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Limitations of ESTs

1. It is very difficult to recover mRNA from some tissues. This results in the non-representation of genes which are only found in these tissues, or which are present in low-abundance.

2. It is known that some important gene regulatory sequences exist within introns. Because ESTs lack intronic sequence, this information is lost.

3. EST sequencing is always "single pass" and so is subject to to a higher rate of sequencing error than is genomic DNA, which is typically sequenced from both ends of the clone (this problem can of course be overcome by confirming ESTs of interest via resequencing or contigging).

4. As they do not include non-transcribed regions of the genome, information about gene regulatory regions (e.g. promoters) will be missing.

5. Since ESTs are developed from mRNA, and highly expressed genes produce many copies of mRNA, there is always redundancy within and between EST libraries. This problem is largely overcome by clustering the ESTs into unigenes.