Abstract
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.
Highlights
Whole genome sequencing is beneficial for the study of samples with limited DNA such as difficult-to-culture microorganisms and the analysis of clinical samples [1,2,3,4,5,6], but most DNA sequencing technologies require nanogram to microgram amounts of DNA for library preparation, while a single bacterial or human cell contains only a few femtograms or picograms of DNA template
We address both the bias in amplification and non-specific amplification by performing Multiple displacement amplification (MDA) in droplets where a sample containing E. coli genomic DNA into millions of ~150 pL droplets which are amplified in parallel, and pooled to generate a single sequencing library
We showed that performing digital droplet MDA (ddMDA) in water-in-oil droplets generated with a simple microfluidic device substantially improved the quality of whole genome amplification compared to a conventional MDA reaction, which was attributed to discretization of template DNA by partitioning into numerous small reaction volumes
Summary
Whole genome sequencing is beneficial for the study of samples with limited DNA such as difficult-to-culture microorganisms and the analysis of clinical samples [1,2,3,4,5,6], but most DNA sequencing technologies require nanogram to microgram amounts of DNA for library preparation, while a single bacterial or human cell contains only a few femtograms or picograms of DNA template. When dealing with the limited amounts of DNA from a single or a few cells, it is necessary to perform whole genome amplification to obtain sufficient material for preparation of a sequencing library [7]. Multiple displacement amplification (MDA) is the most common of several techniques used [8,9] for amplifying limited input DNA [3]. The specific roles of these authors are articulated in the ‘author contributions’ section
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