Single-cell genomic sequencing using Multiple Displacement Amplification

Single microbial cells can now be sequenced using DNA amplified by the Multiple Displacement Amplification (MDA) reaction. The few femtograms of DNA in a bacterium are amplified into micrograms of high molecular weight DNA suitable for DNA library construction and Sanger sequencing. The MDA-generated DNA also performs well when used directly as template for pyrosequencing by the 454 Life Sciences method. While MDA from single cells loses some of the genomic sequence, this approach will greatly accelerate the pace of sequencing from uncultured microbes. The genetically linked sequences from single cells are also a powerful tool to be used in guiding genomic assembly of shotgun sequences of multiple organisms from environmental DNA extracts (metagenomic sequences).     

Improved Multiple Displacement Amplification (iMDA) and Ultraclean Reagents

Background

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA).

Results

A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome.

Conclusions

The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.     

Testing the Reproducibility of Multiple Displacement Amplification on Genomes of Clonal Endosymbiont Populations

The multiple displacement amplification method has revolutionized genomic studies of uncultured bacteria, where the extraction of pure DNA in sufficient quantity for next-generation sequencing is challenging. However, the method is problematic in that it amplifies the target DNA unevenly, induces the formation of chimeric reads and also amplifies contaminating DNA. Here, we have tested the reproducibility of the multiple displacement amplification method using serial dilutions of extracted genomic DNA and intact cells from the cultured endosymbiont Bartonella australis. The amplified DNA was sequenced with the Illumina sequencing technology, and the results were compared to sequence data obtained from unamplified DNA in this study as well as from a previously published genome project. We show that artifacts such as the extent of the amplification bias, the percentage of chimeric reads and the relative fraction of contaminating DNA increase dramatically for the smallest amounts of template DNA. The pattern of read coverage was reproducibly obtained for samples with higher amounts of template DNA, suggesting that the bias is non-random and genome-specific. A re-analysis of previously published sequence data obtained after amplification from clonal endosymbiont populations confirmed these predictions. We conclude that many of the artifacts associated with the use of the multiple displacement amplification method can be alleviated or much reduced by using multiple cells as the template for the amplification. These findings should be particularly useful for researchers studying the genomes of endosymbionts and other uncultured bacteria, for which a small clonal population of cells can be isolated.     

Comparison of Multiple Displacement Amplification (MDA) and Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) in Single-Cell Sequencing

Single-cell sequencing promotes our understanding of the heterogeneity of cellular populations, including the haplotypes and genomic variability among different generation of cells. Whole-genome amplification is crucial to generate sufficient DNA fragments for single-cell sequencing projects. Using sequencing data from single sperms, we quantitatively compare two prevailing amplification methods that extensively applied in single-cell sequencing, multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Our results show that MALBAC, as a combination of modified MDA and tweaked PCR, has a higher level of uniformity, specificity and reproducibility.     

Amplification of a bovine papillomavirus-simian virus 40 chimera.

A chimeric plasmid, pBOP, containing bovine papillomavirus (BPV) and the origin of replication from simian virus 40 (SV40) was constructed. The plasmid was established in mouse cells, where it was maintained stably as an autonomous BPV replicon. Lines carrying pBOP were fused to cells of COS-7, a simian line producing SV40 T antigen. Replication dependent on the SV40 origin and having the kinetics and approximate amplitude of an SV40 infection ensued. SV40 replication is therefore dominant over BPV replication, and the SV40 origin can conveniently be used to amplify lower-copy-number plasmids in mammalian cells.     

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.     

