High Throughput Sequencing (Next-Generation Sequencing)
Over the past five years, so called “Next-Generation” sequencing technologies have made accessible data capable of answering questions fundamental to our understanding of life and the factors that govern human health. The combination of the vast increase in data generated, coupled with plummeting costs required to generate these data, has rendered this technology a tractable, general purpose tool for a variety of applications.
- Whole Genomic Sequencing
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The sequencing of the human genome provided an invaluable reference for researchers around the world. The data generated by that effort can be viewed as a critical first installment in a library of information that is pushing the frontiers of medicine. Whole genome sequencing of multitudes of individuals from defined patient cohorts is the next logical passage in this series. Discovery of genotypic elements impacting the phenotypic observations effecting human health is only possible through the systematic, genome-wide analysis within and across nucleic acids derived from individuals demonstrating affliction at the cellular level. Associations of structural variation and rare mutations with phenotypic observations presents a pathway towards personalized medicine capable of transforming the way the world views patient care.
• Illumina
• SOLiD
• Pacific Biosciences - Targeted Resequencing
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Aggressive progress aimed at understanding the critical factors of disease is imminent. While most mutations are present within regulatory, intronic elements of the genome, the vast majority of the mutations that effect human health are located in the roughly one percent of the genome that encode proteins. Dubbed the “exome”, sequencing of these regions alone provides an efficient means to rapidly discover genomic variants that have the greatest impact on disease. Several technologies have been developed to accomplish directed segmentation of the genome, capturing targeted regions in a highly multiplexed manner. These technologies serve as a valuable front end to next generation sequencing, and recognize the efficiency gains of sequencing solely exonic regions.
Additionally, the methods developed for the multiplexed capture of the protein coding regions have further utility in downstream validation and extension studies focusing on discovered variants across high numbers of individuals. These studies are geared at refining the associations of the subtleties of disease states with variant profiles, and gaining statistical confidence in concordance through surveying large numbers of individuals.
• Roche NimbleGen SeqCap EZ
• Agilent SureSelect
• Illumina TrueSeq - Transcriptomics
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The utility of DNA sequencing technologies has been augmented to allow for investigation of RNA expression profiles. Through sequencing of cDNA fragments generated via reverse transcription of RNA, researchers are enabled to interrogate the broad range differential expression of transcripts across cell states. This information is critical towards elucidation of disease progression and the factors that affect it. Increased understanding of disease progression at the molecular level has substantial implications towards the development of personalized diagnoses, treatment plans, and therapeutic targets focused on efficient and effective patient treatment.
A variety of approaches have been created to facilitate the broad scale sequencing of RNA transcripts. A variety approaches are available for this technology, also known as “RNAseq”. These variations are aimed at capturing RNA transcripts derived from a single-cell, and techniques used to ensure strand specificity of sequenced cDNA fragments.
• Illumina TrueSeq RNA
• New England Biolabs NEBNext RNA Sample Prep
• Nugen Encore NGS Library Prep - Epigenetics
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As researches continue to push the discovery frontiers of cellular development and differentiation, technologies allowing for deeper understanding of molecular influences on these processes are required. Epigenetic analysis of DNA modifications presents a means for rapid discovery of transcription factors. Understanding of the effects of methylation profiles and protein-DNA binding offers clues towards the means and processes that regulate expression. These data are of further value towards the development of therapeutics targeted at controlling transcription factors at the molecular level, thus allowing for control of disease genesis and progression.
Overcoming the Bottleneck
The accessibility and affordability of sequence data is enabling science in ways once thought unimaginable. Continued advances have rendered these technologies omnipresent, and the number and variety of applications for which these data can be used continues to grow. The ease of use, low cost, and astronomical throughput of these systems has shifted the burden of technology development towards processes aimed at recovery and tailored processing of samples upfront to sequence analysis. The need for a suite of tools capable of handling large numbers of samples in application specific processes is quickly becoming the bottleneck in realizing the opportunities toward advancement of diagnostics, personalized medicine, and novel therapeutics poised to overcome the increased challenges of modern health care.
Caliper Life Sciences is committed to being the premier provider of tools capable of overcoming sample preparation bottlenecks presented by technological advancements in sequencing technologies. Our suite of automation and microfluidic solutions provide scalability of throughput, flexibility of application, and the ability to evolve with changing sample preparation requirements.