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Two European Labs Turn to Next-Gen Sequencing for BRCA1/2 Testing

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By Julia Karow

As Myriad Genetics continues to offer comprehensive testing of the BRCA1 and BRCA2 breast cancer risk genes in the US using Sanger sequencing and PCR technology, at least two clinical testing labs at university hospitals in Europe have been eyeing next-generation sequencing platforms, which promise faster turnaround times and lower costs.

While the University of Leeds Institute of Molecular Medicine at St. James's University Hospital performs diagnostic sequencing for the BRCA1, BRCA2, TP53, and other genes on the Illumina Genome Analyzer now, the Center for Medical Genetics Ghent at Ghent University Hospital is currently evaluating the 454 platform for BRCA1 and BRCA2 testing.

According to Graham Taylor, a professor at the Leeds Institute of Molecular Medicine and Leeds Teaching Hospitals, his lab has so far analyzed more than 400 cases on the Illumina GA, using a method he and his colleagues published earlier this year in Human Mutation.

Taylor told In Sequence that his lab is currently the only National Health Service laboratory in the UK that offers an accredited diagnostic service using next-generation sequencing. Establishing this service "required a confluence of two disparate cultures: cutting-edge research informatics and robust diagnostics," he said.

In their paper, Taylor and his colleagues sequenced the BRCA1 and BRCA2 genes in 55 individuals at risk of hereditary breast cancer, whose samples had been previously sequenced in a diagnostic laboratory using the Sanger method. In addition, they sequenced the TP53 gene in 10 individuals with Li-Fraumeni syndrome, where parts of the gene had been previously sequenced, and they analyzed four established tumor cell lines.

For BRCA1 and BRCA2, the scientists amplified the coding regions in 22 long PCR products, constructed barcoded Illumina sequencing libraries, and sequenced 10 samples per flow cell lane on the Illumina GAII, using 51-base-pair single reads.

To align the reads and call sequence variants, they used NextGene and Mutation Surveyor software from SoftGenetics as well as Illuminator, an alignment and variant display program they developed in house.

The researchers found that given a read depth of at least 50-fold, they were able detect all mutations in the BRCA1, BRCA2, and TP53 genes. "Our finding of complete concordance between conventional diagnostic sequence data and the Illumina output in 65 patient DNAs and four cell lines suggests that the technology is ready for use in a diagnostic setting," they concluded.

As with Sanger sequencing, they said, large insertions, copy number changes, and rearrangements would be missed by the method, so additional analyses, for example by multiplex ligation-dependent probe amplification, or MLPA, would be required.

But even with the additional MLPA analysis and confirmatory Sanger sequencing for mutations detected, the researchers estimate that, compared to using the Sanger method, "a 50-percent reduction in consumables costs can be achieved" with the new method, "together with even greater reductions in staff time because the data flow is highly automated, meaning much less checking of sequencing output is required," which leads to faster reporting times.

Diagnostic use of the method, they said in the paper, would require robust operating procedures for sample prep, run quality parameters, and large-scale data handling methods to be established, as well as variant calling and exclusion metrics based on sequence quality and depth of coverage.

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"Currently, the greatest barrier to wide-scale implementation is not the technology itself, but rather the downstream informatics capacity," the authors concluded.

Taylor said that compared to PCR/Sanger sequencing — for which he and his colleagues cited a cost of approximately £1,000 ($1,500) per sample in the paper — the cost of testing has decreased by about half using the Illumina platform. Costs should fall further as the researchers improve the workflow, he said, for example by automating library preparation, and as the output per run of the Illumina sequencers increases.

Taylor said that the NGS-based method also offers a "much reduced" turnaround time compared to PCR/Sanger sequencing, and the retest rate has fallen by a factor of more than ten.

He said the lab has expanded testing by next-gen sequencing to other genes now, including the MSH2, MLH1, and MSH6 genes, though it is still taking "great care to validate the sensitivity and specificity of NGS compared to Sanger sequencing."

