Abstract:Single-cell whole-genome sequencing (WGS) is critical for characterizing dynamic intercellular changes in DNA. Current sample preparation technologies for single-cell WGS are complex, expensive, and suffer from high amplification bias and errors. Here, we describe Digital-WGS, a sample preparation platform that streamlines high-performance single-cell WGS with automatic processing based on digital microfluidics. Using the method, we provide high single-cell capture efficiency for any amount and types of cells … Show more
“…However, WGA can introduce an amplification bias [39] and any microbial or cross-contamination among samples could also be magnified [40]. To solve these issues, WGA performed in sub-nanolitre droplets could significantly reduce the amplification bias and contamination [41,42] as this technique reduces competition for the primers and polymerases among the DNA fragments via the partitioning of the template DNA into tiny individual spaces. For example, a study by Shi et al [12] utilised a microfluidic chip to separate microorganisms in samples and amplify their DNA in droplets to efficiently recover the microbial genome with a markedly reduced amplification bias.…”
Section: Removal Of the Host Cells Before Dna Extractionmentioning
Whole genome metagenomic sequencing is a powerful platform enabling the simultaneous identification of all genes from entirely different kingdoms of organisms in a complex sample. This technology has revolutionised multiple areas from microbiome research to clinical diagnoses. However, one of the major challenges of a metagenomic study is the overwhelming non-microbial DNA present in most of the host-derived specimens, which can inundate the microbial signals and reduce the sensitivity of microorganism detection. Various host DNA depletion methods to facilitate metagenomic sequencing have been developed and have received considerable attention in this context. In this review, we present an overview of current host DNA depletion approaches along with explanations of their underlying principles, advantages and disadvantages. We also discuss their applications in laboratory microbiome research and clinical diagnoses and, finally, we envisage the direction of the further perfection of metagenomic sequencing in samples with overabundant host DNA.
“…However, WGA can introduce an amplification bias [39] and any microbial or cross-contamination among samples could also be magnified [40]. To solve these issues, WGA performed in sub-nanolitre droplets could significantly reduce the amplification bias and contamination [41,42] as this technique reduces competition for the primers and polymerases among the DNA fragments via the partitioning of the template DNA into tiny individual spaces. For example, a study by Shi et al [12] utilised a microfluidic chip to separate microorganisms in samples and amplify their DNA in droplets to efficiently recover the microbial genome with a markedly reduced amplification bias.…”
Section: Removal Of the Host Cells Before Dna Extractionmentioning
Whole genome metagenomic sequencing is a powerful platform enabling the simultaneous identification of all genes from entirely different kingdoms of organisms in a complex sample. This technology has revolutionised multiple areas from microbiome research to clinical diagnoses. However, one of the major challenges of a metagenomic study is the overwhelming non-microbial DNA present in most of the host-derived specimens, which can inundate the microbial signals and reduce the sensitivity of microorganism detection. Various host DNA depletion methods to facilitate metagenomic sequencing have been developed and have received considerable attention in this context. In this review, we present an overview of current host DNA depletion approaches along with explanations of their underlying principles, advantages and disadvantages. We also discuss their applications in laboratory microbiome research and clinical diagnoses and, finally, we envisage the direction of the further perfection of metagenomic sequencing in samples with overabundant host DNA.
“…[23][24][25] Labelling specic single cells lysed in droplets with subsequent droplet breakup and RNA sequencing has achieved successful genomic proling with single cell resolution. [26][27][28][29][30] However, it requires a high cost and increases the operation complications. 31,32 Isothermal enzymatic ampli-cation 33 and DNA cascade hybridization 34,35 amplication strategies have been directly performed in the picoliter volume of water-in-oil droplets.…”
Simultaneous quantification of multi-miRNAs in single-cell reveals cellular heterogeneity, and benefits cancer cells subtype discrimination. Though micro-droplet techniques enable successful single cell encapsulation, the isolated and restricted reaction space of...
“… Fig. 8 Other platforms for single-cell nucleic acid analysis: A centrifugal disk chip for single-cell gene expression analysis (Furutani et al 2012 ), B digital microfluidic chip for whole genome sequencing (Ruan et al 2020 ) …”
Section: Platforms and Integrated Systems For Single-cell Nucleic Acid Analysismentioning
confidence: 99%
“…Another emerging hybrid platform for single-cell nucleic acid analysis is digital microfluidics (DMF) that can manipulate droplets by electrowetting-on-dielectric (EWOD) mechanism. By controlling the droplets of cell samples and different reagents required in MDA, Ruan et al ( 2020 ) achieved single-cell genomic detection of CNVs with minimal bin of 150-kb and SNVs with allele dropout rate of 5.2%. The schematics of DMF on-chip operation process and single-cell WGA analysis procedure was shown in Fig.…”
Section: Platforms and Integrated Systems For Single-cell Nucleic Acid Analysismentioning
Single-cell nucleic acid analysis aims at discovering the genetic differences between individual cells which is well known as the cellular heterogeneity. This technology facilitates cancer diagnosis, stem cell research, immune system analysis, and other life science applications. The conventional platforms for single-cell nucleic acid analysis more rely on manual operation or bulky devices. Recently, the emerging microfluidic technology has provided a perfect platform for single-cell nucleic acid analysis with the characteristic of accurate and automatic single-cell manipulation. In this review, we briefly summarized the procedure of single-cell nucleic acid analysis including single-cell isolation, single-cell lysis, nucleic acid amplification, and genetic analysis. And then, three representative microfluidic platforms for single-cell nucleic acid analysis are concluded as valve-, microwell-, and droplet-based platforms. Furthermore, we described the state-of-the-art integrated single-cell nucleic acid analysis systems based on the three platforms. Finally, the future development and challenges of microfluidics-based single-cell nucleic acid analysis are discussed as well.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
