MDA/IMS-MDA/MIDAS

MDA/IMS-MDA/MIDAS

Multiple displacement amplification (MDA) is a method commonly used for sequencing microbial genomes due to its ability to amplify templates larger than 0.5 Mbp, but it can also be used to study genomes of other sizes . In this method, 3’-blocked random hexamer primers are hybridized to the template, followed by synthesis with Phi 29 polymerase. Phi 29 performs strand-displacement DNA synthesis, allowing for efficient and rapid DNA amplification. Deep sequencing of the amplified DNA allows for accurate representation of reads, while sequencing depth provides better alignment and consensus for sequences.

Microwell displacement amplification system (MIDAS) is a variation on MDA where single cells are randomly distributed into hundreds to thousands of nanoliter wells and their genetic material is simultaneously amplified. Sequencing individual cells simplifies the genomic diversity and improves sensitivity in heterogeneous cell populations. In an alternative approach to simplify genomic diversity, IMS-MDA uses immunomagnetic separation for targeted bacterial enrichment.

Pros:

• Templates used for this method can be circular DNA (plasmids, bacterial DNA)
• Can sequence large templates
• Can perform single-cell sequencing or sequencing for samples with very limited starting material

Cons:

• Strong amplification bias. Genome coverage as low as ~6%
• PCR biases can underrepresent GC-rich templates
• Contaminated reagents can impact results

• MDA: Dean F. B., Nelson J. R., Giesler T. L. and Lasken R. S. (2001) Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res 11: 1095-1099
• IMS-MDA: Seth-Smith H. M. et al. (2013) Generating whole bacterial genome sequences of low-abundance species from complex samples with IMS-MDA. Nat Protoc 8: 2404-2412
• MIDAS: Gole J., Gore A., Richards A., Chiu Y. J., Fung H. L., et al. (2013) Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells. Nat Biotechnol 31: 1126-1132

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