This track shows alignments between the expressed sequence tags (ESTs) in GenBank of all Drosophila species and the genome. ESTs are single-read sequences, typically about 500 bases in length, that usually represent fragments of transcribed genes.
This track follows the display conventions for PSL alignment tracks. In dense display mode, the items that are more darkly shaded indicate matches of better quality.
The strand information (+/-) for this track is in two parts. The first + or - indicates the orientation of the query sequence whose translated protein produced the match. The second + or - indicates the orientation of the matching translated genomic sequence. Because the two orientations of a DNA sequence give different predicted protein sequences, there are four combinations. ++ is not the same as --, nor is +- the same as -+.
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, ESTs for all Drosophila species 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 found. Only alignments having a base identity level within 1% of the best and at least 93% base identity to the genomic sequence were kept.
The all-Drosphila EST track is 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.