Single-cell Whole Genome Sequencing Service

Single-cell whole genome sequencing is a new technology for amplifying and sequencing whole genomes at the single-cell level. This technology aims to amplify a trace amount of genomic DNA obtained from single cell lysis with high coverage thus performing high-throughput sequencing by exon capture as well as revealing cell population differences and evolutionary relationships. Single-cell whole genome sequencing plays an increasingly important role in cancer research, embryonic development, assisted reproduction, cell differentiation, immune mechanisms, microbiology, and other research fields.

CD Genomics' single-cell whole genome sequencing service uses the MDA or MALBAC technology to efficiently amplify trace amounts of whole genomic DNA in single cells to obtain high coverage of the complete genome for high-throughput sequencing.

Whole Genome Amplification

Obtaining whole genome amplification products with high coverage and fidelity guarantees accurate and comprehensive sequencing results.

In fact, the DNA contained within a single cell is very small (usually <10 pg/cell), which cannot meet the detection requirements of most sequencers. Therefore, amplification and enrichment must be performed before further sequencing. Whole genome amplification enables amplification of the entire genome and provides powerful support for single-cell genome sequencing.

Multiple displacement amplification (MDA) allows the access of high fidelity DNA fragments using random primers and isothermal amplification, but the major drawbacks of this strategy are due to its unbalanced genome coverage, amplification bias, chimeric sequences, and non-specific amplification.

The MALBAC (Multiple Annealing and Looping Based Amplification Cycle) differs from the previous nonlinear or exponential amplification methods by largely preventing exponential amplification of DNA, thus resolving amplification bias and reducing the amount of template required for genome sequencing from the µg level to the single-cell level.

Workflow of Single-cell Whole Genome Sequencing Service

Advantages of Our Services

Rich project experience

Abundant successful cases, extensive experience in cooperation with hospitals, universities, and research institutes regarding single-cell whole genome sequencing analysis

Analysis content

Standard analysis + advanced analysis + customized analysis

MALBAC or MDA pre-amplification platform

Mature, efficient amplification technology with greater than 80% genome coverage

Rich experience in performance with various species

Human, mouse, zebrafish, microorganisms, etc.

Sample Requirements

Single-cell isolation is completed by customers;

1-1,000 fresh cells, the sample volume should not exceed 1-2 μL

FAQ

Sample delivery requirements for single-cell genomic

MDA amplification: maximum sample volume should not exceed 2 μL

MALBAC amplification: samples should not contain Ca²⁺ and Mg²⁺ and volume should not exceed 1 μL

Single-cell genome samples should be separated between cells to avoid adhesions and cell debris, which may affect amplification quality

Single-cell genome coverage assessment

After library construction of single cell whole genome samples, a small sample data (e.g., human, 2 G data) will be tested and a 30X coverage assessment will be performed, based on which the customer will decide whether to add this sample or resend it for library construction.

Principles and pros and cons of the single-cell genome amplification strategy

1) MDA

Invented by the Laskin team in 2001, MDA uses random hexamer primers and φ29 DNA polymerase for the reaction. This polymerase has strong strand replacement properties and can amplify DNA fragments of 50~100 kb under isothermal conditions. At the same time, φ29 DNA polymerase has high replication fidelity due to its 3'-5' nucleic acid exonuclease activity and proofreading activity. As a result, the MDA strategy reserved higher genome coverage.

2) MALBAC

The MALBAC amplification process reduces the sequence preference of exponential amplification. The 5' end of the amplification primer contains a universal sequence of 27 bp, while the 3' end is a random sequence of 8 bp, which can be combined with the template at a low temperature of 15~20℃. After 8~12 cycles of quasilinear amplification, these loops can be amplified exponentially.

The advantage of the MALBAC strategy is that the sequence bias is reproducible between different cells, which are suitable for CNV analysis due to its better homogeneity of amplification. The weakness of MALBAC is that it uses a polymerase of lower fidelity than φ29, so MALBAC is expected to contain more false positives when detecting SNV. In addition, due to its repeatability sequence preference, a loss in genome detection may reveal in the low amplified regions during amplification.