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Introduction

IGB can be used to visualize data from Affymetrix showing the locations of GeneChip Expression Array

Table of Contents

Probe set alignments - link.psl files

IGB can display Affymetrix probe sets aligned to onto a reference genome .

Chip design procedures have varied somewhat from chip to chip, but, in general, probes on Affymetrix's standard commercial expression microarrays (such as the Human U133 chip) are designed to match the 3' regions of known or computed mRNA sequences.  These probes are typically 18 to 25 bases long depending on the chip.

In standard arrays, probes are grouped conceptually into probe sets, groups of probes that are expected to measure expression for individual known or computationally-deduced mRNA molecules. 

Probe sequences are typically selected from the 3' regions of these design sequences in order to maximize the amount of sample mRNA to be measured.  (This so-called "3-prime bias" will most likely change as sample preparation protocols improve over time.) 

These design sequences may be identical to known mRNA sequences in GenBank, or they may have been produced computationally by merging ESTs or mRNA sequences into a single sequence, sometimes called a "consensus" sequence. 

IGB can be used to visualize the location of design sequences and probes within the genomic sequence.  Seeing where these sequences are located within the genome can be extremely useful when several overlapping probe sets recognize diverse mRNAs originating from the same gene.  Around 60% of human genes produce multiple variants, and these diverse variants often exhibit very complex configurations of exons and introns.  As a result, it often helps to be able to view a diagram of these complex structures together with probes in order to determine which individual mRNAs are being detected by a given probe set. 

To make this easier, IGB shows where the design sequences and their probes align to the genome together with alignments between the genomic sequence and known or predicted mRNAs.  By showing probes, known mRNAs, and design sequences in the same view, IGB makes it possible to determine quickly which known mRNA a probe set could detect.

However, it is important to note that these genome alignments can only be as good as the genomic sequence on which they are based.  Genomes vary in their level of quality – for example, the human and fruit fly genomes are very high quality, having gone through several releases involving many refinements.  More recently-sequenced genomes (such as mouse or rat) are not yet as reliable.  For the less-refined genomes, there may be many examples of design or mRNA sequences that simply do not align anywhere to the genomic sequence or, if they do align, do so imperfectly.  Readers interested in understanding more about these issues can read more about this topic at the following link:   http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=12149135/.

- it can show probe set design sequences aligned onto a genome with the locations of the probes indicated as blocks.

 

Probe sets visualized in IGB.Image Added

 

When IGB was first developed at Affymetrix, the company distributed probe set alignment files for its catalog 3' IVT arrays. In recent years, however, they've stopped updating these files. So for some genomes, the alignment files you can find on the Affymetrix Web site reference obsolete reference genomes. If you need to work with more up-to-date genomes, we recommend you create your own alignment files or request them from the IGB team. For some genomes, we've added probe set alignments to the main IGBQuickLoad.org site. Mainly we've done this at the request of individual researchers, and so if you would like to request an array, let us know.  If probe set alignments are available from our site, you'll typically find them in a folder named Affymetrix under the Data Sources section of the Data Access panel. 

You can also make your own probe set alignment files using blat, tabix, and a python script we wrote. For more information, see this Bitbucket repository.

 

About probe set alignment visualizations

Depending on when the arrays were designed, Affymetrix typically used expressed sequences from GenBank to select probes for probe sets - these expressed sequences were sometimes called "exemplar" or "consensus" sequences. They then selected individual probes from regions near the 3' end of the expressed sequence. Affymetrix (as of 2014) distributes probe and target sequences on their Web site, where "target sequences" contain the 3' end regions from which the probes were selected.

Probe set visualizations in IGB show the alignments of target, exemplar, or consensus sequences onto the genome. They also show the locations of probes that were selected from the design sequence. See the preceding figure for an example.

Because probes were selected from the expressed sequences, sometimes a probe will be shown as split across an intron. Also, sometimes probes overlap. And sometimes probes may be missing. If the target sequence contains a region that can't align onto the reference, and if this unaligned sequence contains a probe, then that probe will not be shown.

If you have questions about what you see in a probe set alignment, let us know.

 

Why this is useful

Often multiple, seemingly redundant probe sets interrogate one gene. This situation mainly arises when a gene has multiple, alternative three-prime ends due to alternative splicing or alternative termination sites. If an experiment identifies genes where redundant probe sets are differentially expressed with different fold-changes or in opposite directions, this can indicate that the treatment affects splicing as well as the overall abundance of RNAs arising from the gene.

Thus if you observe redundant probe sets that give different or contradictory results, it's a good idea to view them in IGB and compare their alignment to annotated genes and transcripts.