Multi Step Selection in Ig H Chains is Initially Focused on CDR3 and Then on Other CDR Regions

the choice of appropriate clones (clonal selection), and the internal evolution within clones, induced by somatic hyper-mutations, where high affinity mutants are selected for. When a final population of immunoglobulin sequences is observed, the genetic composition of this population is affected by a combination of these two processes. Different immune induced diseases can result from the failure of regulation of clonal selection or of the regulation of the within clone affinity maturation. In order to understand each of these processes separately, we propose a mixed lineage tree/sequence based method to detect within clone selection as defined by the effect of mutations on the average number of offspring. Specifically, we measure the imbalance in the number of leaves in lineage trees branches following synonymous and non-synonymous (NS) mutations. If a mutation is positively selected, we expect the number of leaves in the sub-tree below this mutation to be larger than in the parallel sub-tree without the mutation. The ratio between the number of leaves in such branches following NS mutations can be used to measure selection within a clone. We apply this method to the sampled Ig repertoire from multiple healthy volunteers and show that within clone selection is positive in the CDR2 region and either positive or negative in the CDR3 and FWR3 regions. Selection occurs already at the IgM isotype level mainly in the DH gene region, with a strong negative selection in the join region. This is followed in the later memory stages in the CDR2 region. We have not studied here the FWR1 and CDR1 regions. An important advantage of this method is that it is very weakly affected by the baseline mutation model or by sampling biases, as are most synonymous to NS mutations ratio based methods.