Systematic Characteristic Exploration of the Chimeras Generated in Multiple Displacement Amplification through Next Generation Sequencing Data Reanalysis

Background

The chimeric sequences produced by phi29 DNA polymerase, which are named as chimeras, influence the performance of the multiple displacement amplification (MDA) and also increase the difficulty of sequence data process. Despite several articles have reported the existence of chimeric sequence, there was only one research focusing on the structure and generation mechanism of chimeras, and it was merely based on hundreds of chimeras found in the sequence data of E. coli genome.

Method

We finished data mining towards a series of Next Generation Sequencing (NGS) reads which were used for whole genome haplotype assembling in a primary study. We established a bioinformatics pipeline based on subsection alignment strategy to discover all the chimeras inside and achieve their structural visualization. Then, we artificially defined two statistical indexes (the chimeric distance and the overlap length), and their regular abundance distribution helped illustrate of the structural characteristics of the chimeras. Finally we analyzed the relationship between the chimera type and the average insertion size, so that illustrate a method to decrease the proportion of wasted data in the procedure of DNA library construction.

Results/Conclusion

131.4 Gb pair-end (PE) sequence data was reanalyzed for the chimeras. Totally, 40,259,438 read pairs (6.19%) with chimerism were discovered among 650,430,811 read pairs. The chimeric sequences are consisted of two or more parts which locate inconsecutively but adjacently on the chromosome. The chimeric distance between the locations of adjacent parts on the chromosome followed an approximate bimodal distribution ranging from 0 to over 5,000 nt, whose peak was at about 250 to 300 nt. The overlap length of adjacent parts followed an approximate Poisson distribution and revealed a peak at 6 nt. Moreover, unmapped chimeras, which were classified as the wasted data, could be reduced by properly increasing the length of the insertion segment size through a linear correlation analysis.

Significance

This study exhibited the profile of the phi29MDA chimeras by tens of millions of chimeric sequences, and helped understand the amplification mechanism of the phi29 DNA polymerase. Our work also illustrated the importance of NGS data reanalysis, not only for the improvement of data utilization efficiency, but also for more potential genomic information.     

Generation of Viable Plant-Vertebrate Chimeras

The extreme dependence on external oxygen supply observed in animals causes major clinical problems and several diseases are related to low oxygen tension in tissues. The vast majority of the animals do not produce oxygen but a few exceptions have shown that photosynthetic capacity is physiologically compatible with animal life. Such symbiotic photosynthetic relationships are restricted to a few aquatic invertebrates. In this work we aimed to explore if we could create a chimerical organism by incorporating photosynthetic eukaryotic cells into a vertebrate animal model. Here, the microalgae Chlamydomonas reinhardtii was injected into zebrafish eggs and the interaction and viability of both organisms were studied. Results show that microalgae were distributed into different tissues, forming a fish-alga chimera organism for a prolonged period of time. In addition, microscopic observation of injected algae, in vivo expression of their mRNA and re-growth of the algae ex vivo suggests that they survived to the developmental process, living for several days after injection. Moreover microalgae did not trigger a significant inflammatory response in the fish. This work provides additional evidence to support the possibility that photosynthetic vertebrates can be engineered.     

Pluripotency of Homozygous-Diploid Mouse Embryos in Chimeras

Pluripotency of mouse uniparental cells (complete homozygous-diploid gynogenetic) produced by embryo manipulation was examined in aggregation chimeras with normally fertilized embryos. A male pronucleus was removed from fertilized eggs by micromanipulation and eggs were diploidized with cytochalasin B. Uniparental cells that developed to 4-cell or more advanced stages were aggregated with normally fertilized 8-cell embryos and transferred to the pseudopregnant female uteri to develop to term. Among the pups, 1 female and 3 males were identified as overt chimeras by their coat color and pigmentation of the retina. Using electophoretic analysis of the isozymes, the contribution of uniparental cells in these chimeras was confirmed by findings in the major organs such as liver, brain, small intestine, kidney, spleen, heart and testis. The female chimera produced offspring derived from oocytes of uniparental origin. Our experiments verified the pluripotency of microsurgically produced mouse uniparental cells.     

PNA-DNA Chimeras Forming Quadruplex Structures

Abstract

¹H-NMR, CD, and UV spectroscopy have been used to investigate the structure of PNA/DNA chimeras forming quadruplex structures. In particular, we synthesized ^5′TGGG^3′-t (1) and ^5′TGG^3′-gt (2), where lower and upper case letters indicate PNA and DNA residues, respectively. CD spectrum and all NMR data of (1) are typical of quadruplexes involving four parallel strands. UV melting profile of (1) indicates that its thermal stability is quite similar to that observed for the reference structure [d(TGGGT)]₄. ¹H-NMR spectrum for ^5′TGG^3′-gt (2) shows that this oligonucleotide is not able to fold into a single, well-defined species.     

