Tag Archives: microarray

Analysis of gene expression timecourse data using maSigPro

ANXA11 expression in human smooth muscle aortic cells post-ILb1 exposure

ANXA11 expression in human smooth muscle aortic cells post-ILb1 exposure

About a year ago, I did a little work on a very interesting project which was trying to identify blood-based biomarkers for the early detection of stroke. The data included gene expression measurements using microarrays at various time points after the onset of ischemia (reduced blood supply). I had not worked with timecourse data before, so I went looking for methods and found a Bioconductor package, maSigPro, which did exactly what I was looking for. In combination with ggplot2, it generated some very attractive and informative plots of gene expression over time.

I was very impressed by maSigPro and meant to get around to writing a short guide showing how to use it. So I did finally, using RMarkdown to create the document and here it is. The document also illustrates how to retrieve datasets from GEO using GEOquery and annotate microarray probesets using biomaRt. Hopefully it’s useful to some of you.

I’ll probably do more of this in the future, since publishing RMarkdown to RPubs is far easier than copying, pasting and formatting at WordPress.

Create your own gene IDs! No wait. Don’t.

Here’s a new way to abuse biological information: take a list of gene IDs and use them to create a completely fictitious, but very convincing set of microarray probeset IDs.

This one begins with a question at BioStars, concerning the conversion of Affymetrix probeset IDs to gene names. Being a “convert ID X to ID Y” question, the obvious answer is “try BioMart” and indeed the microarray platform ([MoGene-1_0-st] Affymetrix Mouse Gene 1.0 ST) is available in the Ensembl database.

However, things get weird when we examine some example probeset IDs: 73649_at, 17921_at, 18174_at. One of the answers to the question notes that these do not map to mouse.

The data are from GEO series GSE56257. The microarray platform is GPL17777. Description: “This is identical to GPL6246 but a custom cdf environment was used to extract data. The cdf can be found at the link below.”

Uh-oh. Alarm bells.
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Microarrays, scan dates and Bioconductor: it shouldn’t be this difficult

When dealing with data from high-throughput experimental platforms such as microarrays, it’s important to account for potential batch effects. A simple example: if you process all your normal tissue samples this week and your cancerous tissue samples next week, you’re in big trouble. Differences between cancer and normal are now confounded with processing time and you may as well start over with new microarrays.

Processing date is often a good surrogate for batch and it was once easy to extract dates from Affymetrix CEL files using Bioconductor. It seems that this is no longer the case.
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A brief note: R 3.0.0 and bioinformatics

Today marks the release of R 3.0.0. There will be plenty of commentary and useful information at sites such as R-bloggers (for example, Tal’s post).

Version 3.0.0 is great news for bioinformaticians, due to the introduction of long vectors. What does that mean? Well, several months ago, I was using the simpleaffy package from Bioconductor to normalize Affymetrix exon microarrays. I began as usual by reading the CEL files:

f <- list.files(path = "data/affyexon", pattern = ".CEL.gz", full.names = T, recursive = T)
cel <- ReadAffy(filenames = f)

When this happened:

Error in read.affybatch(filenames = l$filenames, phenoData = l$phenoData,  : 
  allocMatrix: too many elements specified

I had a relatively-large number of samples (337), but figured a 64-bit machine with ~ 100 GB RAM should be able to cope. I was wrong: due to a hard-coded limit to vector length in R, my matrix had become too large regardless of available memory. See this post and this StackOverflow question for the computational details.

My solution at the time was to resort to Affymetrix Power Tools. Hopefully, the introduction of the LONG vector will make Bioconductor even more capable and useful.

It’s #overlyhonestmethods come to life!

Retraction Watch reports a study of microarray data sharing. The article, published in Clinical Chemistry, is itself behind a paywall despite trumpeting the virtues of open data. So straight to the Open Access Irony Award group at CiteULike it goes.

I was not surprised to learn that the rate of public deposition of data is low, nor that most deposited data ignores standards and much of it is low quality. What did catch my eye though, was a retraction notice for one of the articles from the study, in which the authors explain the reason for retraction.
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Simple plots reveal interesting artifacts

I’ve recently been working with methylation data; specifically, from the Illumina Infinium HumanMethylation450 bead chip. It’s a rather complex array which uses two types of probes to determine the methylation state of DNA at ~ 485 000 sites in the genome.

The Bioconductor project has risen to the challenge with a (somewhat bewildering) variety of packages to analyse data from this chip. I’ve used lumi, methylumi and minfi, but will focus here on just the latter.

