Genome-Wide Detection of Single-Nucleotide and Copy-Number Variations of a Single Human Cell

Kindred cells can have different genomes because of dynamic changes in DNA. Single cell sequencing is needed to characterize these genomic differences but has been hindered by whole-genome amplification bias, resulting in low genome coverage. Here we report a new amplification method: Multiple Annealing and Looping Based Amplification Cycles (MALBAC) that offer high uniformity across the genome. Sequencing MALBAC amplified DNA achieves 93% genome coverage ≥1x for a single human cell at 25x mean sequencing depth. We detected digitized copy number variations (CNVs) of a single cancer cell. By sequencing three kindred cells, we were able to call individual single nucleotide variations (SNVs) with no false positives observed. We directly measured the genome-wide mutation rate of a cancer cell line and found that purine-pyrimidine exchanges occurred unusually frequently among the newly acquired SNVs.Chemistry and Chemical Biolog

Protein structure prediction: Do hydrogen bonding and water-mediated interactions suffice?

The many-body physics of hydrogen bond formation in alpha-helices of globular proteins was investigated using a simple physics-based model. Specifically, a context-sensitive hydrogen bond potential, which depends on residue identity and degree of solvent exposure, was used in the framework of the Associated Memory Hamiltonian codes developed previously but without using local sequence structure matches (“memories”). Molecular dynamics simulations employing the energy function using the context-sensitive hydrogen bond potential alone (the “amnesiac” model) were used to generate low energy structures for three alpha-helical test proteins. The resulting structures were compared to both the X-ray crystal structures of the test proteins and the results obtained using the full Associated Memory Hamiltonian previously used. Results show that the amnesiac Hamiltonian was able to generate structures with reasonably high structural similarity (Q ~ 0.4) to that of the native protein but only with the use of predicted secondary structure information encoding local steric signals. Low energy structures obtained using the amnesiac Hamiltonian without any a priori secondary structure information had considerably less similarity to the native protein structures (Q ~ 0.3). Both sets of results utilizing the amnesiac Hamiltonian are poorer than when local-sequence structure matches are used

Probing Meiotic Recombination and Aneuploidy of Single Sperm Cells by Whole-Genome Sequencing

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.Chemistry and Chemical Biolog

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

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.Chemistry and Chemical Biolog

Theoretical studies of biomolecular self-assembly near equilibrium and far from equilibrium

The physical sciences have played a pre-eminent role in the advance of biology not only by providing advanced techniques, but also by providing simple concepts for navigating through the complexity of biological systems. One area where simple physics concepts help understanding complicated biological phenomena is the study of protein folding. By presenting the framework of a simple funneled energy landscape, folding is no longer a paradox from the physics point of view. In the following chapters, we present the investigations of both thermodynamics (predicting native structure) and kinetics (predicting phi -values) of protein folding on the basis of energy landscape theory. On the other hand, the discovery of assembly using biological molecular machinery presents new challenges to statistical mechanics combining the aspects of complexity and far-from-nonequilibrium behavior. In the fifth chapter, a study of nonequilibrium dynamic assembly inspired by microtubule dynamics in cell is presented. The theory provide a general scheme for studying nonequilibrium assembly in one dimension. Chapter 2 is based on the material as it appears in Biochemistry 45: 6458-6466 (2006). The dissertation author was the primary investigator and author of this paper. Chapter 3 is based on the material as it appears in Proc. Natl. Acad. Sci. 104: 3159-3164 (2007). The dissertation author was the primary investigator and author of this paper. Chapter 4 is based on the material as it appears in J. Am. Chem. Soc. 128: 5168-5176 (2006). The dissertation author was the primary investigator and author of this paper. Chapter 5 is based on the material as it appears in Physical Biology, 3: 83-92 (2006). The dissertation author was the primary investigator and author of this pape

Theoretical studies of biomolecular self-assembly near equilibrium and far from equilibrium

The physical sciences have played a pre-eminent role in the advance of biology not only by providing advanced techniques, but also by providing simple concepts for navigating through the complexity of biological systems. One area where simple physics concepts help understanding complicated biological phenomena is the study of protein folding. By presenting the framework of a simple funneled energy landscape, folding is no longer a paradox from the physics point of view. In the following chapters, we present the investigations of both thermodynamics (predicting native structure) and kinetics (predicting phi -values) of protein folding on the basis of energy landscape theory. On the other hand, the discovery of assembly using biological molecular machinery presents new challenges to statistical mechanics combining the aspects of complexity and far-from-nonequilibrium behavior. In the fifth chapter, a study of nonequilibrium dynamic assembly inspired by microtubule dynamics in cell is presented. The theory provide a general scheme for studying nonequilibrium assembly in one dimension. Chapter 2 is based on the material as it appears in Biochemistry 45: 6458-6466 (2006). The dissertation author was the primary investigator and author of this paper. Chapter 3 is based on the material as it appears in Proc. Natl. Acad. Sci. 104: 3159-3164 (2007). The dissertation author was the primary investigator and author of this paper. Chapter 4 is based on the material as it appears in J. Am. Chem. Soc. 128: 5168-5176 (2006). The dissertation author was the primary investigator and author of this paper. Chapter 5 is based on the material as it appears in Physical Biology, 3: 83-92 (2006). The dissertation author was the primary investigator and author of this pape

Single-cell RNA-seq study determines the ontogeny of macrophages in glioblastomas

Abstract A large-scale single-cell RNA-seq analysis of tumor-associated macrophages in gliomas has unveiled a new aspect of the complex tumor microenvironment and new biomarkers

Rare event of histone demethylation can initiate singular gene expression of olfactory receptors

In mammals, the sense of odors relies on the peculiar expression pattern of olfactory receptors (ORs). Each single neuron chooses one, and only one, from all ∼1,400 OR genes that are present in a mouse genome. In neurobiology, a long-standing mystery is how such singularity can be achieved. We show theoretically that a simple kinetic scheme of OR activation followed by feedback can be solely responsible for the observed singularity, as long as the two timescales—slow activation by epigenetic modification and fast feedback by transcriptional regulation—are well separated. Our work provides the theoretical underpinning behind the choice of ORs, and demonstrates how the nervous system utilizes the kinetics of epigenetic changes to direct neurogenesis.Chemistry and Chemical Biolog