PRJNA471611
Somatic hypermutation generates mutational clusters and a high  inter-clonal mutational diversity of the IgVH repertoire in a monoclonal B-cell line
The features of point mutations accumulating in the IgVH alleles of somatically mutating EBV transformed lymphoblastoid cell line (RF) were studied using Sanger and deep sequencing methods and lineage tree analysis.We performed ''next generation sequencing'' (NGS) of cDNA derived from the productive IgVH3-9 allele of cellular sub-clones of the monoclonal RF parental culture. Control libraries included plasmid vector of the IgVH3-9 sequence, and cDNA of the Gapdh and Cmu genes. The experimental libraries exhibited a mean of 25 novel mutations per library versus a mean value of only 0.5 mutations per the control libraries. Both sequencing methods exhibited preferential targeting of ~1.5-2.5 fold of the mutations to the underlined C/G of the canonical tetra- and tri-nucleotide hotspot motifs indicating that a majority of the mutations were likely due to activation-induced cytidine deaminase (AID), a critical enzyme for somatic hypermutation (SHM).Lineage tree analyses by Sanger and NGS, showed mean 'lower and upper boundaries' of mutation frequencies (MF) of ~0.22, and 0.82-1.0 mutation/cell respectively which were accumulated during the estimated ~23 generations required for the growth of the RF clones.Sub-groups of low and high-abundant mutations were differentiated by NGS. The abundant-mutational group contained in some of the RF clones a sub-set of independently repeated and overlapping mutational-clusters (MC), comprising of up to 5 discrete hypermutable sites. The MC subgroup was generated in a successive order in some of the highly developed lineage trees, and their mutational frequencies exceeded by up to ~100 folds those of the sub-group of the low-abundant mutations.Elucidation of the pathway(s) generating the MC category may extend our understanding of the dynamics and mechanisms of targeting of SHM to the rearranged IgVH genes. Both Sanger and NGS, demonstrate high variability of the clonal patterns of their lineage trees, and a high range of their mean inter-clonal MF rates.We suggest that the above features of the monoclonal RF B cell line, are shared by normal germinal center B cells, and enable them to generate by SHM a huge antibody repertoire to successfully cope with foreign pathogens.