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Title: Digital droplet multiple displacement amplification (ddMDA) for whole genome sequencing of limited DNA samples

Here, 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.
 [1] ;  [2] ;  [2] ;  [2] ;  [3]
  1. Sandia National Lab. (SNL-CA), Livermore, CA (United States); Illumina, Inc., San Diego, CA (United States)
  2. Sandia National Lab. (SNL-CA), Livermore, CA (United States)
  3. Univ. of Michigan, Ann Arbor, MI (United States)
Publication Date:
Report Number(s):
Journal ID: ISSN 1932-6203
Grant/Contract Number:
R01 DE020891; AC04-94AL85000
Accepted Manuscript
Journal Name:
Additional Journal Information:
Journal Volume: 11; Journal Issue: 5; Journal ID: ISSN 1932-6203
Public Library of Science
Research Org:
Sandia National Lab. (SNL-CA), Livermore, CA (United States); Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
Sponsoring Org:
USDOE National Nuclear Security Administration (NNSA)
Country of Publication:
United States
genomic libraries; bacterial genomics; genome sequencing; microfluidics; fungal genomics; DNA sequencing; DNA libraries; comparative genomics; 59 BASIC BIOLOGICAL SCIENCES
OSTI Identifier:
Alternate Identifier(s):
OSTI ID: 1303157