Spectratyping plots

The immune repertoire within a sample is represented in each spectratyping plot by the range of CDR3 lengths and their pattern of distribution. Reads for identified clones are arranged along the X‑axis according to the variable gene identity and the Y‑axis according to the CDR3 nucleotide length. The variable gene order reflects the gene position within the IGH locus. Dots are separated vertically along the Y‑axis by 3 nucleotides (one codon), the higher up the Y‑axis the longer the CDR3 region. Circle size indicates the frequency of a particular variable gene-CDR3 nucleotide length combination within the dataset. Circle color represents a forth metric specific to each graph (for example, Shannon Diversity, evenness, clone frequency). Key repertoire metrics are displayed along the lower margin of the plot.

In Ion Reporter™ Software, the spectratyping plots are interactive, allowing you to adjust the data and access clone details. Drag the ends of the horizontal bar below the X‑axis to limit the region (v-genes) to view in the plot. Drag the ends of the color range up or down to limit the clones that are viewed. Hover your cursor over any dot to view the details of an individual clone. Click to restore the default plot view, click to download a static image of the plot (if you have adjusted the plot view, the adjusted plot is downloaded).

Note: T-cell Leukemia (Jurkat) Total RNA is derived from a cell line consisting of a single T‑cell clonotype. Running the Oncomine™ TCR Beta‑LR Assay on Jurkat Total RNA should detect a single clonotype (a single spot on spectratyping plot).

Sequencing of a B cell line such as Ramos will reveal a single dominating clonal lineage, indicated by a single spot on the spectratyping plot.

IGH V-gene usage and mutation rate

Spectratyping plot highlighting frequency of mutated bases over the variable gene of identified clones. Circle color indicates the average frequency of mutated bases for clones having a particular variable gene-CDR3 nucleotide length combination. B cells that have undergone isotype switching tend to have a higher frequency of somatic hypermutation than B cells expressing IgM or IgD isotypes, which tend to represent naive B cells. Systematic differences with respect to reference may indicate the presence of polymorphism within the variable gene that is not captured by the IMGT database. You can further partition the clones by isotype in Ion Reporter™ Software, with the buttons that above the interactive spectratyping plot for Oncomine™ BCR IGH‑LR Assay analyses.

IGH V-gene usage and evenness

Spectratyping plot highlighting evenness of identified clone sizes (Normalized Shannon Entropy). Circle color indicates the evenness of clone sizes for clones having a particular variable gene-CDR3 nucleotide length combination. Values range from 0 to 1, with 1 indicating most even clone sizes. In this representation, portions of the repertoire containing highly expanded clones appear white. You can further partition the clones by isotype in Ion Reporter™ Software, with the buttons that above the interactive spectratyping plot for Oncomine™ BCR IGH‑LR Assay analyses.

IGH V-gene usage and Shannon Diversity

Spectratyping plot highlighting Shannon Diversity (entropy) of identified clones. Circle color indicates the Shannon Diversity of clones having a particular variable gene-CDR3 nucleotide length combination. Portions of the repertoire containing highly expanded clones typically have a corresponding low Shannon Diversity value. You can further partition the clones by isotype in Ion Reporter™ Software, with the buttons that above the interactive spectratyping plot for Oncomine™ BCR IGH‑LR Assay analyses.

IGH V-gene usage and largest clone frequency

Spectratyping plot highlighting the frequency of the largest clone for each variable gene-CDR3 nucleotide length combination. Circle color indicates the frequency of the largest clone having a particular variable gene-CDR3 nucleotide length combination. Dark color indicates the presence of expanded clones. This is a polyclonal repertoire that lacks highly expanded clones. You can further partition the clones by isotype in Ion Reporter™ Software, with the buttons that above the interactive spectratyping plot for Oncomine™ BCR IGH‑LR Assay analyses.

IGH V-gene usage and number of clones

Spectratyping plot highlighting the number of clones that are identified for each variable gene-CDR3 nucleotide length combination. Circle color indicates the number of clones having a particular variable gene-CDR3 nucleotide length combination. Specific variable genes may more frequently participate in VDJ recombination, leading to an enrichment in distinct clones for those variable genes. You can further partition the clones by isotype in Ion Reporter™ Software, with the buttons that above the interactive spectratyping plot for Oncomine™ BCR IGH‑LR Assay analyses.