . 3
1.2 Affymetrix data - after preprocessing . . . . . . . . . . . . . . 3
1.3 ExpressionSet and ExpressionSetIllumina . . . . . . . . . . . 4
1.4 Two colour arrays, NChannelSet, RGList, MAList . . . . . . . 4
1.5 Loading data from ArrayExpress . . . . . . . . . . . . . . . . 5

2 Making the report more informative by adding a factor of inter-

est . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

3 Extended use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.1 Spatial layout of the array . . . . . . . . . . . . . . . . . . . . 5
3.2 Mapping of the reporters . . . . . . . . . . . . . . . . . . . . 6
3.3 RNA quality . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Introduction
The arrayQualityMetrics package produces, through a single function call, a compre-
hensive HTML report of quality metrics about a microarray dataset [1, 2, 3]. The
quality metrics are mainly on the per array level, i. e. they can be used to assess the
relative quality of different arrays within a dataset. Some of the metrics can also be
used to diagnose batch effects, and thus the quality of the overall dataset.
The report can be extended to contain further diagnostics through additional argu-
ments, and we will see examples for this in Section 3.
The aim of the arrayQualityMetrics package is to produce information that is relevant
for your decision making - not, to make the decision. It will often be applied to two,
somewhat distinct, use cases: (i) assessing quality of a “raw” dataset, in order to
Introduction

Figure 1: Left: An example plot from the report. The plot shows the arrays (points) in a two-dimensional
plot area spanned by the first two axes of a principal component analysis (PCA). By moving the mouse over
the points, the corresponding array’s metadata is displayed in the table to the right of the plot. By clicking
on a point, it can be selected or deselected. Selected arrays are indicated by larger points or wider lines in the
plots and by ticked checkboxes in the array table shown in the right panel. Arrays can also be (de)selected
by clicking the checkboxes. Initially, when the report is loaded (or reloaded) by the browser, all arrays are
selected that were called outliers by at least one criterion.

get feedback on the experimental procedures that produced the data; (ii) assessing
quality of a normalised dataset, in order to decide whether and how to use the dataset
(or subsets of arrays in it) for subsequent data analysis.
Different types of microarray data (one colour, two colour, Affymetrix, Illumina) are
represented by different object classes in Bioconductor. The function arrayQuality
Metrics will work in the same way for all of them. Further information about its
arguments can be found in its manual page.
When the function arrayQualityMetrics is finished, a report is produced in the di-
rectory specified by the function’s outdir argument. By default, a directory with a
suitable name is created in the current working directory. This directory contains an
HTML page index.html that can be opened by a browser. The report contains a se-
ries of plots explained by text. Some of the plots are interactive (see Figure 1). Tech-
nically, this is achieved by the use of SVG (scalable vector graphics) and JavaScript,
and it requires that you use a recent (HTML5 capable) web browser1 . Other plots, 1
If in doubt, please
where interactivity is less relevant, are provided as bitmaps (PNG format) and are see the notes about
also linked to PDF files that provide high resolution versions e. g. for publication. browser compatibility
at the top of the
Plus (+) or minus (-) symbols at the begin of different section headings of the report report; or contact the
Bioconductor mailing
(as in the left panel of Figure 1) indicate that you can show or hide these sections
list.
by clicking on the heading. After (re)loading, all sections are shown except for the
Outlier detection barplots, which are hidden and can be expanded by clicking on
them.
Metadata about the arrays is shown at the top of the report as a table (see Figure 1).
It is extracted from the phenoData slot of the data object supplied to arrayQuali
tyMetrics. It can be useful to adjust the contents this slot before producing the
report, and to make sure it contains the right quantity of information to make an
informative report - not too much, not too little.

2
Introduction

In the case of AffyBatch input, some Affymetrix specific sections are added to the
standard report. Also for other types of arrays, sections can be added to the standard
report if certain metadata are present in the input object (see Section 3).
The function arrayQualityMetrics also produces an R object (essentially, a big
list) with all the information contained in the report, and this object can be used by
downstream tools for programmatic analysis of the report. This is discussed in the vi-
gnette Advanced topics: Customizing arrayQualityMetrics reports and programmatic
processing of the output

1 Basic use
1.1 Affymetrix data - before preprocessing
If you are working with Affymetrix GeneChips, an AffyBatch object is the most ap-
propriate way to import your raw data into Bioconductor. Starting from CEL files,
this is typically done using the function ReadAffy from the affy package2 . Here, we 2
For more informa-
use the dataset MLL.A, an object of class AffyBatch provided in the data package tion on how to pro-
duce an AffyBatch
ALLMLL.
from your data,
library("ALLMLL") please see the doc-
umentation of the
data("MLL.A")
affy package.