MDA技术在转基因检测中的研究与应用

PCR技术是转基因植物及其产品检测的主要方法,但是因为其对模板纯度和质量有较高要求,对于低质量模板难以获得稳定的检测结果。本研究利用MDA技术可将低至10 copies的微量DNA样品扩增至可用PCR方法稳定检出;进一步利用MDA技术对转基因玉米有证标准物质Bt11粉末的单颗粒痕量样品释出DNA进行扩增,结合PCR扩增检测,玉米粉末颗粒中内源参照基因的检出率达到70%以上,转基因特异性序列检出与内源参照基因检出的比值,与标准物质标称值基本相符,为建立基于MDA技术转基因植物及其产品粉末颗粒等痕量样品检测方法奠定了基础,同时也为加工产品中复合性状转基因作物的检测提供了潜在的解决方案。     

Multiple steps during the formation of beta-lactoglobulin fibrils

In this study, the heat induced fibrilar aggregation of the whey protein beta-lactoglobulin is investigated at low pH and at low ionic strength. Under these circumstances, tapping mode atomic force microscopy results indicate that the fibrils formed have a periodic structure with a period of about 25 nm and a thickness of one or two protein monomers. Fibril formation is followed in situ using light scattering and proton NMR techniques. The dynamic light scattering results show that the fibrils that form after short heating periods (up to a few hours) disintegrate upon slow cooling, whereas fibrils that form during long heating periods do not disintegrate upon subsequent slow cooling. The NMR results show that even after prolonged heating an appreciable fraction of the protein molecules is incorporated into fibrils only when the beta-lactoglobulin concentration is above approximately 2.5 wt %. The data imply multiple steps during the heat induced formation of beta-lactoglobulin fibrils at low pH and at low ionic strength: (partly) denatured protein monomers are either incorporated into fibrils or form instead a low molecular weight complex that is incapable of forming fibrils. Fibril formation itself also involves (at least) two steps: the reversible formation of linear aggregates, followed by a slow process of "consolidation" after which the fibrils no longer disintegrate upon slow cooling.     

Multiple Displacement Amplification for Generating an Unlimited Source of DNA for Genotyping in Nonhuman Primate Species

We evaluated a whole genome amplification method—multiple displacement amplification (MDA)—as a means to conserve valuable nonhuman primate samples. We tested 148 samples from a variety of species and sample sources, including blood, tissue, cell-lines, plucked hair and noninvasively collected semen. To evaluate genotyping success and accuracy of MDA, we used routine genotyping methods, including short tandem repeat (STR) analysis, denaturing gradient gel electrophoresis (DGGE), Alu repeat analysis, direct sequencing, and nucleotide detection by tag-array minisequencing. We compared genotyping results from MDA products to genotypes generated from the original (non-MD amplified) DNA samples. All genotyping methods showed good results with the MDA products as a DNA template, and for some samples MDA improved genotyping success. We show that the MDA procedure has the potential to provide a long-lasting source of DNA for genetic studies, which would be highly valuable for the primate research field, in which genetic resources are limited and for other species in which similar sampling constraints apply.     

Pathfinding during spinal tract formation in the chick-quail chimera analysed by species-specific monoclonal antibodies.

In order to analyse the spinal tract formation at early stages of development in avian embryos, chick-quail spinal cord chimeras were prepared and species-specific monoclonal antibodies (MAb) were developed. MAbs CN, QN and CQN uniquely stained chick, quail, and both chick and quail nervous tissues, respectively. All three antibodies appeared to bind to the same membrane molecule, but to different epitopes. Cord reversal revealed the features of axonal growth of both cord interneurons and dorsal root ganglion cells. Quail cord interneurons grew along an originally ventral marginal layer in the quail cord transplanted in a reversed position, then turned toward the ventral side at the boundary between the graft and the host, and grew along the host chick ventral marginal layer. Central axons of dorsal root ganglia were restricted to the ventrolateral region of the cord which originally formed the dorsal funiculus. These results suggest that cord interneurons and dorsal root ganglion cells actively select to grow along specific regions of the cord and that spinal tract formation appears to be determined by cord cells, and not by sclerotome cells.     

