Xiaoliang Sunney Xie

Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients

Significance In a few milliliters of blood from a cancer patient, one can isolate a few circulating tumor cells (CTCs). Originating from the primary tumor, CTCs seed metastases, which account for the majority of cancer-related deaths. We demonstrate the analyses of the whole genome of single CTCs, which are highly needed for personalized treatment. We discovered that copy number variations (CNVs), one of the major genomic variations, are specific to cancer types, reproducible from cell to cell, and even from patient to patient. We hypothesize that CNVs at certain genomic loci are selected for and lead to metastasis. Our work shows the prospect of noninvasive CTC-based cancer diagnostics. Circulating tumor cells (CTCs) enter peripheral blood from primary tumors and seed metastases. The genome sequencing of CTCs could offer noninvasive prognosis or even diagnosis, but has been hampered by low single-cell genome coverage of scarce CTCs. Here, we report the use of the recently developed multiple annealing and looping-based amplification cycles for whole-genome amplification of single CTCs from lung cancer patients. We observed characteristic cancer-associated single-nucleotide variations and insertions/deletions in exomes of CTCs. These mutations provided information needed for individualized therapy, such as drug resistance and phenotypic transition, but were heterogeneous from cell to cell. In contrast, every CTC from an individual patient, regardless of the cancer subtypes, exhibited reproducible copy number variation (CNV) patterns, similar to those of the metastatic tumor of the same patient. Interestingly, different patients with the same lung cancer adenocarcinoma (ADC) shared similar CNV patterns in their CTCs. Even more interestingly, patients of small-cell lung cancer have CNV patterns distinctly different from those of ADC patients. Our finding suggests that CNVs at certain genomic loci are selected for the metastasis of cancer. The reproducibility of cancer-specific CNVs offers potential for CTC-based cancer diagnostics.

Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes.

RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling, but is hampered by sequence-dependent bias and inaccuracy at low copy numbers intrinsic to exponential PCR amplification. We developed a simple strategy for mitigating these complications, allowing truly digital RNA-Seq. Following reverse transcription, a large set of barcode sequences is added in excess, and nearly every cDNA molecule is uniquely labeled by random attachment of barcode sequences to both ends. After PCR, we applied paired-end deep sequencing to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance is measured based on the number of unique barcode sequences observed for a given cDNA sequence. We optimized the barcodes to be unambiguously identifiable, even in the presence of multiple sequencing errors. This method allows counting with single-copy resolution despite sequence-dependent bias and PCR-amplification noise, and is analogous to digital PCR but amendable to quantifying a whole transcriptome. We demonstrated transcriptome profiling of Escherichia coli with more accurate and reproducible quantification than conventional RNA-Seq.

Probing meiotic recombination and aneuploidy of single sperm cells by whole-genome sequencing.

Single-Cell Sequencing With the rapid progress in sequencing technologies, single-cell sequencing is now possible, promising insight into how cell-to-cell heterogeneity affects biological behavior. Achieving adequate genome coverage remains a challenge because single-cell sequencing relies on genome amplification that is prone to sequence bias. Zong et al. (p. 1622) report a new amplification method: multiple annealing and looping-based amplification cycles that allowed 93% genome coverage for a human cell. This coverage facilitated accurate detection of point mutations and copy number variations. Lu et al. (p. 1627) used the method to sequence 99 sperm cells from a single individual. Mapping the meiotic crossovers revealed a nonrandom distribution with a reduced recombination rate near transcription start sites. A whole-genome amplification method with reduced bias yields a personal meiotic recombination map. Meiotic recombination creates genetic diversity and ensures segregation of homologous chromosomes. Previous population analyses yielded results averaged among individuals and affected by evolutionary pressures. We sequenced 99 sperm from an Asian male by using the newly developed amplification method—multiple annealing and looping-based amplification cycles—to phase the personal genome and map recombination events at high resolution, which are nonuniformly distributed across the genome in the absence of selection pressure. The paucity of recombination near transcription start sites observed in individual sperm indicates that such a phenomenon is intrinsic to the molecular mechanism of meiosis. Interestingly, a decreased crossover frequency combined with an increase of autosomal aneuploidy is observable on a global per-sperm basis.

Vibrational spectroscopic imaging of living systems: An emerging platform for biology and medicine.

