MALBAC

MALBAC

Multiple annealing and looping–based amplification cycles (MALBAC) is intended to address some of the shortcomings of MDA . In this method, MALBAC primers randomly anneal to a DNA template. A polymerase with displacement activity at elevated temperatures amplifies the template, generating “semi-amplicons.” As the amplification and annealing process is repeated, the semi-amplicons are amplified into full amplicons that have a 3’ end complimentary to the 5’ end. As a result, full-amplicon ends hybridize to form a looped structure, inhibiting further amplification of the looped amplicon, while only the semi-amplicons and genomic DNA undergo amplification. Deep sequencing of the full-amplicon sequences allows for accurate representation of reads, while sequencing depth provides improved alignment for consensus sequences.

Pros:

• Can sequence large templates
• Can perform single-cell sequencing or sequencing for samples with very limited starting material
• Full-amplicon looping inhibits over-representation of templates, reducing PCR bias
• Can amplify GC-rich regions
• Uniform genome coverage
• Lower allele drop-out rate compared to MDA

Cons:

• Polymerase is relatively error prone compared to Phi 29
• Temperature-sensitive protocol
• Genome coverage up to ~90%, but some regions of the genome are consistently underrepresented

• MALBAC: Zong C., Lu S., Chapman A. R. and Xie X. S. (2012) Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science 338: 1622-1626

1. Dey S. S., Kester L., Spanjaard B., Bienko M. and van Oudenaarden A. (2015) Integrated genome and transcriptome sequencing of the same cell. Nat Biotechnol 33: 285-289
2. Li N., Wang L., Wang H., Ma M., Wang X., et al. (2015) The Performance of Whole Genome Amplification Methods and Next-Generation Sequencing for Pre-Implantation Genetic Diagnosis of Chromosomal Abnormalities. J Genet Genomics 42: 151-159
3. Ning L., Li Z., Wang G., Hu W., Hou Q., et al. (2015) Quantitative assessment of single-cell whole genome amplification methods for detecting copy number variation using hippocampal neurons. Sci Rep 5: 11415
4. Upton K. R., Gerhardt D. J., Jesuadian J. S., Richardson S. R., Sanchez-Luque F. J., et al. (2015) Ubiquitous L1 mosaicism in hippocampal neurons. Cell 161: 228-239
5. Zhang C. Z., Adalsteinsson V. A., Francis J., Cornils H., Jung J., et al. (2015) Calibrating genomic and allelic coverage bias in single-cell sequencing. Nat Commun 6: 6822