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MIDAS. (a) Each slide contains 16 arrays of 255 microwells each. Cells, lysis solution, denaturing buffer, neutralization buffer and MDA master mix were each added to the microwells with a single pipette pump. Amplicon growth was then visualized with a fluorescent microscope using a real-time MDA system. Microwells showing increasing fluorescence over time were positive amplicons. The amplicons were extracted with fine glass pipettes attached to a micromanipulation system. (b) Scanning electron microscopy of a single E. coli cell displayed at different magnifications. This particular well contains only one cell, and most wells observed also contained no more than one cell. (c) A custom microscope incubation chamber was used for real time MDA. The chamber was temperature and humidity controlled to mitigate evaporation of reagents. Additionally, it prevented contamination during amplicon extraction because the micromanipulation system was self-contained. An image of the entire microwell array is also shown, as well as a micropipette probing a well. (d) Complex three-dimensional MDA amplicons were reduced to linear DNA using DNA polymerase I and Ampligase. This process substantially improved the complexity of the library during sequencing.
Source publication
Genome sequencing of single cells has a variety of applications, including characterizing difficult-to-culture microorganisms and identifying somatic mutations in single cells from mammalian tissues. A major hurdle in this process is the bias in amplifying the genetic material from a single cell, a procedure known as polymerase cloning. Here we des...
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... The format of the arrays, including well size, pattern and spacing, was optimized to achieve efficient cell loading, optimal amplification yield and convenient DNA extraction. Each slide consisted of 16 arrays, each containing 255 microwells of 400 μm in diameter, allowing for parallel amplification of 16 separate heterogeneous cell populations (Fig. 1a). All liquid handling procedures (cell seeding, lysis, DNA denaturation, neutralization and addition of amplification master mix) required one pump of a pipette per step per array, minimizing the labor required for hundreds of amplification reactions. This system requires less of each amplification and library construction reagent than ...
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... the 1 cell per 10 well case, no more than 0.5% of the wells should contain more than 1 cell. We confirmed that the cells were indeed being seeded at the expected distribution using fluorescent microscopy after staining cells with SYBR Green I ( Supplementary Fig. 1). We thus decided to load cells at a density of 1 cell per 10 wells, ensuring that 99.5% of generated amplicons would arise from a single cell. ...
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... ensuring that 99.5% of generated amplicons would arise from a single cell. The remaining empty wells served as internal negative controls, allowing easy detection and elimination of contaminated samples. We further confirmed proper microbial and mammalian cell seeding in microwells at the 1 cell per 10 well level by scanning electron microscopy (Fig. 1b, Supplementary Fig. ...
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... seeding of cell populations into each microwell array, we performed limited multiple displacement amplification on the seeded single cells in the partitioned microwells, each with a physically separated (save for a thin aqueous layer atop the arrays) volume of ~12 nL, in a temperature and humidity controlled chamber (Fig. 1c, Supplementary Fig. 1). We used SYBR Green I to visualize the amplicons growing using an epifluorescent microscope ( Supplementary Fig. 3). A random distribution of amplicons across the arrays was observed with ~10% of the wells containing amplicons, further confirming the parallel and localized amplification within individual microwells as well as the ...
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... the arrays was observed with ~10% of the wells containing amplicons, further confirming the parallel and localized amplification within individual microwells as well as the stochastic seeding of single cells 19 . After amplification in the microwells, we used a micromanipulation system to extract amplicons from individual wells for sequencing (Fig. 1c). We estimated that the masses of the extracted amplicons ranged from 500 picograms to 3 ...
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... to generate high-complexity libraries from MDA amplicons, resulting in poor genomic coverage (data not shown). To address this issue, we used random hexamers and DNA Polymerase I to first convert the hyperbranched amplicons into unbranched double- stranded DNA molecules, which allowed effective library construction using in vitro transposition (Fig. 1d). In addition, we used a small reaction volume to further increase the efficiency of library construction 20 ...
