DNA nanoball sequencing – Knowipedia – The Next Generation of Global Knowledge

**DNA Nanoball Sequencing**

**Definition**
DNA nanoball sequencing is a high-throughput, cost-effective next-generation sequencing (NGS) technology that utilizes rolling circle amplification to generate compact DNA nanoballs for massively parallel sequencing. This method enables the sequencing of large genomes with high accuracy and efficiency by leveraging patterned arrays and combinatorial probe-anchor synthesis.

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## Introduction

DNA nanoball sequencing (DNBseq) is an advanced next-generation sequencing technology developed to provide high-throughput, accurate, and cost-efficient genomic data. It is distinguished by its unique approach of creating DNA nanoballs (DNBs) through rolling circle amplification (RCA) of circularized DNA templates. These nanoballs are then immobilized on patterned arrays and sequenced using combinatorial probe-anchor synthesis (cPAS), enabling massive parallelization and high data output.

Originally developed and commercialized by Complete Genomics and later refined by BGI (Beijing Genomics Institute), DNA nanoball sequencing has become a prominent alternative to other NGS platforms such as Illumina’s sequencing by synthesis and Pacific Biosciences’ single-molecule real-time sequencing. Its applications span whole-genome sequencing, exome sequencing, transcriptomics, and epigenomics.

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## Historical Background

The evolution of DNA sequencing technologies has been marked by continuous efforts to increase throughput, reduce costs, and improve accuracy. Traditional Sanger sequencing, while highly accurate, was limited by low throughput and high cost. The advent of next-generation sequencing (NGS) technologies in the early 2000s revolutionized genomics by enabling massively parallel sequencing.

DNA nanoball sequencing emerged as a novel approach within this landscape. Complete Genomics pioneered the technology in the late 2000s, introducing a method that combined rolling circle amplification with patterned arrays to generate dense, uniform DNA nanoballs. BGI later adopted and enhanced the technology, branding it as DNBseq, and integrating it into their sequencing platforms.

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## Principles of DNA Nanoball Sequencing

### Rolling Circle Amplification and DNA Nanoball Formation

The core innovation of DNA nanoball sequencing lies in the generation of DNA nanoballs. The process begins with the fragmentation of genomic DNA, which is then ligated to adapters and circularized to form single-stranded DNA circles. These circular templates serve as substrates for rolling circle amplification (RCA), an isothermal enzymatic process that produces long single-stranded DNA concatemers composed of tandem repeats of the original circular template.

The RCA product spontaneously folds into compact, highly uniform DNA nanoballs approximately 200 nanometers in diameter. These nanoballs contain hundreds of copies of the template sequence, providing a high local concentration of DNA for sequencing.

### Patterned Arrays and Nanoball Immobilization

DNA nanoballs are deposited onto patterned arrays—glass slides or flow cells with nanometer-scale wells or spots arranged in a highly ordered grid. The uniform size and shape of the nanoballs allow for dense and even loading onto these arrays, minimizing overlapping signals and enhancing signal-to-noise ratio.

The patterned arrays facilitate precise spatial localization of each nanoball, enabling parallel sequencing of millions to billions of DNA molecules simultaneously.

### Combinatorial Probe-Anchor Synthesis (cPAS)

Sequencing of DNA nanoballs is performed using combinatorial probe-anchor synthesis, a sequencing-by-ligation method. In cPAS, a primer (anchor) hybridizes to a known adapter sequence on the nanoball. Fluorescently labeled probes complementary to the target sequence adjacent to the anchor are then introduced.

Through cycles of hybridization, ligation, imaging, and cleavage, the sequence of bases adjacent to the anchor is determined. The combinatorial nature of the probes allows for efficient decoding of multiple bases per cycle, increasing throughput and reducing reagent consumption.

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## Workflow of DNA Nanoball Sequencing

1. **Sample Preparation**
Genomic DNA is extracted and fragmented into appropriate sizes (typically 200-400 base pairs). Fragmented DNA is end-repaired, A-tailed, and ligated to specialized adapters containing sequences necessary for circularization and sequencing.

2. **Circularization**
Adapter-ligated DNA fragments are circularized using ligase enzymes, forming single-stranded DNA circles that serve as templates for RCA.

3. **Rolling Circle Amplification**
Circular DNA templates undergo RCA, producing long single-stranded DNA concatemers that fold into DNA nanoballs.

