This track shows alignments between human expressed sequence tags (ESTs) in GenBank and the genome that show signs of splicing when aligned against the genome. ESTs are single-read sequences, typically about 500 bases in length, that usually represent fragments of transcribed genes.
To be considered spliced, an EST must show evidence of at least one canonical intron, i.e. the genomic sequence between EST alignment blocks must be at least 32 bases in length and have GT/AG ends. By requiring splicing, the level of contamination in the EST databases is drastically reduced at the expense of eliminating many genuine 3' ESTs. For a display of all ESTs (including unspliced), see the human EST track.
This track follows the display conventions for PSL alignment tracks. In dense display mode, darker shading indicates a larger number of aligned ESTs.
The strand information (+/-) indicates the direction of the match between the EST and the matching genomic sequence. It bears no relationship to the direction of transcription of the RNA with which it might be associated.
The description page for this track has a filter that can be used to change the display mode, alter the color, and include/exclude a subset of items within the track. This may be helpful when many items are shown in the track display, especially when only some are relevant to the current task.
To use the filter:
This track may also be configured to display base labeling, a feature that allows the user to display all bases in the aligning sequence or only those that differ from the genomic sequence. For more information about this option, click here. Several types of alignment gap may also be colored; for more information, click here.
To make an EST, RNA is isolated from cells and reverse transcribed into cDNA. Typically, the cDNA is cloned into a plasmid vector and a read is taken from the 5' and/or 3' primer. For most — but not all — ESTs, the reverse transcription is primed by an oligo-dT, which hybridizes with the poly-A tail of mature mRNA. The reverse transcriptase may or may not make it to the 5' end of the mRNA, which may or may not be degraded.
In general, the 3' ESTs mark the end of transcription reasonably well, but the 5' ESTs may end at any point within the transcript. Some of the newer cap-selected libraries cover transcription start reasonably well. Before the cap-selection techniques emerged, some projects used random rather than poly-A priming in an attempt to retrieve sequence distant from the 3' end. These projects were successful at this, but as a side effect also deposited sequences from unprocessed mRNA and perhaps even genomic sequences into the EST databases. Even outside of the random-primed projects, there is a degree of non-mRNA contamination. Because of this, a single unspliced EST should be viewed with considerable skepticism.
To generate this track, human ESTs from GenBank were aligned against the genome using blat. Note that the maximum intron length allowed by blat is 750,000 bases, which may eliminate some ESTs with very long introns that might otherwise align. When a single EST aligned in multiple places, the alignment having the highest base identity was identified. Only alignments having a base identity level within 0.5% of the best and at least 96% base identity with the genomic sequence are displayed in this track.
This track was produced at UCSC from EST sequence data submitted to the international public sequence databases by scientists worldwide.
Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL. GenBank: update. Nucleic Acids Res. 2004 Jan 1;32(Database issue):D23-6.
Kent WJ. BLAT - the BLAST-like alignment tool. Genome Res. 2002 Apr;12(4):656-64.