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Lander-Waterman’s coverage bound establishes the total number of reads required to cover the whole genome of size G bases. In fact, their bound is a direct consequence of the well-known solution to the coupon collector’s problem which proves that for such genome, the total number of bases to be sequenced should be O(G ln G). Although the result lea...
Citations
... Several approaches have been developed to fulfill these requirements based on diverse immobilizing technologies, including active optical, acoustic, and electrical fields, [4][5][6][7][8][9] as well as passive hydrodynamic/mechanical constrictions and interface microengineering. [10][11][12][13][14][15] The combination of most tools above with microfluidics enhances the controllability and throughput of microscale cell capture during the manipulation process and has great potential in searching for novel and pioneering insights into single-cell omics. [16,17] Despite significant advances, the current approaches still have several challenges to overcome. ...
Methodological improvement to single‐cell manipulation is critical for exploring the fundamentals of cellular life and unraveling biological complexity. Although micro‐manipulation technologies capable of precise cell localization have been widely established, scaling existing platforms for highly efficient single‐cell immobilization without sacrificing cell viability and sample quantity has proven challenging. Here, a highly efficient single‐cell trapping and arraying approach is introduced by advancing the performance of a microfluidic mechanical trapping chip. The chip can achieve representative single‐cell capture with over 99% efficiency and at least a 75% success rate of perfect capture, a precisely controlled single‐cell array, absolute sequential cell captures without cell loss, and the maintenance of high cell viability during the whole manipulation process. This approach enables diverse single‐cell trapping, large‐scale arraying manipulations, and dynamic cellular and molecular analysis, and offers a path toward the development of high‐performance single‐cell systems.
... Megabase-scale copy number variants (CNVs) can be detected using read-depth methods even with one or a few million short reads [6][7][8] . Several studies of human single cortical neurons showed Megabase-scale CNVs, although the precise frequency of these changes remains unclear 7,[9][10][11][12] . These CNVs may be more frequent in younger than aged healthy brains 7,8 , arising in embryonic neurogenesis in mouse 8 , with complex structural variants (SVs) also arising in human neurogenesis 13 . ...
... We note several previous comparisons of WGA methods, with the broad consensus being that MDA is not suitable for calling CNVs by read depth, as there is bias due to over-amplification of certain regions, although it is more accurate at the single-base level [17][18][19][20][21][22][23] . There have been several attempts to reduce MDA amplification bias by performing reactions in nanoliter-scale volumes 10,24,25 . MDA performed after the single-cell genome is partitioned into~50,000 droplets (droplet MDA, or dMDA) in the Samplix X-Drop device was reported to yield more even amplification 26 . ...
The presence of somatic mutations, including copy number variants (CNVs), in the brain is well recognized. Comprehensive study requires single-cell whole genome amplification, with several methods available, prior to sequencing. Here we compare PicoPLEX with two recent adaptations of multiple displacement amplification (MDA): primary template-directed amplification (PTA) and droplet MDA, across 93 human brain cortical nuclei. We demonstrate different properties for each, with PTA providing the broadest amplification, PicoPLEX the most even, and distinct chimeric profiles. Furthermore, we perform CNV calling on two brains with multiple system atrophy and one control brain using different reference genomes. We find that 20.6% of brain cells have at least one Mb-scale CNV, with some supported by bulk sequencing or single-cells from other brain regions. Our study highlights the importance of selecting whole genome amplification method and reference genome for CNV calling, while supporting the existence of somatic CNVs in healthy and diseased human brain.
... Furthermore, these iPS-derived neurons had larger CNVs in their genome than those of fibroblasts from which the iPS cells were induced, suggesting that these mutations occurred in each neuron during the cell differentiation process. Although aneuploidy reported in mouse neurons tends to be fewer in human neurons, the occurrence of large-scale mutations was reported in 13%-41% of the cortical neurons in the human brain (Cai et al., 2014;Gole et al., 2013;McConnell et al., 2013). Consistent with CNVs in neurons examined by this iPSC study, 45 neurons had 2.5-75 megabases CNVs from 110 neurons dissected from three postmortem human brains (McConnell et al., 2013). ...
