No-Amp Targeted Sequencing Used to Interrogate Disease-Associated Repeat Expansion

A new publication released in PLOS One from scientists at the Mayo Clinic offers a great look at our CRISPR/Cas9-based, amplification-free targeted sequencing method and its utility for accurately sizing a clinically important repeat expansion.
“Amplification-free long-read sequencing of TCF4 expanded trinucleotide repeats in Fuchs Endothelial Corneal Dystrophy” comes from lead author Eric Wieben, senior author Michael Fautsch, and collaborators. This is the second group to use the amplification-free technique for this disease; the first performed their work on a PacBio RS II System, while this team used the newer Sequel System.
What makes the disease such an interesting target for this approach? While Fuchs endothelial corneal dystrophy (FECD), a late-onset degenerative eye disease, affects just 4% of Caucasians in the U.S., more than 75% of those cases can be traced to an expansion of a CAG repeat found in the TCF4 gene. That makes FECD “the most common disease that is attributable to the expansion of a trinucleotide repeat,” according to the paper. Intriguingly, Mayo Clinic investigators have found that a fraction of patients with the repeat expansion don’t develop the disease; in this project, they aimed to test their hypothesis that interruptions in the repeat sequence may explain the phenomenon.
But identifying interruptions required sequencing the entire length of the repeat expansion, something that could not be done with PCR amplification due to its likelihood of introducing confounding artifacts in sequencing data. The team turned to PacBio’s recently launched amplification-free protocol (we call it No-Amp), which uses the CRISPR/Cas9 system to target specific sequences of interest. “This method permits the enrichment and direct sequencing of targeted sequences without PCR amplification,” the scientists write, adding that SMRT Sequencing technology “also permits the generation and analysis of full-length sequences from even expanded repeats.”
To evaluate this method, scientists compared results to those from an STR assay and from Southern blots. The data were highly concordant: all of the amplification-free “size estimates for sub-pathological length repeats match the STR results within 1 repeat triplet,” they report. Also, “the sequencing was successful in identifying a previously described interruption within an unexpanded allele and provided sequence data on expanded alleles greater than 2000 bases in length,” the team notes.
While this study found no novel repeat interruptions that might explain why certain individuals do not develop FECD, it did generate some interesting results. First, two samples were found to have “novel variation in the AGG repeats that immediately precede the CAG repeats,” which could help scientists hone their hypothesis about these patients. Another intriguing discovery was the heterogeneity in repeat lengths of the expanded allele. “Given that these samples were not PCR amplified, this suggests somatic instability of the expanded repeat sequence and consequent mosaicism within the population of leukocytes used for the analysis of each specimen,” the scientists report. “In contrast, there is very little heterogeneity of subpathogenic alleles (<40 repeats).”