Improved Multiple Displacement Amplification (iMDA) and Ultraclean Reagents

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.

Template-Dependent Multiple Displacement Amplification for Profiling Human Circulating RNA

“…While these issues are negligible when using large amounts of template, both concerns become significant shortcomings when working with low or ultralow amounts of template (typically <1 ng) since they compress the product space of target molecules and/or aggravate qualitative biases (2,6,7,26). While reagent contamination could be minimized or even eliminated through the use of clean enzymes and reagents (27,28), TIA remains a consistent issue in MDA, despite many attempts to overcome it. The current study provides a simple, but efficient solution in which the use of a specially designed primer (C28) (Figure 1) eliminates TIA.…”

Evaluation of convenient pretreatment protocols for RNA virus metagenomics in serum and tissue samples

“…Several attempts have been performed to reduce amplification introduced bias (Motley et al, 2014;Rosseel et al, 2013). Although the sequencing library preparation kit (Illumina Nextera XT) used in this study is not completely PCR-free, it supports ultra-low DNA input of only a single nanogram which eliminates the need for extra sequence independent genome amplification step.…”

High-resolution single-cell sequencing of malaria parasites

“…To further optimize our protocol for clinical samples, we made two further modifications prior to analyzing a third clinical sample collected in Chikhwawa, Malawi (MAW0). First, since amplification of small amounts of DNA is extremely sensitive to DNA contamination in reagents [28,29], we elected to autoclave PBS (Lonza) used to "capture" sorted cells. Second, we processed half of the samples with a PCR-free library preparation method with the goal of lowering amplification bias introduced after MDA.…”