Receptor Displacement in the Cell Membrane by Hydrodynamic Force Amplification through Nanoparticles

We introduce an intrinsically multiplexed and easy to implement method to apply an external force to a biomolecule and thus probe its interaction with a second biomolecule or, more generally, its environment (for example, the cell membrane). We take advantage of the hydrodynamic interaction with a controlled fluid flow within a microfluidic channel to apply a force. By labeling the biomolecule with a nanoparticle that acts as a kite and increases the hydrodynamic interaction with the fluid, the drag induced by convection becomes important. We use this approach to track the motion of single membrane receptors, the Clostridium perfringens ε-toxin (CPεT) receptors that are confined in lipid raft platforms, and probe their interaction with the environment. Under external force, we observe displacements over distances up to 10 times the confining domain diameter due to elastic deformation of a barrier and return to the initial position after the flow is stopped. Receptors can also jump over such barriers. Analysis of the receptor motion characteristics before, during, and after a force is applied via the flow indicates that the receptors are displaced together with their confining raft platform. Experiments before and after incubation with latrunculin B reveal that the barriers are part of the actin cytoskeleton and have an average spring constant of 2.5 ± 0.6 pN/μm before vs. 0.6 ± 0.2 pN/μm after partial actin depolymerization. Our data, in combination with our previous work demonstrating that the ε-toxin receptor confinement is not influenced by the cytoskeleton, imply that it is the raft platform and its constituents rather than the receptor itself that encounters and deforms the barriers formed by the actin cytoskeleton.     

Course of pH during the formation of amoxicillin by a suspension-to-suspension reaction

Amoxicillin can be produced in an enzymatic suspension-to-suspension reaction in which the substrate(s) and product(s) are mainly present as solid particles, while the reaction takes place in the liquid phase. During these suspension-to-suspension reactions different subprocesses take place, such as dissolution/crystallization of substrates and products, enzymatic synthesis of the product(s), and undesired enzymatic hydrolysis of substrates and/or products. All these subprocesses are influenced by pH and also influence the pH because the reactants are weak electrolytes. This paper describes a quantitative model for predicting pH and concentrations of reactants during suspension-to-suspension reactions. The model is based on mass and charge balances, pH-dependent solubilities of the reactants, and enzyme kinetics. For the validation of this model, the kinetically controlled synthesis of amoxicillin from 6-aminopenicillanic acid and D-(p)hydroxyphenylglycine methyl ester was studied. The pH and the dissolved concentrations took a very different course at different initial substrate amounts. This was described quite reasonably by the model. Therefore, the model can be used as a tool to optimize suspension-to-suspension reactions of weak electrolytes.     

Mechanism of self-regulation and the inotropic reaction of the rat myocardium during aging

The experiments on rat papillary muscles revealed that in ageing rats myocardium has a decreased distension ability. The curves of shortening value/length and force/velocity for the myocardium of old animals are shifted downwards. The alterations in isotonic contraction parameters had a distinct age differentiation, while the age did not affect the inotropic effects of increased frequency, paired stimulation and external calcium. It is suggested that changes in biomechanical properties of ageing myocardium are associated with alterations in calcium transport in sarcoplasmic reticulum.     

Quantifying the Displacement of Mismatches in Multiple Sequence Alignment Benchmarks

Multiple Sequence Alignment (MSA) methods are typically benchmarked on sets of reference alignments. The quality of the alignment can then be represented by the sum-of-pairs (SP) or column (CS) scores, which measure the agreement between a reference and corresponding query alignment. Both the SP and CS scores treat mismatches between a query and reference alignment as equally bad, and do not take the separation into account between two amino acids in the query alignment, that should have been matched according to the reference alignment. This is significant since the magnitude of alignment shifts is often of relevance in biological analyses, including homology modeling and MSA refinement/manual alignment editing. In this study we develop a new alignment benchmark scoring scheme, SPdist, that takes the degree of discordance of mismatches into account by measuring the sequence distance between mismatched residue pairs in the query alignment. Using this new score along with the standard SP score, we investigate the discriminatory behavior of the new score by assessing how well six different MSA methods perform with respect to BAliBASE reference alignments. The SP score and the SPdist score yield very similar outcomes when the reference and query alignments are close. However, for more divergent reference alignments the SPdist score is able to distinguish between methods that keep alignments approximately close to the reference and those exhibiting larger shifts. We observed that by using SPdist together with SP scoring we were able to better delineate the alignment quality difference between alternative MSA methods. With a case study we exemplify why it is important, from a biological perspective, to consider the separation of mismatches. The SPdist scoring scheme has been implemented in the VerAlign web server (http://www.ibi.vu.nl/programs/veralignwww/). The code for calculating SPdist score is also available upon request.