Next Generation Sequencing (NGS) library construction is a workflow bottleneck. The processes of library construction for DNA and RNA sequencing are complex, error prone, costly, and in general are me consuming. Current commercial products offer multistep, multi-hour sample processing that can be cost and labor prohibitive. These issues have created a demand for a simple, rapid, and cost-effective library construction products that offer 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 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 single size selection step occurs after PCR. The PCR free and pre-fragmented DNA protocols are less than 30 minutes.
The kit contains three master mixes and includes adapters, which maximally promotes product flexibility across multiple applications. With master-mixes that are stable for multiple days at ambient temperatures, automation on liquid handling platforms is effortless due to the simple workflow. The technology has been validated on a variety of human sample types and applications with input ranges of 10ng-100ng. The sequencing data is either equivalent or superior to other competing library products, with high mapping rates and excellent performance across variable GC content. 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 & Christopher Mason
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Workflow & Components
Single Tube Additive Reagent Library Prep:
- 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:
1. Fragmentation
2. Library Construction
3. PCR
Two remaining tubes:
1. Stock Adapter
2. Adapter Dilution Buffer
RhinoSeq Reagents
Fragmentation Master Mix Characteristics:
- Single volume for 5– 100ng inputs
- Adjustable for controlled fragment distribution
- No buffer/enzyme pipetting or mixing
- Single tube buffer/enzyme mix
- Identical conditions for microbial and human gDNA fragmentation
FRAGMENTATION MASTER MIX REAGENT
SINGLE TUBE – SINGLE VOLUME
Various input amounts of Human gDNA were fragmented using the RhinoSeq kit to generate short (left) or long (right) fragment distributions. After fragmentation, samples were 2X SPRI® bead cleanup and analyzed using an Agilent High Sensitivity DNA Assay™.
Various input amounts of Human gDNA were fragmented to generate short (left) or long (right) fragment distributions and libraries constructed using the RhinoSeq kit. Short insert libraries were size selected for greater than 180bp and long insert libraries were dual size selected with SPRI® beads. Libraries were analyzed using an Agilent High Sensitivity DNA Assay™.
LIBRARY PREP MASTER MIX REAGENT
SINGLE TUBE – SINGLE VOLUME – 12 MIN PREP
Nucleotide content over a 150bp window of sequencing read illustrates similar start-site complexity between Covaris (left) and random enzymatic fragmentation (right) using the RhinoSeq kit.
MICROBIAL SEQUENCING
WHOLE GENOME
Third Party- Independent Tester
MICROBIAL COMMUNITY
HUMAN WGS
DEEP COVERAGE
Joseph Boland – Independent Tester
NCI/NIH, Leidos Biomedical Research Inc.
SHALLOW COVERAGE
HUMAN FFPE
HLA TYPING
Conclusion
The RhinoSeq kit allows for DNA library prep that is both straighforward and rapid. Total library processing time (less than 50 minutes) is faster than other commercial kits (three to four times faster). The simplicity of the five tube two-step protocol, three-step with PCR, enables 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, easy to automate, and can be applied to a variety of sequencing applications.
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