Agilent innovations help unleash imaginations of researchers. Projects in Agilent Laboratories, our central advanced research facility, begin as an exploration of a very high-risk technology, that if successful, would really drive the fundamental questions that researchers could ask and answer. When Agilent business units continue R&D and commercialize products based on Labs research, innovations typically represent 10, 100 and even 1,000 fold improvements in performance or ease of use over the previous solutions.
Agilent R&D teams include broad and deep technical expertise because to solve the problems that really change the game requires innovation at the interfaces across multiple science and engineering disciplines. We strive to stay enough ahead of where researchers are today so that we can develop the new technology into a robust product in the timeframe for their next-generation research challenges. Our teams collaborate extensively with thought leaders in academic, government and industrial research venues so that Agilent’s R&D investments will fuel the right products at the right time for emerging market needs. These relationships also augment and validate the value of our research.
One example of Agilent’s significant R&D focus is development of world-class oligonucleotide synthesis, which is at the foundation of target enrichment in simplifying DNA sequencing workflows. We provide oligos from the kilo scale to fractions of femtomoles – and in multiplexed mixtures from 2 to 55,000 unique sequences in oligo libraries. Whereas traditional methods can synthesize very good 40- and 60-mer oligos, only Agilent can supply researchers with highest quality oligos of length up to 200-mers, continually advancing genomics and the understanding it will bring.
Although the evolution of powerful instrumentation is revolutionizing genome sequencing, users still face fundamental challenges: full genome sequencing that is still relatively expensive; sample preparation that is costly, time-consuming and complex; and enormous amounts of data that require unprecedented levels of storage and analysis.
Agilent offers a portfolio of workflow products to make genomic experiments run faster, better and/or more cost effectively. We help scientists get their data quickly and move on to their next follow-up experiment. In this article, we highlight workflow contributions that Agilent is providing to help advance the rapidly evolving field of DNA sequencing, today and into the future
Targeting specific genomic regions
Life science researchers can target genomic regions of interest with the Agilent SureSelect Target Enrichment System. This platform enables researchers to increase the speed and cost-efficiency of next-gen sequencing workflows, conduct studies that were previously unfeasible due to the rarity of DNA sample, sequence a larger number of samples and increase throughput with automation. We developed the SureSelect Target Enrichment System by combining the depth of our in situ array technology with innovative bioreagents from Agilent’s Stratagene division and our state-of-the-art lab automation capabilities.
The Agilent SureSelect Target Enrichment System is based on an in-solution design that uses biotinylated RNA “baits” to fish targets out of a “pond” of DNA fragments. Customer-specified libraries are generated in solution, of up to 55,000 biotinylated RNA probes targeting up to 6.8Mb of genomic regions (~10Mb before repeat masking), delivered in single tubes. The capture probes are 120 base pairs long, the longest currently on the market for this application. This length increases the probe effectiveness at capturing DNA that contains unknown mutations, such as single nucleotide polymorphisms, insertions or deletions. Because the protocol is tube-based, researchers can readily automate and scale the platform for studies from 10 reactions through thousands, providing unmatched flexibility for researchers.
Researchers using the Agilent SureSelect Target Enrichment System are discovering links between specific genes and mutations to syndromes such as cleft palate and diseases such as leukemia.(1)
Our proprietary capability to make very long, high quality DNA and RNA cost effectively has enabled us to provide new capabilities beyond microarrays. These include the Agilent SureSelect Target Enrichment System and custom manufacturing of oligos to supply the pharmaceutical and biotech industry’s needs for emerging RNA-based therapeutics. When researchers realize they can access tens of thousands of very high quality oligos at less than one cent per base, they begin to envision experiments that previously would not have been considered possible.
A DNA oligo with a specific base sequence must be synthesized serially, one nucleotide at a time, as shown in the figure below. To achieve high fidelity oligos, the synthesis needs to address deblock and coupling yields as addition of each base extends the oligo polymer. Typically, while coupling can be high and any errors compensated by capping, the deblock step has been a limitation. Because this step is reversible, it can lead to misincorporation of bases resulting in random deletions. In addition, the depurination side reaction needs to be eliminated to achieve long lengths; it has been the main limitation preventing routine synthesis of oligos more than 100 base pairs.
Online sequence design
Researchers can create custom Agilent SureSelect Target Enrichment System kits specifically for their studies on Agilent’s web-based design tool, eArray, the same capability Agilent uses for catalog kits. Whether the goal is to capture a specific set of exons on the X chromosome or a number of defined regions of interest based on genome-wide association studies, eArray contains many key genomes and allows biologists to upload their own sequences. Customized experiments can be designed without up-front design fees or the need to invest in specialized bioinformatics tools. eArray’s design algorithms have been proven to work over a diverse set of genomic locations, such as small and large exons, short and long contiguous genomic targets, genome targets within repeat areas and non-coding DNA.
Also available is the SureSelect Target Enrichment System Human All Exon Kit that targets all human exons in a single tube. It covers 1.22 percent of human genomic regions corresponding to the NCBI Consensus CDS Database (CCDS), including more than 700 human miRNAs from the Sanger v13 database and more than 300 additional human non-coding RNAs in a single tube.
