EST's

Expressed sequence tags are a cost efficient and rapid way of identifying genes.

The majority of the EST sequences (~10,000) will be picked from a cDNA library produced from Lumbricus rubellus maintained in a controled environment. The remainder will be five targeted sets of ~1000 sequences generated from subtractive libraries for each of the five pollutants used in the project.

The cDNA are amplified by PCR then DNA sequenators are used to determin sequences and create the chromatograms. The base calling package "phred" is used to produce sequence files containing information pertaining to "hi quality" regions of the chromaotgrams. These files are then masked for vector sequence, poly A tails and leading linker sequences are removed by the software package "cross_match" to produce text format sequence files.

The sequences are clustered using "CLOBB" and consensi sequences for the clusters are constructed using the software package " phrap". Sequences (consensi for the clusters, individual reads for the singletons) are then 'blasted' using the NCBI's blast search engine.
The following blasts are implemented : BlastX vrs non-redundant database
BlastN vrs non-redundant database
BlastN vrs dbest