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Belgian Team Reports Protocol to Increase NimbleGen SeqCap EZ Multiplexing Capacity

NEW YORK (GenomeWeb) – Researchers from the University of Antwerp this week reported details of a new method to boost the multiplexing capacity of Roche Nimblegen's SeqCap EZ in-solution sequence capture technology up to 96 samples.

Although SeqCap EZ offers high target enrichment efficiency for both exome and small panel enrichment, its standard workflow only provides labeling of samples with single index adapter, limiting the technology's maximum multiplexing capacity to 24 samples per sequencing experiment, the researchers wrote in a paper appearing in PLOS One.

When it comes to small target sizes, this limitation results in excessive coverage and unnecessary costs, they noted.

To overcome the issue, the team developed a method that allows for multiplexing up to 96 samples by combining SeqCap EZ enrichment with dual-indexed sequencing libraries based on homemade dual-index adapters and blocking oligonucleotides.

As a proof-of-principle experiment, the investigators multiplexed and sequenced 34 DNA samples that were obtained from blood and enriched using a custom NimbleGen SeqCap EZ Choice kit, incorporating their own dual-index adapters and blocking oligos where appropriate as they followed the company's DNA sample preparation and library enrichment protocols.

Pre-enrichment pooling of 4, 8, 10, and 12 samples was also evaluated in order to help cut experimental costs.

The researchers were able to successfully boost the sample capacity of the SeqCap EZ technology to 96, demonstrating that the dual-indexing of sequence libraries was compatible with their modified protocol.

"Analysis of the sequencing data revealed that all included indices were recognized and were well distributed over the pools and the respective samples within a pool," they wrote. A minor variation in distribution fell within the range of standard pipetting inaccuracies.

The study results also showed that the homemade blocking oligos against the dual-index adapter sequences prevented the enrichment of aspecific targets without compromising the enrichment stability over the target region, they added.

In addition, the data demonstrated that pre-enrichment pooling of up to 12 samples was possible and did not lead to significant data loss per sample.

Specifically, sequencing data was compared with SNP array data previously obtained for 30 of the 34 samples. A total of 17 SNPs were shared between both datasets and were concordant in all samples.

Overall, the new protocol represents a "robust and high-throughput screening alternative of target regions up to 7 Mb, reducing experimental costs, limiting the risk for incidental findings, and increasing sensitivity and specificity rates," the scientists concluded.