Neurons born during the fetal period have extreme longevity and survive until the death of the individual because the human brain has highly limited tissue regeneration. The brain is comprised of an enormous variety of neurons each exhibiting different morphological and physiological characteristics and recent studies have further reported variations in their genome including chromosomal abnormalities, copy number variations, and single nucleotide mutations. During the early stages of brain development, the increasing number of neurons generated at high speeds has been proposed to lead to chromosomal instability. Additionally, mutations in the neuronal genome can occur in the mature brain. This observed genomic mosaicism in the brain can be produced by multiple endogenous and environmental factors and careful analyses of these observed variations in the neuronal genome remain central for our understanding of the genetic basis of neurological disorders.
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... The primary workflow of this technique involves several key steps: isolation of single cells, whole-genome amplification, detection of the amplified genome, error correction to minimize potential inaccuracies, and determination of the genetic relationships among individual cells (38). In response to the need for the isolation of individual cells, various methods have been developed, including microfluidic and bead-based methods (39) and microwell dilution (40). Currently, using droplets or micromechanical valves in microfluidic device has become more prevalent (41,42). ...
Tuberculosis is a major infectious disease caused by Mycobacterium tuberculosis infection. The pathogenesis and immune mechanism of tuberculosis are not clear, and it is urgent to find new drugs, diagnosis, and treatment targets. A useful tool in the quest to reveal the enigmas related to Mycobacterium tuberculosis infection and disease is the single-cell sequencing technique. By clarifying cell heterogeneity, identifying pathogenic cell groups, and finding key gene targets, the map at the single cell level enables people to better understand the cell diversity of complex organisms and the immune state of hosts during infection. Here, we briefly reviewed the development of single-cell sequencing, and emphasized the different applications and limitations of various technologies. Single-cell sequencing has been widely used in the study of the pathogenesis and immune response of tuberculosis. We review these works summarizing the most influential findings. Combined with the multi-molecular level and multi-dimensional analysis, we aim to deeply understand the blank and potential future development of the research on Mycobacterium tuberculosis infection using single-cell sequencing technology.
... However, only a few high AUCs showing singlecell data exhibited reliable CNA calls ( Supplementary Fig. 5c), and most single-cell MDA products exhibited high false-positive CNA detections compared to bulk CNA profiles. Single-cell bMDA, MDA, and MDA kit groups showed high false-positive CNA calls; thus, the false detections were mainly due to a fundamental limitation of MDA-based chemistry 38,39 , rather than the modified primer in bMDA. ...
Determining mutational landscapes in a spatial context is essential for understanding genetically heterogeneous cell microniches. Current approaches, such as Multiple Displacement Amplification (MDA), offer high genome coverage but limited multiplexing, which hinders large-scale spatial genomic studies. Here, we introduce barcoded MDA (bMDA), a technique that achieves high-coverage genomic analysis of low-input DNA while enhancing the multiplexing capabilities. By incorporating cell barcodes during MDA, bMDA streamlines library preparation in one pot, thereby overcoming a key bottleneck in spatial genomics. We apply bMDA to the integrative spatial analysis of triple-negative breast cancer tissues by examining copy number alterations, single nucleotide variations, structural variations, and kataegis signatures for each spatial microniche. This enables the assessment of subclonal evolutionary relationships within a spatial context. Therefore, bMDA has emerged as a scalable technology with the potential to advance the field of spatial genomics significantly.
... We note several previous comparisons of WGA methods, with the broad consensus being that MDA is not suitable for calling CNVs by read depth, as there is bias due to over-amplification of certain regions, although it is more accurate at the single-base level [17][18][19][20][21][22][23] . There have been several attempts to reduce MDA amplification bias by performing reactions in nanoliter-scale volumes 10,24,25 . MDA performed after the single-cell genome is partitioned into ~50,000 droplets (droplet MDA, or dMDA) in the Samplix X-Drop device was reported to yield more even amplification 26 . ...
