Supplementary Figure 2: Validation of identity thresholds for clonality identification metric.
Grouped lineages of ASCs between sample replicates, cell populations and individual subjects are shown under different analytical requirements: (a) Circos plots are used to display connectivity between: a single CD138- ASC sample split into 3 separate fractions; a CD138+ ASC sample from the same individual (FLU 4, gray); and one CD138- ASC sample from a different individual (FLU 1, blue). Percentages in the upper left corner of each Circos plot indicate the level of CDR3 sequence similarity used to assign membership within the same clone. The two right-hand plots also require identical junction regions, as identified by the 3bp before and after the VH-D split and the 3bp before and after the D-JH split. The clone labeled 1 in the 85% threshold plot is split into multiple clones (1a, 1b, and 1c) when the identical junction requirement is used. (b) Plot of lineage sizes of two replicates of CD138- ASCs from FLU 4 based on 85% shows a high correlation between the sizes of the largest clones in each data set. Deviation from similar sized clones within the pair of data sets primarily occurs in small clones, which would be more prone to reflect sampling and/or sequencing error. The high correlation of large clones within replicates further validates the ability to identify expanded clones in the circulating B cell and ASC populations. Blue numbers indicate number of sequences per clone (non-log) for very small clones.
