Improved Multiple Displacement Amplification (iMDA) and Ultraclean Reagents

BackgroundNext-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA).ResultsA specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome.ConclusionsThe iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.

[1]  Kathryn E. Dagnall,et al.  Comparison of the effects of sterilisation techniques on subsequent DNA profiling , 2007, International Journal of Legal Medicine.

[2]  T. Kunkel,et al.  DNA polymerase fidelity and the polymerase chain reaction. , 1991, PCR methods and applications.

[3]  Roger S Lasken,et al.  Genomic DNA amplification by the multiple displacement amplification (MDA) method. , 2009, Biochemical Society transactions.

[4]  Anne-Brit Kolstø,et al.  Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—One Species on the Basis of Genetic Evidence , 2000, Applied and Environmental Microbiology.

[5]  John Hackett,et al.  The Perils of Pathogen Discovery: Origin of a Novel Parvovirus-Like Hybrid Genome Traced to Nucleic Acid Extraction Spin Columns , 2013, Journal of Virology.

[6]  W. Thilly,et al.  Fidelity of DNA polymerases in DNA amplification. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Alexander Sczyrba,et al.  Decontamination of MDA Reagents for Single Cell Whole Genome Amplification , 2011, PloS one.

[8]  Mitch Leslie,et al.  Cell biology. The power of one. , 2011, Science.

[9]  Wendy S. Schackwitz,et al.  One Bacterial Cell, One Complete Genome , 2010, PloS one.

[10]  Sergei L. Kosakovsky Pond,et al.  Windshield splatter analysis with the Galaxy metagenomic pipeline. , 2009, Genome research.

[11]  Paul C. Blainey,et al.  Digital MDA for enumeration of total nucleic acid contamination , 2010, Nucleic acids research.

[12]  M. Leslie.  Single-cell tech primer. , 2011, Science.

[13]  Thierry Grange,et al.  An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications , 2010, PloS one.

[14]  L. Blanco,et al.  Fidelity of phi 29 DNA polymerase. Comparison between protein-primed initiation and DNA polymerization. , 1993, The Journal of biological chemistry.

[15]  S. Kingsmore,et al.  Comprehensive human genome amplification using multiple displacement amplification , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Fabian Grubert,et al.  A procedure for highly specific, sensitive, and unbiased whole-genome amplification , 2008, Proceedings of the National Academy of Sciences.

[17]  Michael Donley,et al.  UV irradiation and autoclave treatment for elimination of contaminating DNA from laboratory consumables. , 2010, Forensic science international. Genetics.

[18]  Jerilyn A. Walker,et al.  Human DNA quantitation using Alu element-based polymerase chain reaction. , 2003, Analytical biochemistry.

[19]  G. Nevinsky,et al.  The algorithm of estimation of the K m values for primers of various structure and length in the polymerization reaction catalyzed by Klenow fragment of DNA polymerase I from E.coli , 1989, FEBS letters.

[20]  Sallie W. Chisholm,et al.  Whole Genome Amplification and De novo Assembly of Single Bacterial Cells , 2009, PloS one.

[21]  A. Hopwood,et al.  Validation of a dual cycle ethylene oxide treatment technique to remove DNA from consumables used in forensic laboratories. , 2010, Forensic science international. Genetics.

[22]  X. Xie,et al.  Genome-Wide Detection of Single-Nucleotide and Copy-Number Variations of a Single Human Cell , 2012, Science.

[23]  R. O’Neill,et al.  Abundant Human DNA Contamination Identified in Non-Primate Genome Databases , 2011, PloS one.

[24]  Kaisa Silander,et al.  Whole genome amplification with Phi29 DNA polymerase to enable genetic or genomic analysis of samples of low DNA yield. , 2008, Methods in molecular biology.

[25]  D. Stenger,et al.  Nucleic Acid Amplification Strategies for DNA Microarray-Based Pathogen Detection , 2004, Applied and Environmental Microbiology.

[26]  D. Shoemaker,et al.  High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. , 1991, Gene.

[27]  J. Weber,et al.  DNA Extraction Columns Contaminated with Murine Sequences , 2011, PloS one.

[28]  Kun Zhang,et al.  Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells , 2013, Nature Biotechnology.

[29]  Rameen Beroukhim,et al.  Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification. , 2004, Nucleic acids research.

[30]  G. Walker,et al.  Strand displacement amplification--an isothermal, in vitro DNA amplification technique. , 1992, Nucleic acids research.

[31]  Eric Buel,et al.  Quantification of DNA in forensic samples , 2003, Analytical and bioanalytical chemistry.

[32]  David J. Ecker,et al.  Ibis T5000: a universal biosensor approach for microbiology , 2008, Nature Reviews Microbiology.