Now that the data are loaded, we can call arrayQualityMetrics3 . 3

For this vignette, in
order to save compu-
library("arrayQualityMetrics") tation time, we only
arrayQualityMetrics(expressionset = MLL.A[, 1:5], call the function on
outdir = "Report_for_MLL_A", the first 5 arrays; in
your own application,
force = TRUE,
you can call it on the
do.logtransform = TRUE) complete data object.

This is the simplest way of calling the function. We give a name to the directory
(outdir) and we overwrite the possibly existing files of this directory (force). Finally,
we set do.logtransform to logarithm transform the intensities. You can then view
the report by directing your browser to the file index.html in the directory whose
name is indicated by outdir.

1.2 Affymetrix data - after preprocessing

We can call the RMA algorithm on MLL.A to obtain a preprocessed dataset. The
preprocessing includes background correction, between array intensity adjustment
(normalisation) and probeset summarisation. The resulting object nMLL is of class
ExpressionSet and contains one value (expression estimate) for each gene for each
array.

3
Introduction

nMLL = rma(MLL.A)

We can then call again the function arrayQualityMetrics.

arrayQualityMetrics(expressionset = nMLL,
outdir = "Report_for_nMLL",
force = TRUE)

We do not need to set do.logtransform as after rma the data are already logarithm
transformed.

1.3 ExpressionSet and ExpressionSetIllumina

If you are working on one colour arrays other than Affymetrix genechips, you can
load your data into Bioconductor as an ExpressionSet object 4 , or if you work with 4
See the documenta-
Illumina data and the beadarray package, as an ExpressionSetIllumina object. You tion of the Biobase
package.
can then proceed exactly as above.

1.4 Two colour arrays, NChannelSet, RGList, MAList

The package limma imports a wide range of data formats used for two colour arrays
and produces objects of class RGList or MAList. When presented with an object of
these classes, arrayQualityMetrics tries to convert them into an NChannelSet and
then proceeds with calling its NChannelSet method.
Alternatively, you can create an NChannelSet to contain your data “from scratch”.
The documentation of the Biobase package gives instructions on how to do so.
The arrayQualityMetrics function expects the assayData slot of the NChannelSet
object to contain the elements R and G, for the “red” and the “green” intensities.
Optionally, it can contain elements Rb and Gb for associated “background” intensi-
ties. As an alternative to all that, the arrayQualityMetrics function also accepts
NChannelSet objects with a single slot exprs, and will then simply behave like it does
for (single-colour) ExpressionSet objects.
As an example, we consider the dataset CCl4 from the data package CCl4 and
normalize it using the variance stabilization method available in the package vsn.
library("vsn")
library("CCl4")
data("CCl4")
nCCl4 = justvsn(CCl4, subsample = 15000)
arrayQualityMetrics(expressionset = nCCl4,
outdir = "Report_for_nCCl4",
force = TRUE)

4
Introduction

1.5 Loading data from ArrayExpress

You can use the ArrayExpress package [4] to download datasets from the EBI’s Ar-
rayExpress database. The resulting ExpressionSet, AffyBatch or NChannelSet objects
can be directly fed into arrayQualityMetrics.

2 Making the report more informative by adding a

factor of interest
A useful feature of arrayQualityMetrics is the possibility to show the results in the
context of an experimental factor of interest, i. e. a categorical variable associated with
the arrays such as hybridisation date, treatment level or replicate number. Specifying
a factor does not change how the quality metrics are computed. By setting the
argument intgroup to contain the names of one or multiple columns of the data
object’s phenoData slot5 , a bar on the side of the heatmap with colours representing 5
This is where Bio-
the respective factors is added. Similarly, the colours of the boxplots and density conductor objects
store array annotation
plots reflect the levels of the first of the factors named by intgroup.
We use the nMLL example again, and create artificial array metadata factors condi
tion and batch (see Section 3.3 for a more realistic example).

pData(nMLL)$condition = rep(letters[1:4], times = 5)

pData(nMLL)$batch = rep(paste(1:4), each = 5)

arrayQualityMetrics(expressionset = nMLL,
outdir = "Report_for_nMLL_with_factors",
force = TRUE,
intgroup = c("condition", "batch"))

3 Extended use
Some of the quality metrics that the package can compute require specific information
about the features on the arrays. To use these, you need to make sure that this
information is provided in your input object. We use the nCCl4 example again.

