Skewing in sequence read direction. The number of reads performed in a forward or reverse orientation were tabulated for each phred unfiltered, erroneous sequence for which a total of >20 independent sequences were acquired in a lane. Percent forward reads is plotted on the ordinate versus number of sequences acquired in the abscissa. If read direction during sequencing was random, data points would be anticipated to fall within a binomial distribution centered on the value obtained for correct sequence reads. Plotted curves indicate calculated boundaries of the upper and lower limits of values between which 98% of sequences should be found. These were calculated using the Vassar binomial calculator http://faculty.vassar.edu/lowry/binomialX.html with p = probability of forward read among correct sequences, n = number of reads (abscissa), and defining the number of positive events for which the probability of identifying more events (upper curve) or less events (lower curve) is <1%. Plots for full length sequences with a single error for the 5C.C7, OT-1, and DO11.10 TCR (A-C), and corresponding plots for sequences with multiple errors (D-F) are shown.