Ensembl aims to associate micro array probe set identifiers to Ensembl transcript models in a two-step procedure.
In the first step individual probes (oligonucleotides) are mapped to the genome sequence. The Ensembl analysis and annotation pipeline uses the exonerate sequence comparison and alignment tool (Slater et al., 2005) and tolerates only 1 bp mismatch between the probe and the genome sequence assembly. Probes that hit to more than 100 locations, e.g. suspected Alu repeats, are discarded. Individual probes are grouped into probe sets and we require that more than half of the probes of a probe set hit the genome sequence. The probe set size is thereby dynamically calculated as the median of all probe matches from all probe sets on a particular microarray.
Ensembl 'ContigView' displays individual probes that match to the current assembly. If not displayed by default, microarray probe set tracks are available from the 'Features' menu in the yellow menu bar of the 'Detailed View' panel. Pointing at a particular probe in a probe set track displays a pop-up window, which reports the 'Probe length', the 'Match length', as well as the 'Match status'.
'Match status' 'Full match' reports a perfect match, while 'Mismatch' indicates a single base pair mismatch between the probe set and the genome sequence assembly. Probes with more than 1 bp mismatch, as well as probes spanning exon-intron boundaries are not placed and not reported. Please note that all sequences on Ensembl 'FastaView' pages, which are available following the 'Details...', links in the pop-up windows or directly clicking onto probes in the corresponding track, are genome sequences but not probe sequences. While 'Probe length' directly indicates the oligonucleotide length, a combination of 'Match length' and 'Match status' allows conclusions about the location of the mismatch. If 'Match status' is 'Mismatch' and 'Probe length' equals 'Match length' the mismatch is within the probe sequence. If 'Match length' is one bp smaller than 'Probe length', the mismatch occurred at an end position.
In the subsequent second step, we aim to associate microarray probe sets with Ensembl transcript predictions (ENST...). For successful transcript mapping, at least 50% of the probes in a probe set need to match the underlying transcript cDNA sequence extended by a 2 kb window following the most 3' exon. The probe set size is taken from the individual microarray specifications rather than being dynamically calculated. A 3' extension of transcript sequences is required as Ensembl predicts all its transcript models strongly on the basis of experimental evidence. Since there is currently no guarantee that UTR regions in RefSeq or EMBL mRNA records are complete there is also no guarantee that Ensembl predicted a full-length 3' UTR. Microarray probes however tend to be designed against 3' UTR regions, mostly on the basis of EST evidence from NCBI UniGene. Since for most species in Ensembl, EST evidence is not directly exploited for Ensembl gene builds (ENSG..., ENST...), a 2 kb extension appears as a sensible means to successfully map microarray probe sets to Ensembl transcripts.
Guy St. C. Slater and
Ewan Birney
Automated generation of heuristics for biological sequence comparison.
BMC Bioinformatics. 2005 Feb 15;6(1):31
doi:10.1186/1471-2105-6-31
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