American Society of Hematology

Figure 6

$Figure 6. Direct TCRβ CDR3 sequencing captures all of the TCR diversity information present in a conventional spectratype. (A) Comparison of standard TCRβ spectratype data and calculated TCRβ CDR3 length distributions for sequences using representative TCR Vβ gene segments and present in CD4+CD45RO+ cells from male donor 1. CDR3 length is plotted along the x-axis and the number of unique CDR3 sequences with that length (GA sequence data) or the relative intensity of the corresponding peak in the spectratype is plotted along the y-axis. Reducing the information contained in the GA sequence data to a frequency histogram of the unique CDR3 sequences with different lengths within each Vβ family readily reproduces all of the information contained in the spectratype data. The length of the differently colored segments within each bar of the histograms indicates the fraction of unique CDR3 sequences that were observed 1 to 5 times (black), 6 to 10 times (blue), 11 to 100 times (green), or more than 100 times (red). (B) A representative “virtual spectratype” of TCRβ CDR3 sequences extracted from CD4+CD45RO+ T cells from donor 1 that use the Vβ10 gene segment. The CDR3 sequences using Vβ10 were sorted by CDR3 length into a frequency histogram, and the sequences within each length bin were then color-coded on the basis of their Jβ use. The inset shows all of the CDR3 sequences using Vβ10 and Jβ2-6, and having a length of 39 nt, as well as the number of times that each of these sequences was observed in the data. The origin of the nucleotides in each sequence is color-coded as follows: Vβ gene segment, red; template-independent N nucleotide, black; Dβ gene segment, blue; Jβ gene segment, green.$

Direct TCRβ CDR3 sequencing captures all of the TCR diversity information present in a conventional spectratype. (A) Comparison of standard TCRβ spectratype data and calculated TCRβ CDR3 length distributions for sequences using representative TCR Vβ gene segments and present in CD4⁺CD45RO⁺ cells from male donor 1. CDR3 length is plotted along the x-axis and the number of unique CDR3 sequences with that length (GA sequence data) or the relative intensity of the corresponding peak in the spectratype is plotted along the y-axis. Reducing the information contained in the GA sequence data to a frequency histogram of the unique CDR3 sequences with different lengths within each Vβ family readily reproduces all of the information contained in the spectratype data. The length of the differently colored segments within each bar of the histograms indicates the fraction of unique CDR3 sequences that were observed 1 to 5 times (black), 6 to 10 times (blue), 11 to 100 times (green), or more than 100 times (red). (B) A representative “virtual spectratype” of TCRβ CDR3 sequences extracted from CD4⁺CD45RO⁺ T cells from donor 1 that use the Vβ10 gene segment. The CDR3 sequences using Vβ10 were sorted by CDR3 length into a frequency histogram, and the sequences within each length bin were then color-coded on the basis of their Jβ use. The inset shows all of the CDR3 sequences using Vβ10 and Jβ2-6, and having a length of 39 nt, as well as the number of times that each of these sequences was observed in the data. The origin of the nucleotides in each sequence is color-coded as follows: Vβ gene segment, red; template-independent N nucleotide, black; Dβ gene segment, blue; Jβ gene segment, green.

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