MDA/IMS-MDA
MDA/IMS-MDA
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.
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
- Bigdeli S., Dettloff R. O., Frank C. W., Davis R. W. and Crosby L. D. (2015) A simple method for encapsulating single cells in alginate microspheres allows for direct PCR and whole genome amplification. PLoS One 10: e0117738
- Ottolini C. S., Newnham L. J., Capalbo A., Natesan S. A., Joshi H. A., et al. (2015) Genome-wide maps of recombination and chromosome segregation in human oocytes and embryos show selection for maternal recombination rates. Nat Genet 47: 727-735
- Macaulay I. C., Haerty W., Kumar P., Li Y. I., Hu T. X., et al. (2015) G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nat Methods 12: 519-522
- Leung M. L., Wang Y., Waters J. and Navin N. E. (2015) SNES: single nucleus exome sequencing. Genome Biol 16: 55
- Zhang C. Z., Spektor A., Cornils H., Francis J. M., Jackson E. K., et al. (2015) Chromothripsis from DNA damage in micronuclei. Nature 522: 179-184