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

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.

Microfluidics for genome-wide studies involving next generation sequencing

“…The droplets were collected in a micro-centrifuge tube and amplified for 8–10 h. The careful adjustment of fragment concentration improved the WGA uniformity and suppressed non-specific amplification. Nishikawa et al ., 66 Sidore et al ., 67 and Rhee et al 68 . generated similar results to those of eMDA.…”

Whole genome amplification and sequencing of low cell numbers directly from a bacteria spiked blood model

“…Lower reaction volumes have been shown to lower the effect of the bias, however significant scaling down from the current 5 l starting material would be technically difficult. Use of digital droplet MDA has been shown to give a more uniform coverage across the genome and lower none specific amplifcation 44 , however this requires specialised equipment. For this study amplification bias was compensated for bioinformatically, using a process of digital normalisation 33 , which removed over represented reads, and added an additional error removal stage by removing very low level k-mers.…”

Building a lineage from single cells: genetic techniques for cell lineage tracking

“…MDA generates 15–20 μg of DNA from a single nucleus 78 , and MDA-amplified single-cell DNA is sufficiently high quality for calling somatic retrotransposition events 81 and SNVs 17,112 . Several groups have recently described methods for partitioning MDA reactions into nanolitre-sized droplets 113–115 or by using microfluidic devices 116 , which increases the uniformity of amplification and reduces reagent costs. A second approach, degenerate oligonucleotide priming PCR (DOP-PCR), involves the fragmentation of the genome into small pieces, followed by amplification with random priming 117 .…”