The presence of somatic mutations, including copy number variants (CNVs), in the brain is well recognized. Comprehensive study requires single-cell whole genome amplification, with several methods available, prior to sequencing. We compared PicoPLEX with two recent adaptations of multiple displacement amplification (MDA): primary template-directed amplification (PTA) and droplet MDA, across 93 human brain cortical nuclei. We demonstrated different properties for each, with PTA providing the broadest amplification, PicoPLEX the most even, and distinct chimeric profiles. Furthermore, we performed CNV calling on two brains with multiple system atrophy and one control brain using different reference genomes. We found that 38% of brain cells have at least one Mb-scale CNV, with some supported by bulk sequencing or single-cells from other brain regions. Our study highlights the importance of selecting whole genome amplification method and reference genome for CNV calling, while supporting the existence of somatic CNVs in healthy and diseased human brain.
... The cell count was less than one cell in ≈90% of the beads, indicating that the beaded fibers could be used for cloning. Compared to the conventional use of microwells in cloning, [27] hydrogel fibers do not require special equipment. In addition, they are easily handled because single cells can be encapsulated in beads and hardened using Ca-alginate hydrogels. ...
Core‐shell hydrogel fibers are widely used in cell culture applications. A simple and rapid method is presented for fabricating core‐shell hydrogel fibers, consisting of straight or beaded core fibers, for cell culture applications. The core fibers are prepared using interfacial polyelectrolyte complexation (IPC) with chitosan and DNA. Briefly, two droplets of chitosan and DNA are brought in contact to form an IPC film, which is dragged to prepare an IPC fiber. The incubation time and DNA concentration are adjusted to prepare straight and beaded IPC fibers. The fibers with Ca²⁺ are immersed in an alginate solution to form calcium alginate shell hydrogels around the core IPC fibers. To the best of the knowledge, this is the first report of core‐shell hydrogel fibers with IPC fiber cores. To demonstrate cell culture, straight hydrogel fibers are applied to fabricate hepatic models consisting of HepG2 and 3T3 fibroblasts, and vascular models consisting of human umbilical vein endothelial cells and 3T3 fibroblasts. To evaluate the effect of co‐culture, albumin secretion, and angiogenesis are evaluated. Beaded hydrogel fibers are used to fabricate many size‐controlled spheroids for fiber and cloning applications. This method can be widely applied in tissue engineering and cell analysis.
... Among these methods, MDA using phi29 DNA polymerase (phi29DNApol) [30,31] and random primers [32] is the most commonly used method nowadays, generating sufficient quantities of replicated DNA with high fidelity, less amplification bias and large fragment size. Researchers have also developed improved MDA methods to further optimize the performance, including microwell MDA [33], emulsion MDA (eMDA) [34] and microchannel MDA (μcMDA) [35,36]. However, chimeric sequences (i.e. ...
Motivation:
Multiple displacement amplification (MDA) has become the most commonly used method of whole genome amplification, generating a vast amount of DNA with higher molecular weight and greater genome coverage. Coupling with long-read sequencing, it is possible to sequence the amplicons of over 20 kb in length. However, the formation of chimeric sequences (chimeras, expressed as structural errors in sequencing data) in MDA seriously interferes with the bioinformatics analysis but its influence on long-read sequencing data is unknown.
Results:
We sequenced the phi29 DNA polymerase-mediated MDA amplicons on the PacBio platform and analyzed chimeras within the generated data. The 3rd-ChimeraMiner has been constructed as a pipeline for recognizing and restoring chimeras into the original structures in long-read sequencing data, improving the efficiency of using TGS data. Five long-read datasets and one high-fidelity long-read dataset with various amplification folds were analyzed. The result reveals that the mis-priming events in amplification are more frequently occurring than widely perceived, and the propor tion gradually accumulates from 42% to over 78% as the amplification continues. In total, 99.92% of recognized chimeric sequences were demonstrated to be artifacts, whose structures were wrongly formed in MDA instead of existing in original genomes. By restoring chimeras to their original structures, the vast majority of supplementary alignments that introduce false-positive structural variants are recycled, removing 97% of inversions on average and contributing to the analysis of structural variation in MDA-amplified samples. The impact of chimeras in long-read sequencing data analysis should be emphasized, and the 3rd-ChimeraMiner can help to quantify and reduce the influence of chimeras.