3.1 Spatial layout of the array

To plot the spatial distributions of the intensities of the arrays, arrayQualityMet
rics needs the spatial coordinates of the features on the chip. For AffyBatch or
BeadLevelList, this information is automatically available without further user input.
For the other types of objects, two columns corresponding to X and Y coordinates
of the features are required in the featureData slot of the object. These columns
should be named "X" and "Y". If the arrays are split into blocks, rows and columns,
then the function addXYfromGAL (please check its manual page for details) can used

5
Introduction

to convert the row, column and blocks indices into absolute "X" and "Y" coordinates
on the array. In the example of the dataset CCl4, the coordinates of the spots are in
the columns named "Row" and "Column" of the featureData (the slot of the object
containing the annotation of the probes). We copy this information into columns
named "X" and "Y" respectively
featureData(nCCl4)$X = featureData(nCCl4)$Row
featureData(nCCl4)$Y = featureData(nCCl4)$Column

The next call to arrayQualityMetrics with this refined version of nCCl4 (see Sec-
tion 3.3) will now include this information in the report, and the spatial distribution
of the intensities will be shown.

3.2 Mapping of the reporters

The report can also include an assessment of the effect of the target mapping of the
reporters. You can define a featureData column named hasTarget that indicates, by
logical TRUE, if the reporter matches a known transcript, and by FALSE, if not. In the
CCl4 example, many of the reporter names are RefSeq identifiers, while others are
not. Thus, we let hasTarget indicate whether the name begins with "NM".
featureData(nCCl4)$hasTarget = (regexpr("^NM", featureData(nCCl4)$Name) > 0)
table(featureData(nCCl4)$hasTarget)

##
## FALSE TRUE
## 33296 10332

The next call to arrayQualityMetrics with this refined version of nCCl4 (see Sec-
tion 3.3) will now include this information in the report, and the spatial distribution
of the intensities will be shown.

3.3 RNA quality

The RNA hybridized to the arrays in the CCl4 dataset was intentionally made to
good, medium or poor quality, and this is recorded by a so-called RIN value (see
CCl4 vignette).
pd = pData(CCl4)
rownames(pd) = NULL
pd

## Cy3 Cy5 RIN.Cy3 RIN.Cy5

## 1 DMSO CCl4 9.0 9.7
## 2 DMSO CCl4 9.0 5.0
## 3 DMSO CCl4 9.0 2.5
## 4 DMSO CCl4 9.0 2.5

6
Introduction

## 5 CCl4 DMSO 9.7 9.0

## 6 CCl4 DMSO 5.0 9.0
## 7 CCl4 DMSO 2.5 9.0
## 8 DMSO CCl4 9.0 9.7
## 9 CCl4 DMSO 9.7 9.0
## 10 CCl4 DMSO 5.0 9.0
## 11 CCl4 DMSO 2.5 9.0
## 12 DMSO CCl4 9.0 9.7
## 13 DMSO CCl4 9.0 5.0
## 14 DMSO CCl4 9.0 5.0
## 15 DMSO CCl4 9.0 2.5
## 16 CCl4 DMSO 9.7 9.0
## 17 CCl4 DMSO 5.0 9.0
## 18 CCl4 DMSO 2.5 9.0

The RIN is always 9 for the reference (DMSO), the relevant value is that for the test
sample (CCl4).
RIN = with(pd, ifelse( Cy3=="CCl4", RIN.Cy3, RIN.Cy5))
fRIN = factor(RIN)
levels(fRIN) = c("poor", "medium", "good")
pData(nCCl4)$"RNA-integrity" = fRIN

Now we can use this to set the argument intgroup when calling the function ar
rayQualityMetrics.

arrayQualityMetrics(expressionset = nCCl4,
outdir = "Report_for_nCCl4_with_RIN",
force = TRUE,
intgroup = "RNA-integrity")

Boxplots, PCA plot and heatmap in the report will now indicate the values of the
factor RNA-integrity for each array.

Session Info
• R version 4.3.0 RC (2023-04-13 r84269), x86_64-pc-linux-gnu

• Locale: LC_CTYPE=en_US.UTF-8, LC_NUMERIC=C, LC_TIME=en_GB,

LC COLLATE=C, LC_MONETARY=en_US.UTF-8, LC_MESSAGES=en_US.UTF-8,
_
LC_PAPER=en_US.UTF-8, LC_NAME=C, LC_ADDRESS=C, LC_TELEPHONE=C,
LC_MEASUREMENT=en_US.UTF-8, LC_IDENTIFICATION=C

• Time zone: America/New_York

• TZcode source: system (glibc)

