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Probe set alignments - link.psl files

IGB can be used to visualize data from Affymetrix showing the locations of GeneChip Expression Array probe sets and target sequences aligned to a genome.

In most Affymetrix 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. 

These target 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 display location of design sequences and probes within the genomic sequence. Probe set alignments are available as "link.psl" files from the Affymetrix Web site. Some probe set alignments are also available from Affymetrix and IGBQuickLoad data sources in the Available Data sub-panel of the Data Access planel.

Viewing probe sets from IGB DAS

To view probe sets available from IGB DAS2 data source

• Open the Data Access panel
• Open the IGB DAS / affy folder under the Data Sources and Data Sets section of the Data Access tab.
• Click the checkbox next to the array you would like to examine
• zoom to a gene or region of interest and click Load Data.

The probe set, including the target sequence and individual probe, will appear in IGB.

Where to find probe set alignment files

Many are available from the Affymetrix customer support Web site.

To obtain these files, go to the Affymetrx site and look for the array you are interested in.

On the array's support page, look for alignment data files in the "NetAffx Annotation Files" section. Look for files labeled as consensus alignment files or something similar. Note that alignment data files may not be available be available for species with well-characterized genomes.

Affymetrix distributes these files in an Affymetrix-specific format called "link.psl" which IGB can read.

These files consist of two sections:

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

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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.