InquiryWith the continuous development of high-throughput sequencing technology, the application of sequencing technology in the field of scientific research is becoming more and more widespread. It can be found that research has now gradually transitioned from cell-average level studies to single-cell level. Even the biological activities and significance of single cells are studied from multiple levels such as genome, transcriptome or proteome.
Our scWES Service
Single-cell whole exome sequencing refers to high-throughput sequencing after capturing and enriching the DNA sequence of the whole exome with special probes at the single-cell level.
Through R&D and experimental optimization, CD Genomics researchers have successfully launched single-cell exome sequencing (scWES). We use optimized MALBAC/MDA technology to greatly improve the research efficiency of exonic regions in the genome，with a target region coverage of up to 95%. The addition of this technology further improves the single-cell multi-omics research solution launched by CD Genomics.
Applications of scWES
Single-cell exome sequencing can be used to identify and study structural variants within coding regions associated with disease and cell differentiation. Combined with the exome data provided by a large number of public databases, it facilitates a better interpretation of the association between the resulting variant structures and pathogenic mechanisms.
Single-cell exome sequencing technology solves the problems of cellular heterogeneity of tissue samples and library construction of scarce samples. This technology provides new research ideas for in-depth study of cancer occurrence, development mechanisms, diagnosis and treatment at the single cell level.
Comparison of scWGS and scWES
Compared with genome resequencing, exome sequencing has a lower cost and has great advantages for studying SNPs and Indels of known genes. The cost of sequencing single-cell genomes remains high for many samples. Compared to single-cell whole genome sequencing (scWGS), scWES is less expensive and has other advantages (table1 below).
Table 1 Comparison of single-cell genome sequencing technologies
| Technology | Data | Advantages | Disadvantages |
|---|---|---|---|
| scWGS | 30X-50X 90Gb-150Gb |
Genome-wide detection of variant sites | Large amount of sequencing data and high cost |
| Detection of CNV, SV type variants | Data interpretation is difficult | ||
| Low requirement for prior knowledge of disease | Low sequencing depth is not conducive to deep mining of low-abundance variants | ||
| scWES | 100X 13Gb |
Mutations targeting coding regions that alter protein product | Unable to detect variant information in noncoding regions |
| Data analysis is less difficult and easy to interpret | The detection of CNV and SV is not comprehensive and the accuracy is low | ||
| High-depth sequencing to detect low-abundance variants |
Amplification method
Single-cell DNA cannot meet the sample size requirements for next-generation sequencing. To enable single-cell genomic analysis, single-cell amplification techniques have been developed. At present, three amplification methods, MDA, MALBAC and DOP-PCR, are mainly used to amplify the whole genome of single cells. The three amplification methods have their own advantages and disadvantages. Generally, the appropriate amplification method needs to be selected according to the research purpose.
Table 2 Comparison of single-cell genome amplification techniques
| Techniques | DOP-PCR | MDA | MALBAC |
| Product amount | 1~2μg | Several ug ~ tens of μg | 2~4μg |
| Product length | 100bp~1000bp | > 10kb | 300~2000bp |
| High coverage | Low | High | Medium |
| Amplification error rate | High | Low | Medium |
Workflow of Single-cell Whole Exome Sequencing
Sample requirements
1, Single cells isolated in lysate, 5µl of lysate per tube.
2, 3-5 cells of each cell are recommended to facilitate repeat experiments.
3, After putting single cells into lysate, store at -80℃ and transport on dry ice.
4. Store the lysate at -80°C and avoid repeated freezing and thawing.
5. Keep the low temperature environment as much as possible during the operation. The lysate added with single cells should be stored at -80℃ as soon as possible. It is best not to exceed 1 hour. The shorter the intermediate time, the better.
6. Once the sample is frozen at -80°C or on dry ice, it cannot be thawed repeatedly.
FAQ
What do capture specificity and coverage ratio mean in exome capture sequencing?
Exome capture specificity is a genome analysis technology that selects the information of the coding region of interest, and uses the target sequence capture technology to capture all exon sequences in the whole genome and perform high-throughput sequencing. Whereas coverage ratio is the percentage of total bases detected for the sample.
