Sequencing the DNA of a single cell

Last update: August 14, 2016

Overview

Whole genome sequencing on DNA of a single cell has been reported by several authors. Singe cell genome sequencing is of interest in a number of niche applications, such as the measuring of the genomic diversity in one individual’s gamete genomes, the mutation phase determination, and somatic DNA changes in tumors or after exposure to mutagenic agents.

Sequencing library preparation

Sequencing library preparation for single cell DNA sequencing is particularly important, because the small quantity of nucleic acid available requires a specific, random amplification step (to read a general introduction on the sequencing library you can click here). The point is that the initial DNA must be increased to the minimal quantity needed for the library preparation before proceeding to next generation sequencing.

The most common strategy utilized to amplify the whole genome of a single cell can be based on multiple displacement amplification (MDA), PCR, or a combined method (MDA-PCR). MDA makes use of random primers with phi29, a highly processive strand displacing polymerase. While this technique is capable of generating enough amplified material to construct sequencing libraries, it suffers from considerable bias, created by nonlinear amplification. An improvement in using the method of MDA can be done by adding a quasilinear preamplification step that reduced bias (combined MDA-PCR: MDA pre-amplification and PCR-amplification).

Microfluidic systems

The small amount of DNA used in single cell genome sequencing can be handled by the use of microfluidic systems. Microfluidics emerged in the beginning of the 1980s and is used in the development of inkjet printheads, lab-on-a-chip technology, micro-propulsion, micro-thermal technologies and DNA studies. Automated hands-free microfluidic workflows based on small compartmentalization have been designed to efficiently enable efficient cell capture and lysis for up to 96 single cells per run – Fluidigm (South San Francisco, CA).

Breda Genetics srl

Upon request, Breda Genetics is able to offer whole-genome sequencing on a DNA from single cell after MDA-PCR quasilinear amplification. For info and requests, please contact info@bredagenetics.com.

References: PMID: 24502796220869612117004322817899274489712751009726302375,  Wikipedia: Microfluidics.

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