Diffusion of Finite-Size Particles in Confined Geometries

The diffusion of finite-size hard-core interacting particles in two- or three-dimensional confined domains is considered in the limit that the confinement dimensions become comparable to the particle’s dimensions. The result is a nonlinear diffusion equation for the one-particle probability density function, with an overall collective diffusion that depends on both the excluded-volume and the narrow confinement. By including both these effects, the equation is able to interpolate between severe confinement (for example, single-file diffusion) and unconfined diffusion. Numerical solutions of both the effective nonlinear diffusion equation and the stochastic particle system are presented and compared. As an application, the case of diffusion under a ratchet potential is considered, and the change in transport properties due to excluded-volume and confinement effects is examined.     

Experimentally Synthesized Plant Chimeras 3. Qualitative and Quantitative Characteristics of the Flowers of Interspecific Nicotiana Chimeras

The flowers from a series of interspecific periclinal chimerasbetween Nicotiana glauca Grahm. and N. tabacum Su/su L. werequantitatively and qualitatively compared with the flowers oftheir component species and the sexual hybrid. Results indicatethat the epidermal component of the chimeral flowers has thegreatest influence on flower morphology and that each histogenicarrangement results in a unique flower morphology. When comparedto interspecific hybrids a greater diversity of flower shapes,sizes and colours exists with periclinal chimeras, demonstratingthat the experimental synthesis of chimeras between morphologicallydistinct components can be an important source of new phenotypes. Nicotiana glauca Grahm., Nicotiana tabacum L., tobacco, chimeras, graft chimeras, floral morphology, flowers     

Chimeras for mutations induced in dormant tomato seeds

Genetic chimeras of the VFNT cherry tomato line (Lycopersicon esculentum) were produced by mutagenizing seeds with ethyl methane sulfonate (EMS). The chimeras thereby produced were evaluated by progeny-testing the fruits of the genetically mosaic tissue. A total of 2011 M₁ plants was grown from treated seeds and evaluated by screening their 95175 (M₂) progeny for mutations affecting seedling phenotype. Three vigorous and fertile M₁ plants bearing mutant progeny with definitive phenotypes were selected for systematic harvesting and analysis. The specific location of each fruit was noted at harvest time, enabling the mutated sporogenous tissue of the mosaic M₁ plants to be traced. Sectoring appeared in both branch and floral tissues. In several cases, mutant progenies were restricted to individual branches or parts thereof. True-breeding recessive mutants whose monogenic mode of inheritance was later established occasionally segregated within M₁ fruit progenies at frequencies that indicate a non-homogeneous floral meristem origin. The data emphasize the necessity of making a well-distributed harvest of mosaic plants in order to detect as many variants as possible, as mosaic sectors may or may not recur late in ontogeny and may not contribute to sporogenous tissue early in development.     

Construction of Intra-Domain Chimeras of Aminoacyl-tRNA Synthetases

Abstract

To explore new approaches to enzyme engineering, intra-domain chimeras of two aminoacyltRNA synthetases were constructed. Connections were made within the nucleotide folds of these enzymes at sites earlier shown either to be dispensable for activity or able to accomodate oligopeptide insertions. (R.M. Starzyk, T.A. Webster and P. Schimmel, Science 237, 1614 (1987); R.M. Starzyk, J.J. Burbaum and P. Schimmel, Biochemistry, in press). Based on the known structure of one synthetase and structural modeling of the other, the locations of the connection sites allow the possibility of functional “compound” ATP and tRNA binding sites. Of five chimeric genes which were constructed, three direct synthesis of polypeptides that accumulate in vivo. These stable hybrids provide prototypes to which mutagenesis procedures may be applied to produce enzymatically active chimeric synthetases.     

Synthesis and Characterization of Nicotiana-Solanum Graft Chimeras

Intergeneric Nicotiana tabacum L./Solanum laciniatum Ait. graftchimeras were produced from decapitated grafts made betweenthese two graft compatible species. Graft unions were treatedwith or without either the auxin, p-chlorophenoxyacetic acid(p-CPA) or the cytokinin, benzylaminopurine (BAP). While BAPwas inhibitory to shoot formation, p-CPA increased the numberof adventitious shoots and raised the frequencies of shoot-forminggrafts obtained. Approximately 9% (14/151) of the shoots producedat p-CPA-treated graft unions were intergeneric chimeras. Theauxin application significantly increased levels of chimeralshoot recovery numbers thus indicating a direct auxin effecton chimeral shoot production. The types and numbers of chimerasproduced were independent of the scion/stock graft combinationemployed. All chimeras appeared to arise initially as mericlinalor sectorial chimeras, with a proportion of the former ones(5/14) stabilizing into periclinal chimeras. The morphologicalcharacteristics of these latter chimeras were compared withthose of the two parental species. The LII layer of speciesdetermined the characteristics of the vegetative and floralcomponents. However, the LI layer modified the qualitative traitsof both components. The LIII layer on the other hand alteredthe quantitative traits of the vegetative organs and the flower.This layer also determined the growth type, breeding behaviourand inflorescence type of synthesized chimeras. These organizationalfunctions of the LIII layer have not been reported previously. Chimeras, intergeneric chimera synthesis, grafting, graft chimeras, Nicotiana tabacum, Solanum aviculare, p-chlorophenoxyacetic acid, peroxidase isoenzymes     

