Single Cell DNA Sequencing Service
Creative Biolabs provides comprehensive whole genome amplification to discover DNA mutations in single cells. We examine genotype and phenotype from single cells at the same time, exposing the heterogeneity of your samples with unparalleled details. Single-cell DNA sequencing approach can be used to identify complete biomarkers that can assist better stratify patients, detect resistance earlier, and predict recurrence.
Single Cell DNA Sequencing
By bringing the study of genomes to the cellular level, single-cell genomics promises to contribute new perspectives to our understanding of genetics. By dissecting the contributions of individual cells to the biology of ecosystems and species, these techniques are opening up new frontiers. For example, Single-cell genomics can now be used to identify and assemble the genomes of unculturable microorganisms, assess the roles of genetic mosaicism in normal physiology and disease, and determine the contributions of intra-tumor genetic heterogeneity in cancer development and treatment response. This field is based on the ability to analyze a single DNA molecule extracted from a single cell.
Fig.1 Opportunities enabled by single-cell sequencing strategies. (Gawad, 2016)
Single Cell DNA Sequencing Service Workflow
We offer scientific and meticulous design for single cell DNA sequencing to ensure high-quality research results, including nuclear suspensions dissociating, staining, single nuclei flow-sorting, single nuclei amplifying, and DNA sequencing.
Fig.2. Single cell DNA sequencing experimental workflow. (Leung, 2017)
Applications of Single Cell DNA Sequencing Service
Compartmentalizing microbial dark matter.
Sequencing has the ability to overcome the sampling bias, when researchers use culturing methods to isolate microbes. Using the 16S ribosomal RNA sequencing to identify as-yet unculturable bacterial phyla and major archaeal groups has been successful, though the rest of the genomes of those putative new phyla are difficult to assemble because the sequencing data was obtained from samples containing multiple species. Single-cell genomics has the potential to assemble the genomes of low-frequency species found in metagenomic collections, as well as genome assemblies of wholly uncharacterized bacteria.
Identifying genetic mosaicism in multicellular organisms.
The development of cytogenetic methods led to the discovery that cells within the same individual can have varied numbers of chromosomes. Patients with mosaic expression of dominant Mendelian diseases were later identified by atypical patterns of the traditional cutaneous manifestations of numerous disorders. More recently, the development of variant detection tools based on next-generation sequencing has allowed the discovery of a number of novel disorders caused by mosaic SNVs or CNVs.
The sequential acquisition of genetic variations in single cells mediate tumor genesis, maintenance, and evolution. The goal of the large-scale cancer sequencing projects is to catalog these variations in order to better understand tumor biology. Single-cell sequencing research is increasingly being used to analyze intra-tumor genomic heterogeneity at the single-cell level.
|Paper Title||Single-cell mutation analysis of clonal evolution in myeloid malignancies|
|Abstract||In this study, they used single-cell mutational profiling on 146 samples from 123 patients in their investigation. They discovered that AML is dominated by a small number of clones, many of which have co-occurring epigenetic regulator alterations. Signaling gene mutations, on the other hand, frequently occur more than once in different subclones, indicating that clonal diversity is rising. They discovered combinations of mutations that synergized to boost clonal proliferation and dominance by mapping clonal trajectories for each sample.|
|Methods||Single-cell DNA sequencing|
|Result||The discovery of frequent, recurring epigenetic regulator mutations in CH patients and the reduced prevalence of overt myeloid malignancies in CH patients suggest that clonal evolution from disease-initiating clones to leukemic clones is the rate-limiting phase in myeloid transformation. Bulk sequencing analyses have previously been used to predict significant characteristics of clonal evolution; nevertheless, the molecular sequence of events that drive myeloid transformation has not been dissected at a single-cell, clonal level. They employed scDNA-seq to characterize clonal evolution in myeloid malignancies and gain insights into the etiology of myeloid transformation that could not be obtained from bulk sequencing.|
Fig.3 Copy-number evolution in clonal extinction patients. (Miles, 2020)
|Paper Title||Chemoresistance Evolution in Triple-Negative Breast Cancer Delineated by Single-Cell Sequencing|
|Abstract||In this study, they profiled longitudinal samples from 20 TNBC patients during neoadjuvant treatment using single-cell DNA and RNA sequencing, as well as bulk exome sequencing (NAC). Deep exome sequencing revealed ten patients in whom NAC resulted in clonal extinction and ten patients in whose clones survived therapy. They did a more extensive investigation on 8 patients, analyzing 900 cells with single-cell DNA sequencing and 6,862 cells with single-cell RNA sequencing.|
|Methods||Single-cell DNA and RNA sequencing|
|Result||They looked at the genomic and phenotypic evolution of tumor cells in TNBC patients as a result of NAC treatment, and discovered two types of clonal dynamics: extinction and persistence. After treatment, NAC eradicated the tumor cells, leaving only normal diploid cell types, including numerous fibroblasts and immunological cells, in the clonal extinction patients. The clonal persistence patients, on the other hand, had a substantial number of remaining tumor cells with altered genotypes and phenotypes in response to NAC.|
Fig.3 Copy-number evolution in clonal extinction patients. (Kim, 2018)
Frequently Asked Questions
The principle of single-cell genome amplification.
Random six polymer primers and 29 DNA polymerase were used in the reaction, which had robust chain replacement capabilities and could amplify a 50-100kb DNA fragment under isothermal conditions. The fidelity of the 29 DNA polymerase is great due to its 3'-5' exonuclease activity and proofreading activity.
What's the MALBAC?
The Quasilinear amplification mechanism, MALBAC (Multiple Annealing and Looping–Based Amplification Cycles), decreases the sequence preference of exponential amplification. The 5' of amplified primers contains the common 27bp sequence, and the 3' is a random 8bp sequence, which can be coupled with the template at low temperature (1520 C), and then amplify these ring-shaped amplicons after 812 cycles of quasilinear amplification.
Creative Biolabs has accumulated extensive experience in the field of single cell DNA sequencing. We can offer you the customized service with highest quality, accurate data, and a fast turnaround timeline.
- Gawad, C., et al. Single-cell genome sequencing: current state of the science. Nature Reviews Genetics. 2016; 17(3): 175-188.
- Leung, M.L., et al. Single-cell DNA sequencing reveals a late-dissemination model in metastatic colorectal cancer. Genome research. 2017; 27(8): 1287-1299.
- Miles, L.A., et al. Single-cell mutation analysis of clonal evolution in myeloid malignancies. Nature. 2020; 587(7834): 477-482.
- Kim, C., et al. Chemoresistance evolution in triple-negative breast cancer delineated by single-cell sequencing. Cell. 2018; 173(4): 879-893. e13.