Presumed genealogical trees showing examples of sequence divergence within CDR3-related clones.

<p>Each tree is based on clonally-related IgH sequences amplified at different time points from an independent bulk LCL culture derived from EBV-infected naïve B cells. In each case, functional IgH sequences were first aligned with the nearest germline IGH V, D and J alleles to determine the number and location of mutations. Each clonally-related IgH sequence is represented as a box, with the number of incremental mutations shown on each branch; the sequence name and the position of the codon changes (according to the Kabat numbering system) are indicated inside the box while newly acquired mutations are underlined. (A) Prospective analysis of LCL5 identified a sequence (clone w4s6) with zero mutations relative to the nearest germline allele IGHV4-59*01 and a further 9 clonally-related variants (with up to 8 additional changes) which segregated into two major branches. Note that the same mutation at codon 70 was seen in both branch 1 and branch 2 and was therefore assumed to have occurred twice independently. (B) Serial analysis of LCL8 identified a sequence (w6s17) with two mutations relative to the nearest germline allele IGHV3-23*04 and a further 5 clonally-related variants with up to 6 additional sequence changes (note the change in codon 31 involved two substitutions). In this case the nearest germline IGVH allele was a presumed hypothetical intermediate and is therefore represented as a dotted box.