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PopGen's Reflex Method Helps Sequence Single Gene in Many Samples; Potential Use to Build Long Reads


A new targeted enrichment method called Reflex, developed by UK startup Population Genetics Technologies, enables scientists to sequence single genes in several thousand samples at low cost and from small amounts of DNA.

According to Andrew Slatter, one of the lead authors of an article in Nucleic Acids Research this month that describes the method, Reflex is most suitable for sequencing discreet contiguous DNA regions – such as one or two genes – in a large number of samples. As such, it is at the opposite end of the target enrichment spectrum of methods that capture an entire exome – thousands of genes – in a single sample.

The company is also working on a variation of the method to reconstruct long synthetic sequence reads from short reads that is akin to Illumina's Moleculo technology.

Following a recent change in its business model, Population Genetics is making Reflex, which is patent-protected, available as a service to agricultural biotechnology companies. It also plans to license the technology to others who could provide it to end users and to use Reflex in partnerships with cancer diagnostics and infectious disease firms.

Reflex, which was originally conceived by Sydney Brenner, a Nobel laureate and co-founder of Population Genetics, could be especially useful for identifying rare variants in a target region, whether in thousands of individuals, the cells of a tumor, or in a virus population.

The Reflex workflow starts with a long-range PCR reaction, typically 2 to 10 kilobases in length, to amplify the target. This introduces a sequencing adaptor, a sample-specific barcode, and a Reflex sequence at one end.

Long-range PCR amplicons from many samples, typically 384, are then pooled in equal amounts. The next step generates a tiled series of 'daughter PCR products' across the target that each retain a copy of the sample-specific barcode.

In this step, the scientists first generate a PCR product that has the adaptor, barcode, and Reflex sequence copied to both ends, a process that involves the formation of an intramolecular loop, mediated by two inverted Reflex sequences. They then generate the daughter PCR products, which are compatible in length with the read length of a next-gen sequencing platform. Finally, they pool equal amounts of the barcoded daughter PCR products for sequencing.

Adding a second barcode to each pool of 384 samples has allowed the company to sequence more than 3,000 DNA samples in a single Illumina MiSeq run, though the method is independent of the sequencing platform.

Similar to other PCR-based enrichment methods, Reflex requires low nanogram amounts of input DNA per sample. According to the company, it can target members of multigene families with high specificity.

The cost per sample depends on the number of samples analyzed, the size of the target, the number of long-range PCR reactions required, and the sequencing platform used. Most of the work involves optimizing the initial long-range PCR reaction and synthesizing the oligonucleotides to generate the daughter PCR products, according to Slatter.

In their paper, the scientists estimate that sequencing a single 10-kilobase long-range PCR amplicon in 96 samples on an unspecified Illumina platform would cost about $81 per sample, and sequencing it in 9,600 samples would cost about $10 per sample.

"For anything over 100 samples, we are [cost-]competitive," Frank Massam, Population Technologies' chief commercial officer, told In Sequence. "And when you get up to thousands, we're very competitive indeed."

The company is also working on a version of Reflex that would allow it to reconstruct long sequence reads from short reads, similar in principle to Ilumina's Moleculo method. This version, which the firm is about to test in a study with an unnamed industry partner, requires the addition of a second molecular barcode, in addition to the sample barcode, to identify the original molecule from which a sequence read derived.

The approach would work in solution and needs no additional instrumentation, Slatter said, which likely sets it apart from Illumina's Moleculo. "From what we know about [Moleculo], it's based on a limiting dilution of molecules via some kind of compartmentalization," he explained. "The Reflex reaction works via an in-solution intramolecular barcode transfer, so you would not need to compartmentalize."

So far, Population Genetics has used the Reflex method, which it has been offering as a service for more than a year, in four projects with human DNA as well as several projects in plants. These have used Illumina, Ion Torrent, and Roche 454 platforms for sequencing, though the majority of customers have opted for the Illumina MiSeq.

One project, with researchers at the Wellcome Trust Centre for Human Genetics in Oxford, involved sequencing a 4.5-kilobase gene, identified in a genome-wide association study of coronary artery disease, in 3,000 individuals.

David Buck, head of high-throughput genomics at the center, told In Sequence that for this project the Reflex technology "seemed like the perfect solution on paper, able to focus on a single gene and cheaper per sample than any other available technique at the time."

A pilot project to sequence all 3,000 samples in a single MiSeq run "went well," he said, with all but 11 bases of the target gene covered. The full project faced a few challenges related to the adapters used by Population Genetics, which contained a mono-sequence that interfered with prephasing and phasing calculations that the Illumina technology makes based on the first 15 bases, but he said his team found a way around that.

Despite these challenges, "high-quality data was generated for the majority of samples and the project completed successfully," Buck said, adding that a manuscript containing the results is currently under revision at a scientific journal.

"The technology, as long as you can amplify your target region, clearly works and is cheaper than competing technologies," he said, adding that he would use it again for other projects that require sequencing of a single gene, if Population Genetics continues to offer it as a service.

According to Massam, the company recently narrowed its focus to three areas, after "finding that we were getting swamped with too many applications and potential projects" for service work.

Going forward, it will provide services for all its technologies to agricultural biotechnology companies only. Reflex is not Population Genetics' only child: the firm also offers VeriTag, a technology to accurately identify low-frequency variants in mixed tumor samples or virus populations that it published two years ago (IS 5/11/2011); as well as a pre-enrichment pooling , tagging, and sequencing approach for targeted sequencing in larger numbers of samples.

Massam said the company has contracts with several agbio firms worldwide. In 2011, for example, it signed a research agreement with Syngenta Biotechnology, in conjunction with a Series B financing round, to validate the use of its technology in plant breeding studies.

In addition, Population Genetics is looking to partner with cancer diagnostics and infectious disease companies to commercialize the Reflex and VeriTag technologies, either through collaborations or under license.

Massam said the company is in discussions with several potential partners, both in the UK and in the US, some of which are "at an advanced stage."