• Running under: Ubuntu 22.04.2 LTS

7
Introduction

• Matrix products: default

• BLAS: /home/biocbuild/bbs-3.17-bioc/R/lib/libRblas.so

• LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0

• Base packages: base, datasets, grDevices, graphics, methods, stats, utils

• Other packages: ALLMLL 1.39.0, Biobase 2.60.0, BiocGenerics 0.46.0,
CCl4 1.37.0, affy 1.78.0, arrayQualityMetrics 3.56.0, hgu133acdf 2.18.0,
limma 3.56.0, vsn 3.68.0
• Loaded via a namespace (and not attached): AnnotationDbi 1.62.0,
BeadDataPackR 1.52.0, BiocManager 1.30.20, BiocStyle 2.28.0,
Biostrings 2.68.0, DBI 1.1.3, Formula 1.2-5, GenomeInfoDb 1.36.0,
GenomeInfoDbData 1.2.10, GenomicRanges 1.52.0, Hmisc 5.0-1,
IRanges 2.34.0, KEGGREST 1.40.0, KernSmooth 2.23-20, Matrix 1.5-4,
MatrixGenerics 1.12.0, R6 2.5.1, RColorBrewer 1.1-3, RCurl 1.98-1.12,
RSQLite 2.3.1, Rcpp 1.0.10, S4Vectors 0.38.0, XML 3.99-0.14,
XVector 0.40.0, affyPLM 1.76.0, affyio 1.70.0, annotate 1.78.0, askpass 1.1,
backports 1.4.1, base64 2.0.1, base64enc 0.1-3, beadarray 2.50.0, bit 4.0.5,
bit64 4.0.5, bitops 1.0-7, blob 1.2.4, cachem 1.0.7, checkmate 2.1.0, cli 3.6.1,
cluster 2.1.4, codetools 0.2-19, colorspace 2.1-0, compiler 4.3.0, crayon 1.5.2,
data.table 1.14.8, deldir 1.0-6, digest 0.6.31, dplyr 1.1.2, evaluate 0.20,
fansi 1.0.4, farver 2.1.1, fastmap 1.1.1, foreign 0.8-84, gcrma 2.72.0,
genefilter 1.82.0, generics 0.1.3, ggplot2 3.4.2, glue 1.6.2, grid 4.3.0,
gridExtra 2.3, gridSVG 1.7-5, gtable 0.3.3, hexbin 1.28.3, highr 0.10,
htmlTable 2.4.1, htmltools 0.5.5, htmlwidgets 1.6.2, httr 1.4.5,
hwriter 1.3.2.1, illuminaio 0.42.0, interp 1.1-4, jpeg 0.1-10, jsonlite 1.8.4,
knitr 1.42, labeling 0.4.2, lattice 0.21-8, latticeExtra 0.6-30, lifecycle 1.0.3,
magrittr 2.0.3, matrixStats 0.63.0, memoise 2.0.1, munsell 0.5.0, nnet 7.3-18,
openssl 2.0.6, pillar 1.9.0, pkgconfig 2.0.3, plyr 1.8.8, png 0.1-8,
preprocessCore 1.62.0, reshape2 1.4.4, rlang 1.1.0, rmarkdown 2.21,
rpart 4.1.19, rstudioapi 0.14, scales 1.2.1, setRNG 2022.4-1, splines 4.3.0,
stats4 4.3.0, stringi 1.7.12, stringr 1.5.0, survival 3.5-5, svglite 2.1.1,
systemfonts 1.0.4, tibble 3.2.1, tidyselect 1.2.0, tools 4.3.0, utf8 1.2.3,
vctrs 0.6.2, withr 2.5.0, xfun 0.39, xtable 1.8-4, yaml 2.3.7, zlibbioc 1.46.0

References
[1] Audrey Kauffmann, Robert Gentleman, and Wolfgang Huber.
arrayQualityMetrics - a Bioconductor package for quality assessment of
microarray data. Bioinformatics, 25:415–416, 2009.
[2] Audrey Kauffmann and Wolfgang Huber. Microarray data quality control
improves the detection of differentially expressed genes. Genomics, 95:138–142,
2010.

8
Introduction

[3] Matthew N. McCall, Peter N. Murakami, and Rafael A. Irizarry. Assessing

microarray quality. Technical report, Department of Biostatistics, Johns Hopkins
Bloomberg School of Public Health, Baltimore, MD, 2010.
[4] Audrey Kauffmann, Tim F. Rayner, Helen Parkinson, Misha Kapushesky,
Margus Lukk, Alivs Brazma, and Wolfgang Huber. Importing ArrayExpress
datasets into R/Bioconductor. Bioinformatics, 25:2092–2094, 2009.