SageELF Enables Targeted RenSeq Capture for PacBio Sequencing

We’ve had targeted capture on the brain lately as we work with collaborators to put the finishing touches on our HLS-CATCH method for selecting large genomic elements using Cas9 guides and the SageHLS system. So today, we wanted to revisit a great capture paper from scientists at the Earlham Institute and other organizations.

Targeted capture and sequencing of gene-sized DNA molecules” came out in BioTechniques a year ago from lead author Michael Giolai, senior author Matthew Clark, and collaborators. It’s a terrific effort by scientists who know the value of nailing down every piece of a protocol in order to get the most robust, reliable results. For this method, the team focused on sample prep for the PacBio sequencing pipeline, and they made extensive use of the SageELF platform to optimize results.

Scientists began with RenSeq, short for R-gene enrichment sequencing, which was originally developed to help short-read sequencing platforms conquer challenging, highly repetitive sequence regions. In theory, incorporating long-read sequencing would be even more successful. “Here, we demonstrate that the use of RenSeq on DNA fragments up to 7-kb long in combination with PacBio sequencing results in full contig assemblies covering entire R-gene clusters with inter- and intragenic regions,” the team reported in BioTechniques. “Our approach can be used to capture, sequence, and distinguish between similar members of the NB-LRR gene family—key genes in plant immune systems.”

Giolai et al. focused on key steps in the RenSeq capture process: shearing, size selection, and PCR amplification. For more information about how the SageELF instrument makes a difference in this method, check out table 1 and figures 1 and 2 in the paper. Ultimately, the approach allows the PacBio platform to sequence only the longest fragments, generating the longest reads possible for a library and helping distinguish even high-identity sequences.

“This makes the optimized RenSeq protocol not only interesting for very accurate long-read R-gene enrichment,” the scientists noted, “but also as a robust and reproducible technique for the hybridization-based specific capture of fragments up to 7-kb in any genomic context—and it could be used for gap filling, other types of genome finishing, or structural variation verification.”

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