4. **Nanoball Purification and Quantification**
DNA nanoballs are purified to remove unreacted components and quantified to ensure optimal loading density on patterned arrays.

5. **Loading on Patterned Arrays**
Purified nanoballs are loaded onto patterned arrays where they self-assemble into ordered arrays due to their uniform size.

6. **Sequencing by cPAS**
Sequencing is performed through cycles of probe hybridization, ligation, imaging, and cleavage, decoding the sequence of each nanoball.

7. **Data Analysis**
Raw fluorescence images are processed to identify base calls, align reads to reference genomes, and perform variant calling or other downstream analyses.

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## Advantages of DNA Nanoball Sequencing

### High Throughput and Scalability

The dense packing of DNA nanoballs on patterned arrays allows for the simultaneous sequencing of billions of DNA molecules, enabling whole-genome sequencing at population scale.

### Cost-Effectiveness

The use of rolling circle amplification and combinatorial probe-anchor synthesis reduces reagent consumption and instrument complexity, lowering per-base sequencing costs compared to other NGS platforms.

### High Accuracy and Low Error Rates

The redundancy inherent in DNA nanoballs (multiple copies of the template per nanoball) and the sequencing chemistry contribute to high base-calling accuracy and low error rates, particularly for single nucleotide variants.

### Reduced Amplification Bias

Unlike PCR-based amplification methods, RCA used in DNA nanoball sequencing is less prone to amplification bias, resulting in more uniform coverage across the genome.

### Compact Data Footprint

The sequencing data generated is highly efficient, with minimal duplication and high-quality reads, facilitating downstream bioinformatics processing.

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## Limitations and Challenges

### Read Length Constraints

DNA nanoball sequencing typically produces short reads (50-150 base pairs), which can complicate the assembly of complex genomic regions, structural variant detection, and haplotype phasing compared to long-read sequencing technologies.

### Instrumentation and Platform Availability

DNA nanoball sequencing platforms are primarily developed and commercialized by a limited number of providers, which may restrict accessibility and integration into diverse laboratory settings.

### Complexity of Library Preparation

The circularization and rolling circle amplification steps add complexity to library preparation compared to simpler linear amplification methods, potentially increasing turnaround time and requiring specialized expertise.

### Limited Direct Detection of Epigenetic Modifications

Unlike some third-generation sequencing technologies, DNA nanoball sequencing does not inherently detect DNA modifications such as methylation without additional chemical treatments or protocols.

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## Applications

### Whole-Genome Sequencing

DNA nanoball sequencing is widely used for whole-genome sequencing (WGS) in research and clinical settings, enabling comprehensive analysis of genetic variation across populations.

### Exome and Targeted Sequencing

By enriching for coding regions or specific genomic loci, DNA nanoball sequencing facilitates focused studies on disease-associated genes and variants.

### Transcriptome Analysis

RNA sequencing (RNA-seq) using DNA nanoball technology allows for gene expression profiling, alternative splicing analysis, and transcript discovery.

### Metagenomics

The high throughput and accuracy of DNA nanoball sequencing make it suitable for characterizing complex microbial communities in environmental and clinical samples.

### Cancer Genomics

DNBseq is employed in cancer research for mutation detection, copy number variation analysis, and monitoring tumor heterogeneity.

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## Comparison with Other Sequencing Technologies

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## Future Directions

Advancements in DNA nanoball sequencing continue to focus on increasing read length, improving library preparation workflows, and integrating epigenetic detection capabilities. Efforts to combine DNBseq with complementary technologies may enhance structural variant detection and haplotype phasing.

Additionally, the expansion of DNBseq into clinical diagnostics, personalized medicine, and large-scale population genomics projects is anticipated, driven by its cost-effectiveness and scalability.

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## Conclusion

DNA nanoball sequencing represents a significant innovation in next-generation sequencing technologies, offering a unique combination of high throughput, accuracy, and cost efficiency. Its distinctive approach of rolling circle amplification and patterned array sequencing enables large-scale genomic studies with reliable data quality. While it faces challenges related to read length and platform accessibility, ongoing developments promise to expand its applications and impact in genomics research and clinical practice.

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**Meta Description:**
DNA nanoball sequencing is a high-throughput next-generation sequencing technology that uses rolling circle amplification to generate compact DNA nanoballs for efficient and accurate genome sequencing. It offers cost-effective, scalable sequencing suitable for diverse genomic applications.