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Beyond the sequence of the genome, its three-dimensional structure is important in regulating gene expression. To understand cell-to-cell variation, the structure needs to be understood at a single-cell level. Chromatin conformation capture methods have allowed characterization of genome structure in haploid cells. Now, Tan et al. report a method called Dip-C that allows them to reconstruct the genome structures of single diploid human cells. Their examination of different cell types highlights the tissue dependence of three-dimensional genome structures. Science , this issue p. 924 A single-cell chromatin conformation capture method employs transposon-based whole-genome amplification to detect chromatin contacts. Three-dimensional genome structures play a key role in gene regulation and cell functions. Characterization of genome structures necessitates single-cell measurements. This has been achieved for haploid cells but has remained a challenge for diploid cells. We developed a single-cell chromatin conformation capture method, termed Dip-C, that combines a transposon-based whole-genome amplification method to detect many chromatin contacts, called META (multiplex end-tagging amplification), and an algorithm to impute the two chromosome haplotypes linked by each contact. We reconstructed the genome structures of single diploid human cells from a lymphoblastoid cell line and from primary blood cells with high spatial resolution, locating specific single-nucleotide and copy number variations in the nucleus. The two alleles of imprinted loci and the two X chromosomes were structurally different. Cells of different types displayed statistically distinct genome structures. Such structural cell typing is crucial for understanding cell functions.

Cells integrate multiple measurement modalities to navigate their environment. Soluble and substrate-bound chemical gradients and physical cues have all been shown to influence cell orientation and migration. Here we investigate the role of asymmetric hydraulic pressure in directional sensing. Cells confined in microchannels identified and chose a path of lower hydraulic resistance in the absence of chemical cues. In a bifurcating channel with asymmetric hydraulic resistances, this choice was preceded by the elaboration of two leading edges with a faster extension rate along the lower resistance channel. Retraction of the “losing” edge appeared to precipitate a final choice of direction. The pressure differences altering leading edge protrusion rates were small, suggesting weak force generation by leading edges. The response to the physical asymmetry was able to override a dynamically generated chemical cue. Motile cells may use this bias as a result of hydraulic resistance, or “barotaxis,” in concert with chemotaxis to navigate complex environments.

Because of the rapid development of communication and service in Taiwan, competition among telecommunication companies has become ever fiercer. Differences in marketing strategy usually become the key factor in keeping existing customers while attracting new ones. Although electronic word-of-mouth (e-WOM) is one of the most important pieces of information to a consumer making a purchase decision, very few articles on opinion mining have discussed and compared the relationship between multifaceted word-of-mouth (WOM) and marketing strategy. In this paper, we use our Chinese opinion-mining system (Wu et al. in J Supercomput 73:2987–3001, 2017 ) not only to retrieve articles related to 4G and conduct reputation analysis but also to discuss the relation between WOM and marketing strategy. The results show that (1) e-WOM can immediately and directly reflect the results of marketing strategy, and (2) although users are primarily concerned with aspects of price, online speed, and signal quality, for most Taiwanese customers, price is the key in choosing a telecommunication company. Moreover, although this paper used 4G-related articles from June 2014 to June 2015 for analysis, the results are consistent with the Taiwanese telecommunication companies’ current marketing strategy of attracting customers through low pricing.

Single-nucleotide variants (SNVs), pertinent to aging and disease, occur sporadically in the human genome, hence necessitating single-cell measurements. However, detection of single-cell SNVs suffers from false positives (FPs) due to intracellular single-stranded DNA damage and the process of whole-genome amplification (WGA). Here, we report a single-cell WGA method termed multiplexed end-tagging amplification of complementary strands (META-CS), which eliminates nearly all FPs by virtue of DNA complementarity, and achieved the highest accuracy thus far. We validated META-CS by sequencing kindred cells and human sperm, and applied it to other human tissues. Investigation of mature single human neurons revealed increasing SNVs with age and potentially unrepaired strand-specific oxidative guanine damage. We determined SNV frequencies along the genome in differentiated single human blood cells, and identified cell type-dependent mutational patterns for major types of lymphocytes.

Associations have been reported of the seven-repeat (7R) allele of the human dopamine receptor D4 (DRD4) gene with both attention-deficit/hyperactivity disorder and the personality trait of novelty seeking. This polymorphism occurs in a 48-bp tandem repeat in the coding region of DRD4, with the most common allele containing four repeats (4R) and rarer variants containing 2-11. Here we show by DNA resequencing/haplotyping of 600 DRD4 alleles, representing a worldwide population sample, that the origin of 2R-6R alleles can be explained by simple one-step recombination/mutation events. In contrast, the 7R allele is not simply related to the other common alleles, differing by greater than six recombinations/mutations. Strong linkage disequilibrium was found between the 7R allele and surrounding DRD4 polymorphisms, suggesting that this allele is at least 5-10-fold \"younger\" than the common 4R allele. Based on an observed bias toward nonsynonymous amino acid changes, the unusual DNA sequence organization, and the strong linkage disequilibrium surrounding the DRD4 7R allele, we propose that this allele originated as a rare mutational event that nevertheless increased to high frequency in human populations by positive selection.

