Current next Generation Sequencing (NGS) library construction is a bottleneck in the sequencing workflow. The processes of library construction for DNA and RNA sequencing are complex, error prone, costly, and time consuming. Current commercial products are multistep, multi-hour sample processing that tend to be cost and labor prohibitive. These issues have created a demand for a simple, rapid, and cost-effective library construction product that offers users application flexibility while minimizing construction complexity for low to ultra-high throughput sample processing. SeqOnce has developed a novel library technology that is rapid and minimizes sample processing complexity. The five tube RhinoSeq kit is comprised of formatted master mixes for a simple and stable user workflow. The 12 minute library construction uses a single master-mix that when combined with fragmentation and PCR steps produces libraries in less than 50 minutes. A size selection step occurs after PCR and the PCR free protocol is less than 30 minutes.
The RhinoSeq kit contains three master mixes and includes adapters, which promotes product flexibility across multiple applications. With multi-day ambient temperature stable master-mixes, automation on liquid handling platforms is effortless due to the simple workflow. The technology has been validated with multiple samples (ranging from microbial to human) and supports a wide range of nucleic acid types and input range (5ng- 400ng). The resulting sequencing data is either equivalent or superior in quality to other competing library products, with excellent performance across variable GC content genomes. The technology is currently optimized for the Illumina platform and alternative NGS platforms are under evaluation for future development.
Authors: Joseph Dunham, Benjamin Akins, Christopher Angermayer & Timothy Harkins
Continue reading or see the poster here.
Workflow & Components
- Libraries in under 50 minutes
- Two-step protocol, three-step with PCR
- Reagents stable for >24 hours at ambient temperature
- Minimal setup due to master mix format
Five reagent tube kit:
Three master mix tubes for:
- Library Construction
Two remaining tubes:
- Stock Adapter
- Adapter Dilution Buffer
Fragmentation & Library Reagents
Fragmentation Master Mix Characteristics:
- Single volume for 5– 400ng inputs
- Adjustable for controlled fragment distribution
- No buffer or enzyme pipetting or mixing
- Single tube buffer and enzyme formulation
- Identical conditions for microbial and human gDNA fragmentation
FRAGMENTATION MASTER MIX REAGENT
SINGLE TUBE – SINGLE VOLUME
LIBRARY PREP MASTER MIX REAGENT
SINGLE TUBE – SINGLE VOLUME – 12 MIN PREP
Initial testing of the RhinoSeq kit was performed by generating microbial large insert libraries for whole-genome sequencing. Libraries were generated from 50ng of C. difficile, E. coli, and B. pertussis gDNA. As illustrated below the RhinoSeq kit resulted in minimal start site and GC bias.
Third Party- Independent Tester
Shallow Whole Genome Sequencing
The RhinoSeq DNA Library Prep kit allows for DNA library construction that is both straightforward and rapid. The total library processing time (less than 50 minutes) is substantially faster than other commercial kits (three to four times faster). The simplicity of the five tube two-step protocol, three-step with PCR, enables both experienced and inexperienced users to easily prepare libraries for sequencing. The flexibility of this system also allows users to readily scale the number of samples processed and is easy to automate.
Download the poster here.