Imaging with molecular vibrations The vibrational spectra of biomolecules could in principle image cells and tissue without added markers. Practically, several technical problems need to be overcome to achieve sufficient imaging depths, resolution, and data acquisition speed. Cheng and Xie review emerging bioimaging methods for use in the lab and the clinic. Science, this issue p. 10.1126/science.aaa8870 BACKGROUND Biomolecules can serve as natural labels for microscopy by measuring their molecular vibration spectra in living cells and tissues. However, the transition from spectroscopy of molecules in cuvettes to spectroscopic imaging of living systems requires more than putting spectrometers on microscopes. A series of technical challenges must be addressed, such as delivery of light beams for sample excitation and scattering of signals, which limit the probe depth of spectroscopic imaging. There are also trade-offs in how large a spectral window can be measured at a pixel (the amount of chemical information) in a given amount of time (the recording speed). ADVANCES Several technical advances made by different groups have pushed the boundary of the vibrational spectroscopic imaging field in terms of spectral acquisition speed, detection sensitivity, spatial resolution, and penetration depth. Specifically, coherent Raman scattering microscopy has emerged as a high-speed vibrational imaging platform. Single-frequency coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopes have reached a video-rate imaging speed. Multiplex CARS by broadband excitation has reached a speed of 3.5 ms per pixel; multiplex SRS covering a window of 200 wave numbers has reached a speed of 32 μs per pixel. The marriage of high-speed coherent Raman microscopy and Raman-sensitive tags of large cross sections has enabled real-time imaging of small molecules at micromolar concentration. Nanoscale vibrational imaging has been demonstrated by integration of atomic force microscopy and vibrational spectroscopy. Vibrational imaging of deep tissue has been enabled by acoustic detection of overtone transitions or optical detection of diffuse photons. Biological applications of CARS and SRS microscopy have generated new insights into myelin biology, lipid droplet biology, intracellular drug delivery, and single-cell metabolism. Meanwhile, clinical applications of vibrational spectroscopic imaging are enabling molecule-based diagnosis of cancer and heart disease without the need for any exogenous contrast agent. Examples include intravascular vibrational photoacoustic imaging of lipid-laden plaques and spatially offset Raman spectroscopic detection of cancer margins. OUTLOOK There remain two central challenges facing the field. One is to increase the detection sensitivity of vibrational microscopy to micromolar or even nanomolar levels, so that low-concentration biomolecules in a living system can be mapped. The other is to increase the vibrational imaging depth to tens of centimeters for noninvasive molecule-based medical diagnosis. With continuous developments, high-resolution, high-speed vibrational microscopy will cultivate unexpected discoveries in cell biology. The findings may lead to the development of new therapies for currently incurable diseases. Meanwhile, with further improvement of penetration depth and progressive reduction of instrument size, vibrational spectroscopic imaging devices are expected to become fundamental clinical tools for disease diagnosis and therapy effectiveness evaluation. Molecular fingerprints for biology and medicine through imaging. Recent efforts focused on pushing the fundamental limits of vibrational spectroscopic imaging in terms of spectral acquisition speed, detection sensitivity, spatial resolution, and penetration depth. The resulting platforms are enabling transformative applications in functional analysis of single living cells and noninvasive diagnosis of human diseases with biomarker sensitivity. Vibrational spectroscopy has been extensively applied to the study of molecules in gas phase, in condensed phase, and at interfaces. The transition from spectroscopy to spectroscopic imaging of living systems, which allows the spectrum of biomolecules to act as natural contrast, is opening new opportunities to reveal cellular machinery and to enable molecule-based diagnosis. Such a transition, however, involves more than a simple combination of spectrometry and microscopy. We review recent efforts that have pushed the boundary of the vibrational spectroscopic imaging field in terms of spectral acquisition speed, detection sensitivity, spatial resolution, and imaging depth. We further highlight recent applications in functional analysis of single cells and in label-free detection of diseases.

Enhanced Efflux Activity Facilitates Drug Tolerance in Dormant Bacterial Cells

Summary Natural variations in gene expression provide a mechanism for multiple phenotypes to arise in an isogenic bacterial population. In particular, a sub-group termed persisters show high tolerance to antibiotics. Previously, their formation has been attributed to cell dormancy. Here we demonstrate that bacterial persisters, under β-lactam antibiotic treatment, show less cytoplasmic drug accumulation as a result of enhanced efflux activity. Consistently, a number of multi-drug efflux genes, particularly the central component TolC, show higher expression in persisters. Time-lapse imaging and mutagenesis studies further establish a positive correlation between tolC expression and bacterial persistence. The key role of efflux systems, among multiple biological pathways involved in persister formation, indicates that persisters implement a positive defense against antibiotics prior to a passive defense via dormancy. Finally, efflux inhibitors and antibiotics together effectively attenuate persister formation, suggesting a combination strategy to target drug tolerance.

Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy

Significance We developed an optical imaging technique that combines reflected light-sheet illumination with superresolution microscopy, allowing us to image inside mammalian nuclei at subdiffraction-limit resolution and to count biomolecules with single-copy accuracy. Applying this technique to probe the spatial organization of RNA polymerase II-mediated transcription, we found that the majority of the transcription foci consist of only one RNAP II molecule, contrary to previous proposals. By quantifying the global extent of clustering across RNAP II molecules in the nucleus, we provide clear and convincing answers to the controversy surrounding the prevalent existence of “transcription factories.” Moreover, our work presents imaging and analysis tools for the quantitative characterization of nuclear structures, which could be generally applied to probe many other mammalian systems. Superresolution microscopy based on single-molecule centroid determination has been widely applied to cellular imaging in recent years. However, quantitative imaging of the mammalian nucleus has been challenging due to the lack of 3D optical sectioning methods for normal-sized cells, as well as the inability to accurately count the absolute copy numbers of biomolecules in highly dense structures. Here we report a reflected light-sheet superresolution microscopy method capable of imaging inside the mammalian nucleus with superior signal-to-background ratio as well as molecular counting with single-copy accuracy. Using reflected light-sheet superresolution microscopy, we probed the spatial organization of transcription by RNA polymerase II (RNAP II) molecules and quantified their global extent of clustering inside the mammalian nucleus. Spatiotemporal clustering analysis that leverages on the blinking photophysics of specific organic dyes showed that the majority (>70%) of the transcription foci originate from single RNAP II molecules, and no significant clustering between RNAP II molecules was detected within the length scale of the reported diameter of “transcription factories.” Colocalization measurements of RNAP II molecules equally labeled by two spectrally distinct dyes confirmed the primarily unclustered distribution, arguing against a prevalent existence of transcription factories in the mammalian nucleus as previously proposed. The methods developed in our study pave the way for quantitative mapping and stoichiometric characterization of key biomolecular species deep inside mammalian cells.

Genome-wide study of mRNA degradation and transcript elongation in Escherichia coli

An essential part of gene expression is the coordination of RNA synthesis and degradation, which occurs in the same cellular compartment in bacteria. Here, we report a genome‐wide RNA degradation study in Escherichia coli using RNA‐seq, and present evidence that the stereotypical exponential RNA decay curve obtained using initiation inhibitor, rifampicin, consists of two phases: residual RNA synthesis, a delay in the interruption of steady state that is dependent on distance relative to the mRNAs 5′ end, and the exponential decay. This gives a more accurate RNA lifetime and RNA polymerase elongation rate simultaneously genome‐wide. Transcripts typically have a single RNA decay constant along all positions, which is distinct between different operons, indicating that RNA stability is unlikely determined by local sequences. These measurements allowed us to establish a model for RNA processing involving co‐transcriptional degradation, providing quantitative description of the macromolecular coordination in gene expression in bacteria on a system‐wide level.

Stochastic phenotype transition of a single cell in an intermediate region of gene state switching.

Multiple phenotypic states often arise in a single cell with different gene-expression states that undergo transcription regulation with positive feedback. Recent experiments show that, at least in E.coli, the gene state switching can be neither extremely slow nor exceedingly rapid as many previous theoretical treatments assumed. Rather, it is in the intermediate region which is difficult to handle mathematically. Under this condition, from a full chemical-master-equation description we derive a model in which the protein copy number, for a given gene state, follows a deterministic mean-field description while the protein-synthesis rates fluctuate due to stochastic gene state switching. The simplified kinetics yields a nonequilibrium landscape function, which, similar to the energy function for equilibrium fluctuation, provides the leading orders of fluctuations around each phenotypic state, as well as the transition rates between the two phenotypic states. This rate formula is analogous to Kramers theory for chemical reactions. The resulting behaviors are significantly different from the two limiting cases studied previously.

Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization

Significance In in vitro fertilization (IVF), current methods of diagnosing chromosome abnormality and screening for viability of transfer require biopsy of embryos, which affects embryo quality, awaits long-term biosafety test, and requires specialized skills. We demonstrate the principle of noninvasive chromosome screening (NICS), which is based on sequencing the genomic DNA secreted into the culture medium from the embryo, avoiding the need for embryo biopsy and substantially increasing the safety. By characterizing its precision and demonstrating successful live births, we validate that NICS offers the potential of significantly improving the clinical outcome of IVF. Preimplantation genetic screening (PGS) is widely used to select in vitro-fertilized embryos free of chromosomal abnormalities and to improve the clinical outcome of in vitro fertilization (IVF). A disadvantage of PGS is that it requires biopsy of the preimplantation human embryo, which can limit the clinical applicability of PGS due to the invasiveness and complexity of the process. Here, we present and validate a noninvasive chromosome screening (NICS) method based on sequencing the genomic DNA secreted into the culture medium from the human blastocyst. By using multiple annealing and looping-based amplification cycles (MALBAC) for whole-genome amplification (WGA), we performed next-generation sequencing (NGS) on the spent culture medium used to culture human blastocysts (n = 42) and obtained the ploidy information of all 24 chromosomes. We validated these results by comparing each with their corresponding whole donated embryo and obtained a high correlation for identification of chromosomal abnormalities (sensitivity, 0.882, and specificity, 0.840). With this validated NICS method, we performed chromosome screening on IVF embryos from seven couples with balanced translocation, azoospermia, or recurrent pregnancy loss. Six of them achieved successful clinical pregnancies, and five have already achieved healthy live births thus far. The NICS method avoids the need for embryo biopsy and therefore substantially increases the safety of its use. The method has the potential of much wider chromosome screening applicability in clinical IVF, due to its high accuracy and noninvasiveness.