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Comparison of multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) in single-cell sequencing.

ABSTRACT: 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.

SUBMITTER: Chen M

PROVIDER: S-EPMC4259343 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Comparison of multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) in single-cell sequencing.

Chen Minfeng M Song Pengfei P Zou Dan D Hu Xuesong X Zhao Shancen S Gao Shengjie S Ling Fei F

PloS one 20141208 12

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 mul ...[more]

PMID: 25485707

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Multiple annealing and looping based amplification cycles for digital transcriptomics (MALBAC-DT) of single cells from the developing mouse cortex and hippocampus

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2021-01-07 | GSE162509 | GEO

A low cost and input tailing method of quality control on multiple annealing, and looping-based amplification cycles-based whole-genome amplification products.

Project description:BackgroundSingle-cell whole-genome sequencing provides novel insights into the nature of genetic heterogeneity in normal and diseased cells. However, amplification of formalin-fixed tissues with low cell numbers is still problematic and multiple annealing, and looping-based amplification cycles (MALBAC) is a commonly used whole-genome amplification (WGA) method with low cell numbers.MethodsWe developed a low-input tailing method to evaluate the MALBAC-based WGA from sub-nanogram or less quantities of input DNA. The tailing method uses 2100 BioAnalyzer to evaluate the size distribution of MALBAC products, and comparing the tailing with 10380 bp.ResultsCompared with a 22 loci qPCR panel, the tailing method provided a similar WGA evaluation efficiency in 13 samples on one set of study, with lower input, cheaper cost, shorter manual time, and a clear filtering cut off. Later, we demonstrated a strong correlation between tailing size and coverage breadth in another 29 samples on two sets of assays. As a result, the tailing method showed that it could predict whether a sequence breadth achieved 70% or not with 100% accuracy on these three sets of assays. Although further studies are needed, this tailing method is expected to be used as an excellent tool to select high-quality WGA products before library construction.ConclusionsOur tailing method can provide a new WGA quality test to evaluate the WGA efficiency with 100% accuracy (42/42). Compared with qPCR panel, our tailing method needs lower input, cheaper cost, shorter manual time, a clear filtering cut off, and extendable high throughput as well as the same sensitivity.

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Enhanced sequencing coverage with digital droplet multiple displacement amplification.

Project description:Sequencing small quantities of DNA is important for applications ranging from the assembly of uncultivable microbial genomes to the identification of cancer-associated mutations. To obtain sufficient quantities of DNA for sequencing, the small amount of starting material must be amplified significantly. However, existing methods often yield errors or non-uniform coverage, reducing sequencing data quality. Here, we describe digital droplet multiple displacement amplification, a method that enables massive amplification of low-input material while maintaining sequence accuracy and uniformity. The low-input material is compartmentalized as single molecules in millions of picoliter droplets. Because the molecules are isolated in compartments, they amplify to saturation without competing for resources; this yields uniform representation of all sequences in the final product and, in turn, enhances the quality of the sequence data. We demonstrate the ability to uniformly amplify the genomes of single Escherichia coli cells, comprising just 4.7 fg of starting DNA, and obtain sequencing coverage distributions that rival that of unamplified material. Digital droplet multiple displacement amplification provides a simple and effective method for amplifying minute amounts of DNA for accurate and uniform sequencing.

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Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification.

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Single cell transcriptome amplification with MALBAC.

Project description:Recently, Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) has been developed for whole genome amplification of an individual cell, relying on quasilinear instead of exponential amplification to achieve high coverage. Here we adapt MALBAC for single-cell transcriptome amplification, which gives consistently high detection efficiency, accuracy and reproducibility. With this newly developed technique, we successfully amplified and sequenced single cells from 3 germ layers from mouse embryos in the early gastrulation stage, and examined the epithelial-mesenchymal transition (EMT) program among cells in the mesoderm layer on a single-cell level.

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Genome amplification of single sperm using multiple displacement amplification.

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Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA Samples.

Project description:Multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.

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Structural alterations from multiple displacement amplification of a human genome revealed by mate-pair sequencing.

Project description:Comprehensive identification of the acquired mutations that cause common cancers will require genomic analyses of large sets of tumor samples. Typically, the tissue material available from tumor specimens is limited, which creates a demand for accurate template amplification. We therefore evaluated whether phi29-mediated whole genome amplification introduces false positive structural mutations by massive mate-pair sequencing of a normal human genome before and after such amplification. Multiple displacement amplification led to a decrease in clone coverage and an increase by two orders of magnitude in the prevalence of inversions, but did not increase the prevalence of translocations. While multiple strand displacement amplification may find uses in translocation analyses, it is likely that alternative amplification strategies need to be developed to meet the demands of cancer genomics.

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