Applications for Next-Generation DNA Sequencing to Accelerate Quantification and Library Prep

For scientists looking to perform next-generation DNA sequencing, a new range of tools has been developed that can greatly simplify and accelerate library preparation as well as enabling absolute quantification of amplified sequences.

Next-generation sequencing has the potential to transform life science in fields as diverse as human genomics, species discovery, and personalized medicine--any field in which advancement depends on a deeper understanding of genomic variation and transcriptome complexity. Compared to generation I (Sanger) sequencing of the Human Genome Project, generation II technology lowers the cost per base dramatically while producing giga-base results per day. In spite of this cost-effectiveness, the technology faces barriers to widespread adoption, among them the complexity of sample preparation and quantification, particularly when preparing 100,000,000 or 1,000,000,000 amplicons for deep resequencing.

The Fluidigm (South San Francisco, CA, USA) Access Array integrated fluidic circuit (IFC) eases the burden of library preparation for resequencing or long-range polymerase chain reaction (PCR) because it facilitates the amplification and bar-coding of 48 samples in parallel, with minimal time and labor and in as few as three hours with yields that are equimolar and routinely at 100,000,000 or 1,000,000,000. Because every sample may be identified by its barcode, all 48 samples can be multiplexed at the sequencing step. This capability dramatically reduces the time and money required for large-scale projects, opening new vistas of research. For example, scientists interested in large cohort studies can comparatively easily sequence specific genes of interest from hundreds of individuals in just a matter of weeks.

While simple in theory, next-generation sequencing can be very challenging because of the lack of an easy and effective method to quantify the concentration of molecules within sequencing libraries. The Slingshot kit from Fluidigm is the first product to easily determine the optimal sample concentration-before samples advance to sequencing. With SlingShot, digital PCR is used to easily determine the absolute quantity of target sequences, thereby improving sequencing results generally and enabling the sequencing of previously undetectable samples.

By exploiting the unique microfluidic properties of IFCs to detect only amplifiable molecules within the sample mixture--SlingShot has extremely small sample requirements typically offering a 1,000-fold reduction in input volumes. As IFCs are able to count individual molecules--using digital PCR--SlingShot eliminates titration steps at the sequencing stage. SlingShot is also easy-to-use requiring only three liquid-handling steps per library. The unique power of SlingShot can be used to improve the productivity of next-generation sequencing tools from almost any manufacturer.

Fluidigm develops, manufactures, and markets proprietary IFC systems that considerably improve productivity in life-science research. Fluidigm's IFC enable the simultaneous performance of thousands of sophisticated biochemical measurements in extremely minute volumes. These "integrated circuits for biology” are made possible by miniaturizing and integrating liquid-handling components on a single microfluidic device (IFC). Fluidigm's systems, consisting of instrumentation, software, and single-use chips, increase throughput, decrease costs, and enhance sensitivity compared to conventional laboratory systems. Fluidigm products have not been cleared or approved by the U.S. Food and Drug Administration for use as a diagnostic and are only available for research use

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