Figure 2

Clone Acceptance Criteria. This screenshot shows the interface to establish acceptance criteria by setting the maximum allowed number of discrepancies of each type. Different values can be set for discrepancies of low and high confidence. The user sets values for two thresholds – one that triggers a manual review, and one that automatically rejects the clone. Users can also opt to handle conservative and non-conservative amino acids substitutions separately or to treat all amino acid changes as one type. Once the settings are created, users can name the set and store it for future use. In this way, users may create different acceptance criteria for different purposes. Thus, a single collection of clones can be evaluated by different acceptance criteria by invoking these named sets. The criteria shown here are used routinely for determining final acceptance of clones. The numbers in the boxes indicate the absolute number of the indicated type of discrepancy for inclusion in that category. As indicated, this set of criteria does not distinguish between conservative and non-conservative missense mutations. Any clones with 1 or 0 high confidence missense substitution(s) are automatically accepted (as long as they have no other discrepancies that prevent automatic acceptance). Clones with 3 or more high-confidence missense substitutions are automatically rejected; if the clones have 2 they are triaged for additional sequencing or manual analysis. A higher bar is set to automatically reject clones based on low-confidence substitutions (10 or more), because many of these will be resolved with further sequencing. Similarly, this parameter set automatically passes clones only if they have no frameshift discrepancies of any type. Clones with 1 high-confidence or 9 low-confidence frameshift discrepancies or more are automatically rejected. Clones must meet all the pass criteria for automatic acceptance, whereas clones that meet any automatic fail criteria are automatically failed.