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

Rhee, Minsoung; Light, Yooli K.; Meagher, Robert J.; Singh, Anup K.; Kumar-Sinha, Chandan

doi:10.1371/journal.pone.0153699

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

Journal Article · Wed May 04 00:00:00 EDT 2016 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0153699· OSTI ID:1266216

Rhee, Minsoung ^[1]; Light, Yooli K. ^[2]; Meagher, Robert J. ^[2]; Singh, Anup K. ^[2]; Kumar-Sinha, Chandan ^[3]

Sandia National Lab. (SNL-CA), Livermore, CA (United States); Illumina, Inc., San Diego, CA (United States)
Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Univ. of Michigan, Ann Arbor, MI (United States)

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. In spite of its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. 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. As a consequence, 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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Research Organization:: Sandia National Lab. (SNL-CA), Livermore, CA (United States); Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

Sponsoring Organization:: USDOE National Nuclear Security Administration (NNSA)

Grant/Contract Number:: R01 DE020891; AC04-94AL85000

OSTI ID:: 1266216

Alternate ID(s):: OSTI ID: 1303157

Report Number(s):: SAND2016-4583J

Journal Information:: PLoS ONE, Vol. 11, Issue 5; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 31 works

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

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