Abstract
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.
Highlights
Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; many relevant samples are comprised of only a few cells
A widely used method for whole genome amplification is multiple displacement amplification (MDA); MDA relies on priming of target DNA with random primers and the use of the strand-displacing φ29 polymerase to amplify all of the DNA in a given sample [1,2,3]. φ29 DNA polymerase is a highly processive, stranddisplacing polymerase with a very low error rate of 1 in 106-107 nucleotides [4,5]; the error rates of Taq polymerase and Pfu polymerase, both commonly used in PCR are 3 in 104 and 3 in 106, respectively [6,7]
Decontamination of laboratory consumables with ethylene oxide (ETO) To determine the effectiveness of the ETO treatment, laboratory consumables were contaminated with bacterial DNA, whole bacterial cells, or human DNA
Summary
Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; 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. A method for the whole genome amplification of DNA from single cells called MALBAC was reported to perform better than MDA [8]. This method employs several rounds of multiple primer annealing extension cycles with a strand-displacing polymerase followed by PCR. Our iMDA protocol does not require the use of FACS, specialized microfabrication or operating with nanoliter volumes yet provides an ultraclean DNA amplification reaction
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