Whole-Genome Sequencing Of Single Cells—MALBAC

Whole-genome sequencing of single cancer cells using MALBAC identified 35 newly acquired single-nucleotide variations (green triangles).

Sometimes the inherited disease or cancer is just caused by a tiny genetic change in a single cell. But such changes are hard to find like someone throws a stone into the sea, with only tiny alter, also because the existing DNA amplification methods are poorly suited for single-cell genome sequencing, in fact they are better at bulk sequences.

However, a single cell is often all that’s available for applications and vital factors, such as prenatal screening or analysis of circulating tumor cells.

Recently a new amplification method eased out sequence biases that hamper the bulk techniques to achieve uniform single-cell genome coverage. The scientist called the technique as MALBAC, for multiple annealing and looping-based amplification cycles. It yields better sequence coverage than has previously been available for single-cell genome sequencing.

In MALBAC, genomic DNA is copied to form looped products. These loops can’t serve as templates, so in each cycle only the genomic DNA can be copied. The amount of DNA increases linearly rather than exponentially as it would in other amplification methods such as polymerase chain reaction (PCR) or multiple displacement amplification. After five MALBAC cycles, scientists collect the DNA loops and use them as templates for further amplification by PCR.

Scientist did the linear amplification first to avoid most of the amplification bias generated in the first few cycles of PCR. The linear amplification made the process even and smooth when across the genome.

As a result, the genome coverage is more uniform than multiple displacement amplification, the current single-cell standard, but less uniform than bulk sequencing.

In one demonstration, scientists sequenced individual genomes from cells obtained by allowing a single cancer cell to divide 20 times. They achieved 93% coverage of individual genomes and identified 35 newly acquired single-nucleotide mutations. They also determined copy number variations, replication errors that result in abnormal numbers of particular DNA sequences. Data from other amplification methods are too noisy to reliably detect copy number variations.

Compared to bulk DNA sequencing in terms of the uniformity, MALBAC is about halfway home in making single-cell genome sequencing. The new way may is a potentially important advance.