Formation of Human Neuroblastoma in Mouse-Human Neural Crest Chimeras

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人源化小鼠人源细胞检测方法

目的人源化小鼠在人类疾病与再生医学研究中具有广泛应用,为获得一种理想的人源化小鼠检测方法,研究人特异性线粒体序列扩增作为人源化动物模型中人类DNA检测方法的可行性。方法设计人线粒体DNA特异性引物,并用该引物对人脐血造血干细胞嵌合体小鼠进行了检测。结果在人源化动物模型的人类DNA检测中,这种人线粒体DNA的PCR检测方法能够达到理想的特异性与灵敏性,是一种理想的嵌合体小鼠检测方法。结论获得了一种理想的人源化小鼠人源细胞检测方法。     

Constructing Suprachiasmatic Nucleus Chimeras In Vivo

We introduce a new transplantation technique for analyzing suprachiasmatic nucleus (SCN) development and function. Neural precursor (‘stem’) cells are harvested from the brains of mice engineered to express the green fluorescent protein, propagated in culture with growth factors, and injected into the forebrain ventricles of individual embryos in order to gain access to the ventricular germinal zone and the developing brain. Initial data indicate that such cells can incorporate within the SCN and appear to differentiate into astroglial phenotypes. In addition to applications in chronobiology, SCN chimeras may be a potentially powerful model system for analyzing the structural and functional plasticity of implanted precursor cells.     

鸡囊胚细胞嵌合体制作技术研究及其应用前景

家鸡X期囊胚细胞(BCs)嵌合体技术,既是利用转基因技术进行家鸡品种改良和凭借转基因家鸡生物反应器生产医用蛋白等研究领域的关键技术,也是利用BCs冻存家鸡和珍稀鸟类双亲种质资源实现鸟类品种资源多样性保护、利用和挽救珍稀濒危鸟类的重要途径。从家鸡BCs嵌合体制作技术的基本过程：(1) 羽色嵌合体家鸡模型的建立;(2) 囊胚的分离与消化;(3) 受体种蛋的致弱处理;(4) 受体种蛋的开窗（包括部位、方法及封口技术等）;(5) 供体细胞导入受体胚（显微注射或简易操作）;(6) 孵化（常规方法或换壳培养）等几个方面的研究进展、目前存在的问题以及研究方向等进行了系统阐述。Abstract: The technology of producing chicken chimeras using blastodermal cells is very important not only in the field of transgenic chicken bioreactor but also in searching for efficient ways to conserve avian genetic resource. The basic processes for producing chicken chimeras consist of: (1) Setting up the color model; (2) Separating and dissociating of donor embryos; (3) Compromising of the recipient embryos; (4) Windowing and recovering the recipient eggs; (5) Cells injecting; (6) Method of hatching. The progress, obstacles and prospects of producing chicken chimeras via BCs were discussed in this paper.     

花烛离体培养叶色变异株系的相关性状

以花烛(Anthurium andraeanum)间接器官发生途径中再生出的一株花叶变异植株为原始材料, 进行增殖并对得到的3个叶色变异株系的叶色相关性状进行了初步研究。结果表明: 通过愈伤组织器官发生途径和腋芽增殖途径对这一花叶苗进行增殖, 均分离到3种变异株系, 即花叶苗、黄化苗和天鹅绒绿色叶片苗; 天鹅绒绿色苗叶片中的叶绿素含量比正常离体苗的含量低; 叶片解剖结构表明, 叶绿体在叶肉细胞中的分布与其叶片表现型相同, 天鹅绒绿色叶片与正常叶片在解剖结构上无明显差异。花烛原套只具有1层细胞, 无明显的L2层分生结构, 因此叶肉的薄壁细胞完全由向各个方向分裂的原体细胞发育而来, 这种组织结构导致花叶叶片中含有叶绿体的细胞和不含有叶绿体的薄壁细胞呈不规则分布。这种花叶株系可以作为育种材料或直接作为盆栽花烛进行推广。     

Quantitative Analysis of Endocytosis with Cytoplasmic pHluorin Chimeras

The pH‐sensitive green fluorescent protein (GFP) variant pHluorin is typically fused to the extracellular domain of transmembrane proteins to monitor endocytosis. Here, we have turned pHluorin inside‐out, and show that cytoplasmic fusions of pHluorin are effective quantitative reporters for endocytosis and multivesicular body (MVB) sorting. In yeast in particular, fusion of GFP and its variants on the extracellular side of transmembrane proteins can result in perturbed trafficking. In contrast, cytoplasmic fusions are well tolerated, allowing for the quantitative assessment of trafficking of virtually any transmembrane protein. Quenching of degradation‐resistant pHluorin in the acidic vacuole permits quantification of extravacuolar cargo proteins at steady‐state levels and is compatible with kinetic analysis of endocytosis in live cells.