Update: a related post from Oliver Hofmann (@fiamh).
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APIs have let me down part 1/2: ArrayExpress

The API – Application Programming Interface – is, in principle, a wonderful thing. You make a request to a server using a URL and back come lovely, structured data, ready to parse and analyse. We’ve begun to demand that all online data sources offer an API and lament the fact that so few online biological databases do so.

Better though, to have no API at all than one which is poorly implemented and leads to frustration? I’m beginning to think so, after recent experiences on both a work project and one of my “fun side projects”. Let’s start with the work project, an attempt to mine a subset of the ArrayExpress microarray database.
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Trust no-one: errors and irreproducibility in public data

Just when I was beginning to despair at the state of publicly-available microarray data, someone sent me an article which…increased my despair.

The article is:

Deriving chemosensitivity from cell lines: Forensic bioinformatics and reproducible research in high-throughput biology (2009)
Keith A. Baggerly and Kevin R. Coombes
Ann. Appl. Stat. 3(4): 1309-1334

It escaped my attention last year, in part because “Annals of Applied Statistics” is not high on my journal radar. However, other bloggers did pick it up: see posts at Reproducible Research Ideas and The Endeavour.

In this article, the authors examine several papers in their words “purporting to use microarray-based signatures of drug sensitivity derived from cell lines to predict patient response.” They find that not only are the results difficult to reproduce but in several cases, they simply cannot be reproduced due to simple, avoidable errors. In the introduction, they note that:

…a recent survey [Ioannidis et al. (2009)] of 18 quantitative papers published in Nature Genetics in the past two years found reproducibility was not achievable even in principle for 10.

You can get an idea of how bad things are by skimming through the sub-headings in the article. Here’s a selection of them:
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GEO database: curation lagging behind submission?

geometadb

GSE and GDS records in GEOmetadb by date

I was reading an old post that describes GEOmetadb, a downloadable database containing metadata from the GEO database. We had a brief discussion in the comments about the growth in GSE records (user-submitted) versus GDS records (curated datasets) over time. Below, some quick and dirty R code to examine the issue, using the Bioconductor GEOmetadb package and ggplot2. Left, the resulting image – click for larger version.

Is the curation effort keeping up with user submissions? A little difficult to say, since GEOmetadb curation seems to have its own issues: (1) why do GDS records stop in 2008? (2) why do GDS (curated) records begin earlier than GSE (submitted) records?

library(GEOmetadb)
library(ggplot2)

# update database if required using getSQLiteFile()
# connect to database; assumed to be in user $HOME
con <- dbConnect(SQLite(), "~/GEOmetadb.sqlite")

# fetch "last updated" dates for GDS and GSE
gds <- dbGetQuery(con, "select update_date from gds")
gse <- dbGetQuery(con, "select last_update_date from gse")

# cumulative sums by date; no factor variables
gds.count <- as.data.frame(cumsum(table(gds)), stringsAsFactors = F)
gse.count <- as.data.frame(cumsum(table(gse)), stringsAsFactors = F)

# make GDS and GSE data frames comparable
colnames(gds.count) <- "count"
colnames(gse.count) <- "count"

# row names (dates) to real dates
gds.count$date <- as.POSIXct(rownames(gds.count))
gse.count$date <- as.POSIXct(rownames(gse.count))

# add type for plotting
gds.count$type <- "gds"
gse.count$type <- "gse"

# combine GDS and GSE data frames
gds.gse <- rbind(gds.count, gse.count)

# and plot records over time by type
png(filename = "geometadb.png", width = 800, height = 600)
print(ggplot(gds.gse, aes(date,count)) + geom_line(aes(color = type)))
dev.off()

biomaRt and GenomeGraphs: a worked example

As promised a few posts ago, another demonstration of the excellent biomaRt package, this time in conjunction with GenomeGraphs.

Here’s what we’re going to do:

  1. Grab some public microarray data
  2. Normalise and get a list of the most differentially-expressed probesets
  3. Use biomaRt to fetch the genes associated with those probesets
  4. Plot the data using GenomeGraphs

If you want to follow along on your own machine, it will need to be quite powerful. We’ll be processing exon arrays, which requires a 64-bit machine with at least 4 GB (and preferably, at least 8-12 GB) of RAM. As usual, I’m assuming some variant of Linux and that you’re comfortable at the command line.
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