Assessment of PCR bias. The rearranged TCRβ CDR3 regions present in approximately 30 000 T-cell genomes were amplified through 25 cycles of PCR, and the PCR products were split into 2 pools. One pool was amplified an additional 15 cycles, and then the PCR products from the 25-cycle and 40-cycle reactions were sequenced in separate lanes of a GA1 flow cell. Of the TCRβ CDR3 sequences observed in the 25-cycle PCR lane, 97% were also observed in the 40-cycle PCR lane. Each point on the graph represents a single unique CDR3 sequence, plotted according to the number of times that sequence was observed in the data from 25-cycle (abscissa) and 40-cycle (ordinate) PCR reactions, respectively. The density of sequences at each point in the plot is indicated by color, with purple the highest density and red the lowest. The solid line represents a linear regression of the data, and the dotted lines 1 SD above and below the mean.