An association of the dopamine receptor D4 (DRD4) gene located on chromosome 11p15.5 and attention deficit/hyperactivity disorder (ADHD) has been demonstrated and replicated by multiple investigators. A specific allele [the 7-repeat of a 48-bp variable number of tandem repeats (VNTR) in exon 3] has been proposed as an etiological factor in attentional deficits manifested in some children diagnosed with this disorder. In the current study, we evaluated ADHD subgroups defined by the presence or absence of the 7-repeat allele of the DRD4 gene, using neuropsychological tests with reaction time measures designed to probe attentional networks with neuroanatomical foci in D4-rich brain regions. Despite the same severity of symptoms on parent and teacher ratings for the ADHD subgroups, the average reaction times of the 7-present subgroup showed normal speed and variability of response whereas the average reaction times of the 7-absent subgroup showed the expected abnormalities (slow and variable responses). This was opposite the primary prediction of the study. The 7-present subgroup seemed to be free of some of the neuropsychological abnormalities thought to characterize ADHD.

Archaeological, anatomical, linguistic, and genetic data have suggested that there is an old and significant boundary between the populations of north and south China. We use three human genetic marker systems and one human-carried virus to examine the north/south distinction. We find no support for a major north/south division in these markers; rather, the marker patterns suggest simple isolation by distance.

Analysis of the genome of the flavivirus responsible for the 1999 New York City encephalitis epidemic cloned from human brain by reverse-transcription polymerase chain reaction indicates its identity as a lineage I West Nile virus (WNV; WNV-NY 1999) closely related to WNVs previously isolated in the Middle East.

3D genome structures play a key role in gene regulation and cell functions. Characterization of genome structures necessitates single-cell measurements. This has been achieved for haploid cells but remained a challenge for diploid cells. Here we report a single-cell chromatin conformation capture method, termed Dip-C, which combines a transposon-based whole-genome amplification method, called Multiplex End-tagging Amplification (META), to detect many chromatin contacts, and an algorithm to impute the two chromosome haplotypes linked by each contact. We reconstructed the genome structures of single diploid human cells from a lymphoblastoid cell line and from primary blood cells with high spatial resolution, locating specific single-nucleotide and copy-number variations in the nucleus. The two alleles of imprinted loci and the two X chromosomes were structurally different. Cells of different types displayed statistically distinct genome structures. Such structural cell typing is crucial for understanding cell functions.

In human and many other metazoans, both linear genome sequence and three-dimensional nuclear structure can vary between different cells within the same organism. Variations in linear DNA reflects the mutational history of the cell, and those in three-dimensional genome folding help regulate gene expression. Single-cell genomic assays therefore provide valuable means for characterizing the composition and regulation of genomes on a cell-to-cell basis. A sensitive and reproducible method for amplifying the genome of each single cell is the first step for decoding its linear DNA. In Chapter 2 we describe our single-cell whole-genome amplification assay that utilizes droplet microfluidics to produce long amplicons with accurate allele counting capability. This method has reproducibly achieved high genomic coverages of 91.0% ± 3% (average ± standard deviation) for all the cells verified to be non-replicated. To prevent DNA self-looping, we further included 16 transposon sequences, and labeled the two complementary strands of double-stranded DNA to correct for artifacts in the detection of single-nucleotide variations (SNVs). Described in Chapter 3, this assay, termed Multiplexed End-Tagging Amplification (META) of Complementary Strands, has revealed the mutational histories of blood and neuron cells with the highest accuracy known so far. Contrary to the conventional understanding that cell types are characterized by their transcriptomes, we found that SNV patterns are cell-type dependent and can be used to define cell types by principle component analysis. By integrating META with chromosome conformation capture, we re-constructed the genome-wide nuclear structures of single diploid human cells to an unprecedented resolution of 20kb. In Chapter 3 our method of Diploid Chromosome Conformation Capture (Dip-C) is illustrated; it has uncovered the cell-type dependent 3D genome architecture for human peripheral blood mononuclear cells. For the first time, the Waddington epigenetic landscape of human cells can be determined based on 3D genome folding, with valleys representing cell types and the width of each valley specifying cell-to-cell differences. Survey into more cell types at higher spatial resolution will help elucidate the relationships between genome structures and their functions in health and disease.