Single-Cell DNA Sequencing Q&A

1) Isolation of individual cells from the cell population.
2) Extraction, processing and amplification of the genetic material of each isolated cell.
3) Preparation of a sequencing library containing the genetic material of the isolated cells.
4) Sequencing of the library using sequencers

We offer a variety of single-cell sequencing services that can analyze transcriptomic, genomic, epigenomic, and immune repertoire profiling at the resolution of a single cell. We offer a comprehensive service from sample preparation to sequencing analysis. The main difference lies in the NGS library construction strategy and downstream analysis.
Our single-cell RNA sequencing utilizes UMI technology for unbiased amplification sequencing to obtain accurate and comprehensive transcriptome information. Single-cell epigenomic sequencing provides access to DNA methylation and chromatin accessibility analysis (single-cell ATAC sequencing). Our single-cell genome sequencing technology can be used to map the genealogy of embryonic and organ development.

Raw sequencing data needs to be pre-processed. Low-quality reads and those that are unlikely to be from viable cells are discarded and filtered. For example, reads containing low counts and barcodes with few detected genes may represent background noise.
After quality control, sequencing data are normalized by cellular barcodes. Data from single-cell sequencing vary widely between cells, generally because of the low sequencing depth per cell. The data are then subjected to downstream analysis, including feature selection, downscaling and/or visualization. Our goal is to make meaningful biological interpretations of the results through multiple bioinformatic analyses.

Sequencing quality is influenced by several factors, the main ones being the total number of libraries that can be extracted from the cell population and the number of reads detected. The ideal number of cells depends on the expected number of different cell subpopulations or states. The number of reads indicates the sequencing depth, depending on the size of the genome: the higher the read depth, the more reliable the details. Sample and library preparation protocols can also affect the quality of the results.

Fluorescence-activated cell sorting (FACS) involves the use of fluorescent molecules attached to target-specific antibodies to label the cellular proteins on which cell selection is based. This technique allows the selection of cells based on multiple parameters. However, FACS requires >10,000 starting cells and rapid flow may impair cell viability.
Magnetically activated cell sorting (MACS) uses antibody-mediated superparamagnetic nanoparticles to label specific proteins on target cells. However, the purity of MACS isolation depends on the specificity and affinity of the antibody used for labeling.
Laser capture microdissection (LCM) uses a laser to isolate target cells from a solid tissue sample placed on a microscope slide. LCM requires visual inspection of their morphology to identify the target cells. In addition, cells may be sectioned during isolation and UV light may damage DNA and RNA molecules.
Manual cell harvesting or microscopic manipulation requires an inverted microscope in combination with a micropipette to select and isolate target cells. Micro-manipulation has been used for live cultures and embryonic cells. However, it has limited throughput.