Reducing Turnaround with 454

Reducing the turnaround time for BRCA1 and BRCA2 testing was the main motivation for the Center for Medical Genetics Ghent at Ghent University Hospital in Belgium to explore next-generation sequencing.

Three years ago, when the Illumina platform still had a relatively short read length, the center chose to work with the 454 platform and its longer reads, Kim De Leeneer, a graduate student at the center who has been developing the next-gen sequencing assay, told In Sequence. The 454 platform also allowed her to use the same primers the center uses to generate 200- to 300-base pair PCR amplicons for its existing BRCA1/2 test, which uses high-resolution melting analysis, or HRM.

About two years ago, the lab sequenced its first 11 patient samples in a single 454 proof-of-principle run, but sequence coverage between samples differed by as much as 10-fold and the test "was not cost-effective at all," according to De Leeneer.

However, by optimizing multiplexed PCR reactions, the researchers now achieve sequencing coverage that only differs by two-fold between samples, generating 111 amplicons in 16 PCR reactions.

With the Titanium upgrade to the 454 FLX, De Leeneer said, the goal is now to sequence 70 patient samples per run, which takes about two weeks, including sample preparation, plus at least another two weeks for data analysis. This compares with 11 samples the lab currently analyzes per month using its HRM-based test.

The bottleneck is still the data analysis. "We still have some issues there because it's so labor-intensive," De Leeneer said. Right now, the lab is using its own in-house variant identification pipeline, or VIP, which is Linux-based and not very user-friendly. To change that, the researchers are currently evaluating SoftGenetics' NextGene software to see if it performs as well as their own and is easier for technicians to use.

In validating their 454 assay with known samples, the researchers have encountered problems detecting mutations in homopolymers. "Using the criteria we defined to make sure we have a sensitive and specific test, we still cannot detect mutations present in a homopolymeric region," De Leeneer said. A possible solution would be to combine 454 sequencing with HRM on those amplicons that have a homopolymeric stretch.

De Leeneer said she does not expect a 454-based test to reduce the cost of the current HRM assay, which she said is highly optimized, though she did not say how much it costs.

Cost and turnaround time could be further improved by automating sample preparation for the 454, she said, though she cautioned that the multiplexed PCR reactions are very sensitive to pipetting errors.

She said that validation of the 454 test and the new software should be completed by the end of the year. "Then, it's for the lab to decide to switch over or to wait a bit, because we still have a system that is working fine," she said.

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In the meantime, her lab has also brought the Illumina sequencing platform in house. Generally speaking, De Leeneer said, the researchers choose the 454 platform to sequence a "reasonable" number of amplicons in many patient samples, and the Illumina platform to sequence several hundred amplicons in fewer patient samples.

As these and other testing labs are exploring next-gen sequencing platforms, Myriad Genetics is still using Sanger sequencing and multiplexed quantitative PCR assays for its Comprehensive BRACAnalysis test, which is priced at $3,340.

According to technical specifications published on its website, the company PCR-amplifies the patient DNA, running 35 PCR reactions for BRCA1 and 47 for BRCA2. It then sequences the products directly in forward and reverse directions using fluorescent dye-labeled sequencing primers and analyzes the chromatographic tracings for each amplicon using a proprietary computer-based review followed by visual inspection and confirmation. It confirms variants that are potentially clinically significant by another round of PCR/Sanger sequencing. In addition, the company tests for large rearrangements in the two genes using recombination-specific PCR and multiplexed quantitative PCR.

Two years ago, Myriad was testing the 454 platform, initially in plant genome sequencing projects (IS 3/11/2008). At the time, Mark Skolnick, then Myriad's chief scientific officer, told In Sequence that the company was "exploring the feasibility" of using the 454 platform for diagnostic sequencing, though he said changing platforms would involve a lot of effort.

Jerry Lanchbury, Myriad's current CSO, declined to provide an update on these efforts for this article, saying that "it's our policy not to comment on any technical development we might be undertaking."

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