Availability and implementation:
The 3rd-ChimeraMiner is available on GitHub, https://github.com/dulunar/3rdChimeraMiner.
... Furthermore, this approach also significantly reduces the high costs of WGA (Table 1). Previous studies have applied this approach at sub-nanoliter (nL) and picoliter (pL) volumes in microfluidic devices [38,40,[44][45][46][47][48], nanowells [49,50], planar surfaces [51,52], and hydrogels [53], which are compared in detail in Figure 2. Many of these approaches and their devices remain largely unused outside of their respective publications, likely because most microfluidic chips and other platforms are not commercially available; they require complex fabrication and operation [54,55], and are therefore hard to access and implement in other research groups. ...
... Here, we sorted single E. coli cells into 384-well plates to compare SAG amplification bias within total MDA microliter and sub-microliter reactions for the first time. In contrast, previous studies have largely examined volume reduction in the sub-nanoliter to picoliter range [38,44,45,47,[49][50][51]65]. ...
... Furthermore, the issue of lower template specificity also explains why there was an observed trend that larger reaction volumes had more contaminant reads removed after filtering than the smaller reactions ( Figure 4C). Lower specificity, leading to more contamination, is likely due to the increased competition between background contamination and the E. coli single-cell DNA [40,49]. This increase in contamination was also reflected in the higher amplification gain and product yield mentioned previously ( Figure 3A,B), which other studies reported as well [45,47,49]. ...
Microbial single-cell genomics (SCG) provides access to the genomes of rare and uncultured microorganisms and is a complementary method to metagenomics. Due to the femtogram-levels of DNA in a single microbial cell, sequencing the genome requires whole genome amplification (WGA) as a preliminary step. However, the most common WGA method, multiple displacement amplification (MDA), is known to be costly and biased against specific genomic regions, preventing high-throughput applications and resulting in uneven genome coverage. Thus, obtaining high-quality genomes from many taxa, especially minority members of microbial communities, becomes difficult. Here, we present a volume reduction approach that significantly reduces costs while improving genome coverage and uniformity of DNA amplification products in standard 384-well plates. Our results demonstrate that further volume reduction in specialized and complex setups (e.g., microfluidic chips) is likely unnecessary to obtain higher-quality microbial genomes. This volume reduction method makes SCG more feasible for future studies, thus helping to broaden our knowledge on the diversity and function of understudied and uncharacterized microorganisms in the environment.
... Additionally, Phi29 polymerase has both high proofreading activity and strand displacement activity, whereas other DNA polymerases have only strong strand displacement activity (such as Bst and Klenow) or only strong proofreading activity (such as T4). The DNA amplified by MDA has a higher molecular weight (up to 100 kb) and better genome coverage than the output of other scWGA methods [3,11,23,[28][29][30][31]. To further improve the performance of MDA, researchers have developed some customized strategies [32], including microwell MDA (MIDAS) [33], emulsion MDA (eMDA) [34], and microchannel MDA (μcMDA) [35,36]. ...
Multiple displacement amplification (MDA) based on isothermal random priming and high fidelity phi29 DNA polymerase-mediated processive extension has revolutionized the field of whole genome amplification by enabling the amplification of minute amounts of DNA, such as from a single cell, generating vast amounts of DNA with high genome coverage. Despite its advantages, MDA has its own challenges, one of the grandest being the formation of chimeric sequences (chimeras), which presents in all MDA products and seriously disturbs the downstream analysis. In this review, we provide a comprehensive overview of current research on MDA chimeras. We first reviewed the mechanisms of chimera formation and chimera detection methods. We then systematically summarized the characteristics of chimeras, including overlap, chimeric distance, chimeric density, and chimeric rate, as found in independently published sequencing data. Finally, we reviewed the methods used to process chimeric sequences and their impacts on the improvement of data utilization efficiency. The information presented in this review will be useful for those interested in understanding the challenges with MDA and in improving its performance.
