Single-Cell DNA Sequencing

Advances in whole-genome amplification and Novogene’s expertise have made single-cell sequencing easily accessible to researchers. Novogene is one of the few NGS providers with extensive experience in single-cell sequencing technology, including single-cell DNA sequencing. We offer the highest quality services in amplification, library construction, sequencing, and bioinformatics analysis to our customers, and our results have been published in leading scientific journals.

With single-cell DNA sequencing, the genomic heterogeneity of cell populations can be explored at the level of the individual cell. Genetic changes, such as point mutations and copy number variation occurring during disease and normal development processes, are profiled using the minute amounts of DNA from single cells. Applications include analysis of genetic heterogeneity within unicellular and multicellular organisms, detection of chromosomal anomalies in germ line cells, preimplantation genomic screening of embryos, and defining the genetic composition of tumors for developing more targeted therapies.

The Novogene Advantage

• Leader in single-cell genomics: We are one of the few providers of this technology, with the highest ranking in technical capability and experience, and publications in the field.
• Advanced amplification methods: We use state-of-the-art, best-in-class amplification methods including AccuSomatic™ Amplification, which enables the discovery of true somatic single nucleotide variations (SNVs) in single cells that could not be accurately detected by any prior method, and MALBAC to provide uniform data while reducing rates of false positives and false negatives.
• Comprehensive processing: Our single-cell sequencing services include amplification, library construction, sequencing, and bioinformatics analysis.

Project Workflow

Sequencing Strategy

• 350 bp insert DNA library
• HiSeq platform, paired-end 150 bp

Data Quality Guarantee

• Q30 ≥ 80%

Sample Requirements

• We accept fresh single cells and laser captured single cells.
• Sorted single cells should be stored in 1 x PBS buffer (excluding calcium and magnesium) in a total volume of ≤ 2 µL. The stored cells should be freezed in liquid nitrogen and shipped out with dry ice.

Turnaround Time

• Amplification: within 10 working days from verification of sample quality
• Library preparation and sequencing: within 22 working days
• Data analysis: 8 working days

Recommended Sequencing Depth

• For normal sample: effective sequencing depth of 30X
• For tumor sample: effective sequencing depth of 50X

Analysis Pipeline

Table. Representative data quality results of single-cell DNA sequencing from Novogene.

Sample Name	Raw reads	Raw data (G)	Effective (%)	Error (%)	Q 20 (%)	Q 30(%)	GC (%)
G6_10	119752154	34.59	99.80	0.03	95.64	89.85	44.74
G6_11	114546150	33.26	99.80	0.04	94.00	87.43	44.87
G6_12	116002747	33.57	99.80	0.04	95.29	89.95	44.91
G6_9	127417212	37.23	99.70	0.03	96.42	91.15	44.99

Project Example

The following studies utilized Novogene's expertise in single-cell DNA sequencing.

Probing meiotic recombination and aneuploidy of single sperm cells by whole-genome sequencing
Science, 338:1627-1630 (2012)

In this study, Novogene’s single-cell DNA sequencing technology was used to analyze the genomes of individual human sperm following amplification by MALBAC. The results revealed variation in the distribution of recombination events along the genome and provided new insights into meiotic mechanisms.



Figure. Identifying crossover positions in individual sperm cells.

Single-cell exome sequencing identifies mutations in KCP, LOC440040, and LOC440563 as drivers in renal cell carcinoma stem cells
Cell Research 1-4 (2016)

Renal cell carcinoma (RCC), the most common form of adult kidney cancer, has a low mutation rate. In this study, three novel renal cancer stem cell driver mutations were discovered using Novogene’s advanced single-cell exome sequencing technology. With over 140X coverage, 297 somatic SNVs were found, with 141 of these located in coding regions. Three missense mutations in the loci KCP, LOC440563, and LOC440040 were unique to CD133+ RCC cells and have not been reported in RCC before. This study suggests that these three novel mutations could play significant roles in RCC diagnostics and therapeutic treatment.

 



Figure. Identification of driver genes in renal cell carcinoma stem cells via single-cell exome sequencing

Single-cell 5-formylcytosine landscapes of mammalian early embryos and ESCs at single-base resolution
Cell Stem Cell, 20: 1-12 (2017)

5-formylcytosine (5fC), as an oxidized derivative of 5mC (5-methylcytosine), plays an important role in active DNA demethylation, yet remains unexplored at the single-cell level. Here, Novogene’s HiSeq platform was adopted in a novel “CLEVER-seq” (chemical-labeling-enabled C-to-T conversion sequencing) approach, to map the 5fc dynamics at single-cell, single-base resolution. CLEVER-seq identifies the intrinsic 5fC heterogeneity in mouse gametes and early embryos, provides evidence of active DNA methylation in parental and maternal genomes, and indicates that the emergence of promoter 5fC production precedes the up-regulation of gene expression. This new CLEVER-seq displays an important resource for further epigenetic reprogramming in single cells at single base resolution.


Figure. The landscape of DNA formylation in mouse pluripotent stem cells and early embryos.