Assessment of the quality of directional cloning of individual EST libraries. Bins of library quality scores (LQS) at intervals of 0.05 are depicted along the x-axis. The heights of the bars reflect the number of human ESTs derived from libraries with an LQS that falls in a given bin. The LQS of each EST library was determined by calculating the fraction of ESTs from a given library that were deposited in the same orientation as the best-of-UniGene (BOU) representative of the UniGene cluster to which a given EST belonged. In our initial analysis, we assumed that all BOU representatives were correctly oriented (blue bars). As this is not the case, we repeated that analysis by calculating the LQS exclusively from ESTs belonging to UniGene clusters where the BOU representative possessed a defined ORF, indicating that it was correctly oriented (red bars). As a final improvement, we flipped in silico all ESTs annotated as 3' sequencing reads, as these are generally not reoriented before deposit in sequence databases. The result was a bimodal distribution of LQS scores (green bars) that appears to correspond broadly with directional (peak near LQS = 1.0) and non-directional (peak near LQS = 0.5) library generation protocols. A full list of both mouse and human EST libraries and their LQS scores is available at our website .