A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis. We report a liquid-phase, capture-based sequencing and bioinformatics pipeline, Sleeping Beauty (SB) capture hybridization sequencing (SBCapSeq), that facilitates sequencing of transposon insertion sites from single tumor cells in a SB mouse model of myeloid leukemia (ML). SBCapSeq analysis of just 26 cells from one tumor revealed the tumor’s major clonal subpopulations, enabled detection of clonal insertion events not detected by other sequencing methods and led to the identification of dominant subclones, each containing a unique pair of interacting gene drivers along with three to six cooperating cancer genes with SB-driven expression changes.

SignificanceDespite concerted efforts to identify causal genes that drive breast cancer (BC) initiation and progression, we have yet to establish robust signatures to stratify patient risk. Here we used in vivo transposon-based forward genetic screening to identify potentially relevant BC driver genes. Integrating this approach with survival prediction analysis, we identified six gene pairs that could prognose human BC subtypes into high-, intermediate-, and low-risk groups with high confidence and reproducibility. Furthermore, we identified susceptibility gene sets for basal and claudin-low subtypes (21 and 16 genes, respectively) that stratify patients into three relative risk subgroups. These signatures offer valuable prognostic insight into the genetic basis of BC and allow further exploration of the interconnectedness of BC driver genes during disease progression.

Poisson's spot is a diffraction phenomenon producing an intensity maximum at the center of the geometric shadow of circular opaque objects. In an analog of the Poisson spot experiment, we show that a tubular cone of x-rays incident upon a crystalline sample produces diffraction spots or foci, corresponding to Bragg maxima within a transmission shadow. We discuss the beam geometry and the intensity gain recorded at the foci in transmission mode. We describe the geometric growth and decay of the foci over a linear axis with the aid of a movie sequence synchronized with the plotting of a diffractogram. The mean signal of a small central area in each successive camera image provides the intensity data for the diffractogram. © 2010 American Institute of Physics. ͓doi:10.1063/1.3514235͔The characterization and measurement of crystallographic structure are of fundamental importance in many branches of science. Angular dispersive x-ray diffraction 1 employed routinely for such analysis dominates this field. However, the coherently scattered or diffracted x-ray signatures are weaker, by orders of magnitude, in comparison with the interrogating x-ray beam. State of the art commercially available powder diffractometers may employ highly sensitive large-area detectors with high quantum efficiency and low noise operating over relatively long integration periods. 2This conventional approach is not ideal for the development of scanning techniques and direct imaging applications, which would benefit from higher intensity signals, reduced integration periods, and converging diffracted beams. In this paper we report examples of diffraction image sequences, which demonstrate the growth and collapse of x-ray foci along the symmetry axis of a tubular interrogating x-ray beam.3 Our approach embodies an analog, employing x-rays, of the optical phenomenon that produces an intensity maximum, known as Poisson's spot, 4 at the center of the geometric shadow of circular opaque objects. In common with short wavelength Poisson spot techniques employing x-ray zone plates 5 or molecular beams 6 we measure the relative intensity of diffraction maxima at the center of a circular geometric shadow. Unlike these techniques, we employ an annular zone, defined at the intersection of a tubular cone of x-rays and a crystalline sample, enabling constructive on-axis interference to form intense spots or foci. The relative intensity and distribution of the foci correspond to the Bragg maxima determined by the crystalline structure of the sample.Consideration of diffraction patterns composed of individual Debye ring contributions hypothesizes the formation of a Poisson spot analog in Fig. 1. A tubular cone of x-rays with its symmetry axis incident normally upon a planar polycrystalline sample and image plane ͑positioned on the transmission side of the sample͒ will produce a continuum of relatively inclined Debye cones resulting in planar patterns composed of elliptical rings. The resultant circular termini and spot intensity fluctuations are due to rotatio...

30The systematic identification of genetic events driving cellular transformation and tumor progression in the absence 31 of a highly recurrent oncogenic driver mutation is a challenge in cutaneous oncology. In cutaneous squamous cell 32 carcinoma (cuSCC), the high UV-induced mutational burden poses a hurdle to achieve a complete molecular 33 landscape of this disease. Here, we utilized the Sleeping Beauty transposon mutagenesis system to statistically 34 define drivers of keratinocyte transformation and cuSCC progression in vivo in the absence of UV-IR, and identified 35 established tumor suppressor genes, as well as previously unknown oncogenic drivers of cuSCC. Functional analysis 36 confirms an oncogenic role for the ZMIZ genes, and tumor suppressive roles for KMT2C, CREBBP and NCOA2, in the 37 initiation or progression of human cuSCC. Taken together, our in vivo screen demonstrates an extremely 38 heterogeneous genetic landscape of cuSCC initiation and progression, which could be harnessed to better 39 understand skin oncogenic etiology and prioritize therapeutic candidates. 40