HRS cell flow sorting and ultralow-input sequence library validation. (A) Identification of HRS cells for flow cytometric sorting. HRS cells (red) show high forward and side scatter, are positive for CD30, bright for CD40 and CD95, and are typically positive for CD15. Many cases show various degrees of rosetting by T cells, resulting in composite CD5⁺/CD45⁺ immunophenotype. CD20⁺ (light blue) B cells and CD5⁺ (green) T cells with appropriate CD45 and side scatter parameters were sorted for experiment controls. (B) Sorted HRS cells could be visualized on a cytospin using Wright-Giemsa stain to confirm population identity and purity. Original magnification ×100. (C) Comparison of depth of sequence coverage per base between libraries generated with 1 ng (red), 10 ng (blue), and 100 ng (green) of starting genomic DNA from intratumoral T cells. Depth of coverage was comparable between 10 ng and 100 ng DNA input, resulting in 48× vs 52× median coverage, respectively. (D) Each of 2 panels depicts copy number variation analysis results comparing data between 2 sequenced libraries. Exonic probe segments (x-axis) vs copy number change on log₂ scale (y-axis) are plotted for a single representative chromosome (chr 6). Comparing data from a 10-ng low-input library from intratumoral T cell DNA to a 100-ng normal-input library from intratumoral T cell DNA from the same case showed no significant false-positive results; that is, low-input and normal-input libraries are copy neutral (top). Numerous segmental copy number alterations could be seen when data from a 10-ng low-input library from HRS were compared against intratumoral T cells of the same case (bottom), indicating that this method reveals copy number gains and losses. FSC, forward scatter; SSC, side scatter.