DNA Library Prep for DNA Nanoball Technology Sequencing Platforms

Next-generation sequencing (NGS) is advancing translational research and increasing our understanding of human health, from improving diagnostic testing accuracy to the development of precision-based medicines for cancer and infectious disease. As the need for clinical applications within NGS continues to grow, new sequencing technologies and platforms are being developed to keep up with the demand. One such novel sequencing technology platform is based on DNA nanoball (DNB) technology which combines single-stranded circular (ssCir) library construction, generation and loading of DNBs onto patterned nanoarrays, and combinatorial probe anchor synthesis (cPAS) sequencing.¹

While this technology delivers robust data with rapid turnaround times at reduced per-base cost compared to previous sequencing techniques, researchers need to ensure their DNA library preparation solutions, which may come from third parties, are compatible with these novel platforms. Through extensive evaluation, Roche has shown that its DNA library prep kits can generate high-quality libraries for whole-genome sequencing (WGS) on platforms employing this DNB technology.²

The DNB sequencing platform can be a cost-effective alternative to short-read sequencing technologies that provides researchers with an innovative solution for generating reliable data.³

After a double-stranded DNA library is constructed, the steps of the DNB sequencing technology include:^1,3-5

1. Library denaturation
2. Single strand circularization through splint oligo ligation
3. Exonuclease digestion
4. Rolling circle amplification (RCA) with Phi29 DNA polymerase to create DNBs
5. DNB loading onto patterned nanoarrays
6. cPAS sequencing

Researchers have shown that the technology can be used for complete genome association studies and can be utilized to identify rare variants and somatic mutations that are important in clinical settings.³

Comparative studies between the DNB platform and other technologies show that these sequencers produce similar, high-quality data in WGS, single-cell RNA-seq and bulk RNA-seq.^6-8

There are several advantages to using the DNB technology:^{3,4, 6-8}

• Low error rate
• Reduced risk of optical duplicates
• Reduced duplication rates
• Shorter sequencing time
• High base calling accuracy
• Reduced adapter carryover
• Negligent index hopping
• Comparable data outputs at reduced cost/base

While there are several advantages to using DNB, there are a few disadvantages:

• Longer workflow until sequencing-ready library
• Few third party library prep solutions
• Shorter read lengths
• More hands-on-time
• Multiplexing requires complicated adapter set-up

Roche has developed DNA library prep kits and accessory reagents that are compatible with the DNB sequencing platform for library construction.² But researchers need to consider several important parameters before using this workflow.

Selection of an appropriate library prep kit: which will depend on preference for use of mechanically sheared DNA or integrated, low-base enzymatic fragmentation.

Size selection for whole-genome sequencing applications: since a uniform size distribution is vital for the DNB technology, size selection is critical and therefore high DNA inputs ≥500 ng are needed, especially when size selection is employed.

Roche’s library prep solutions provide rapid and high-quality library construction that can significantly improve turnaround time and cost-effectiveness. These prep kits can construct libraries in less than three hours, faster than other sample prep methods.9 Benefits include lower duplication rates and higher sequencing coverage, ability to execute PCR-free workflows and employing qualified automation methods on many of the third-party automation platforms.⁹

Roche’s sample prep solutions for library construction are proven, simple and complete, helping researchers unlock the potential from each of their samples.