Agilent provides specialty reagents and everyday essentials for purifying, quantifying, amplifying and analyzing nucleic acids. Examples for genomics sample prep include PCR enzymes to amplify a single or few copies of a piece of DNA and reverse transcriptases to transcribe single-stranded RNA into double-stranded DNA.
Automated Sample Prep and Sample Enrichment
The Bravo Automated Liquid Handling Platform provides robotics for next-generation sequencing workflows to automate sample preparation and sample enrichment prior to sequencing. Automation enables faster, higher quality and more reproducible sequencing compared to manual preparation, which is more time-consuming and a source of variability.
The Bravo nine-plate position footprint easily fits inside a standard laminar flow hood, enabling automated liquid handling for genomic applications. The unit automatically performs multi-channel pipetting and serial dilutions in 96- and 384-well formats. It also can dispense to a single well. Researchers can customize the deck easily to meet a wide range of applications including general liquid handling, serial dilutions, PCR preparation and cleanup, cell-based assays, filtration assays, magnetic bead based separations.
After a next-generation system has sequenced the sample, researchers need to analyze and extract knowledge from the raw data. They also may need to integrate other heterogeneous data from previous experiments and draw conclusions that cross boundaries of application disciplines, such as genomics, genetics, metabolomics, proteomics and biomarker screening.
The Agilent GeneSpring Analysis Platform offers an expanding suite of integrated software applications for established and emerging technologies. GeneSpring integrates data and results from multiple applications so researchers can quickly analyze, compare and view results from different experiments in a single user interface.
The modular data storage architecture of GeneSpring handles data from applications such as genotyping, microarray-based comparative genomic hybridization, biomarker profiling, ChIP-on-chip location analysis, gene expression profiling, mass spectrometry-based proteomics and next-generation sequencing.
GeneSpring displays data in ways that help researchers conceptualize the information in their data and convey it to their peers. The various types of plots, graphs and diagrams highlight different aspects of the data and allow researchers to extract visual information in multiple ways.
Agilent has been supporting the GeneSpring Analysis Platform for 10 years, expanding our informatics capabilities to enable researchers to accelerate discovery and draw conclusions from studies that intersect scientific boundaries. After researchers analyze their results and compare these with their beginning hypothesis, they can move quickly to design more content with SureSelect and begin their next experiment.
Future directions in data analysis
The GeneSpring platform will continue to be Agilent’s platform of choice to support all the different multi-omics data types researchers will need to relate together. While it will become easier to acquire enormous data sets of raw genomic information, it will be even more critical to provide bioinformatics and applications to help researchers manipulate, share and make sense of all this data.
We’re continuing to extend and advance GeneSpring’s capabilities in statistical analysis, data management and visualization to keep pace with the explosion of DNA sequence information from advancements in next-generation sequencing. Future directions for GeneSpring will enable researchers to investigate and answer an ever-wider range of biological questions.
While GeneSpring provides modeling capabilities to allow researchers to generate and explore biochemical networks, we’re also implementing a pathway level of integration through open source -- adding to and integrating with Cytoscape. Pathway and network diagrams help place statistical results in a biological context. Direct navigation between biological pathways and their associated genes will provide even richer user experience and systems-level insight for future research including emerging applications such as synthetic biology.
Steps toward synthetic biology
Synthetic biology - the redesign of biological systems and their component parts for useful and practical purposes - is an important emerging field. Because it combines multiple disciplines, synthetic biology relies upon many experimental approaches, reagents and measurement solutions. It is already emerging as a new paradigm for chemical manufacturing, and many believe biofuels produced through synthetic biology will help address the energy challenges our world faces. A fundamental enabler of this field will be the ability to synthesize high quality DNA controllably with reproducibly defined sequence and length as addressed earlier. The field also will require significant technology advances for the assembly and characterization of genetic components, networks and genomes – work that leverages Agilent’s strengths in innovation.
Synthetic biology has many parallels to the electronics industry. Standardized, integrated electronic parts, devices and tools have enabled a well-developed, mature industry. Advocates of synthetic biology similarly champion development of tools and processes that will enable standardized, integrated biological parts, devices and networks to efficiently manufacture high-value chemicals, pharmaceuticals, bio-fuels and nanomaterials. Applications in alternative electronics and DNA based computation also are advancing throughout the world.
Many thought leaders in the field believe that when efficient DNA assembly methods and validation tools are developed, gene synthesis will become technologically accessible. In parallel, appropriate oversight measures will need to be defined and in place to ensure that use of such technology is in the interest of mankind. Enabling such capability would greatly benefit a broad range of research areas and significantly influence the direction of these areas well into the 21st century.
Agilent is committed to accelerating advancements in DNA sequencing. We continue to collaborate with scientists and thought leaders to develop innovative solutions to problems of the next year and the next decade. We are charting future directions and supporting the entire innovation process to get inventions out of our laboratories and into products and solutions that create real value for our customers.
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(2) Research With Agilent Oligonucleotide Libraries
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