Sample Prep Finds a Target in Sequencing

Next-generation sequencing is among the fastest-growing markets in the life science instrument market, with 2009 instrument demand estimated to increase more than 40%. The advent of next-generation sequencing has birthed the need for novel approaches to sample preparation (for the purpose of this article, sample preparation encompasses purification, fragmentation, target capture and enrichment prior to sequencing).

One of the applications for which next-generation sequencing is most in demand is targeted resequencing of mammalian genomes. Targeted resequencing is the sequencing of specific genomic regions and targets. It is currently being used for disease research to study genetic mutations and variations. To improve the sample preparation process for next-generation sequencing applications, with a focus on targeted resequencing, Agilent and RainDance Technologies have released new products.

The standard capture and enrichment method for targeted resequencing, PCR, is rife with problems. PCR limits the number of multiple capture reactions that can be run simultaneously before affecting yield and specificity. It is also prone to generating useless data (called artifacts) and can be self-terminating. The foremost problem is that a researcher must run multiple PCR reactions to obtain an ample amount of target product, which is both expensive and time consuming.

One solution to these problems is the adaptor-ligation method. Prior to PCR, a double strand of DNA containing a specific PCR primer is linked to genomic targets, allowing for better target capture results. Drawbacks of this method are that it can be labor intensive for large regions and that researchers are limited to targets that are enzymatically compatible with the primers. Alternatives to PCR include multiplex probe–based DNA methods, in which oligonucelotide probes hybridize and bind with complimentary genomic targets. This method has distinct advantages over PCR, the biggest being that it is efficient and can generate a sufficient amount of product from a single multiplex reaction.

Agilent’s microarray technology led it to enter the sample preparation market for next-generation sequencing with an oligonucleotide probe–based target capture method. Agilent’s Oligo Library Synthesis technology enables the creation of long biotinylated RNA in complex mixtures. Dr. Fred Ernani, product manager for Emerging Genomics Applications at Agilent, explained why the company entered the targeted resequencing market: “Because of next-generation sequencing being a growing market, and because the common problem in the workflow across all the next-generation sequencers in the industry is that they simply aren’t capable of targeting specific areas of interest in genomes, we saw this to be a substantial business to get into.”

Agilent collaborated with the Broad Institute of MIT and Harvard and licensed the Institute’s solution-phase capture technology, leading to the development of the SureSelect Target Enrichment System, which will be released on February 26. The System’s genome partitioning kit includes up to 55,000 biotinylated cRNA probes, buffers and magnetic beads. The System is scalable due to its reaction being both chemical and enzymatic. “The smallest kit size is for 10 reactions, at least in the custom format,” said Dr. Ernani. “The researcher will be able to design a kit and still cost-effectively test 10 samples, then scale up the study by the same technology, but run thousands of samples in a very cost-effective way due to the nature of this kit.”

Agilent offers a free web service for kit customization. In addition, the company also has a standardized catalog offering. “Our kit that will have a catalog offering is more a demonstration of this type of technology. That kit has been designed to capture the exons of the X chromosome,” stated Dr. Ernani. The System uses standard lab equipment, such as microliter plates, and requires 3 µg of gDNA. It is currently designed to fit into the Illumina Genome Analyzer workflow, and Agilent is working on integrating it with Applied Biosystems’ SOLiD System.

Later this year, Agilent will release an oligonucleotide probe–based solid-phase capture system, which uses a microarray. “The array capture method is the more manual method and, at least upon initial release, it will capture the smaller part of the genome. We see it as the cost-effective way to do smaller studies where people may want to iterate their targeting enrichment design,” explained Dr. Ernani. Each microarray will come at a fixed cost and will run a single capture reaction. This makes the product suitable for optimizing reactions, in that experimental design can be done less expensively. The array-capture method requires 20 µg of gDNA. Online microarray customization will also be available.

The market opportunities for sample preparation products for next-generation sequencing can be understood by the variety of approaches that are being introduced. RainDance Technologies entered the target enrichment market in November 2008 with the RDT 1000 for sample preparation on a massive scale. The RDT 1000 is a standalone automated benchtop system that performs target capture and enrichment in a disposable microfluidic chip. According to Steve Becker, vice president of Operations at RainDance, “the only way to accurately identify mutations that are associated with disease is to examine a large number of individuals—in the range of hundreds to thousands.”

RainDance’s approach to next-generation sequencing sample prep is PCR based. Using the adaptor-ligation method, the PCR reactions take place in a disposable microfluidic chip that produces oil-based microdroplets containing uniform amplified targets that can then be sequenced within any workflow. “The RDT 1000 generates approximately 1.5 million PCR reactions per sample using the RainStorm microdroplet technology. Each droplet measures 22 pL in volume and is capable of producing in excess of one million copies of a single target sequence in each droplet,” said Mr. Becker. “What separates the RDT 1000 process from other droplet-based PCR systems is the fact that any given droplet can contain a different primer pair, resulting in targeted amplification of thousands of genomic loci with many replicate reactions for each locus.” The system overcomes many of PCR’s problems due to the reaction size. For example, adverse interactions between primer pairs and kinetic problems that arise during PCR multiplexing both contribute to unnecessary rate limiting. These factors are avoided with the RDT 1000 due to the individual reactions that take place within the microdroplets.

The automated and nonautomated approaches, and the different capture methods used for sample prep for next-generation sequencing, show the opportunities that exist in the ancillary markets to next-generation sequencing. Sample preparation will continue to advance in order to keep up with the evolving needs of the next-generation sequencing market. “The capabilities of [next generation sequencing] instruments are going from 35 base reads, to 50, to 75 base reads. They put out a lot more data and as these instruments change, so do the needs of the users of these instruments,” said Dr. Ernani.

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