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In Chronic Myeloid Leukemia Study, SMRT Sequencing Detects Resistance Mutations Early, New Splice Isoforms and More

Thursday, March 26, 2015

Scientists from Uppsala University report in a recent paper that using the Iso-Seq™ method with SMRT® Sequencing allowed them to detect and monitor mutations in the BCR-ABL1 fusion gene for patients with chronic myeloid leukemia (CML). Screening mutations in this region is important for determining the point at which these patients become resistant to tyrosine kinase inhibitor (TKI) therapies, and is currently performed in the clinic using Sanger sequencing, quantitative RT-PCR, and other assays.

The paper, “Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing,” was published last month in BMC Cancer from lead author Lucia Cavelier and collaborators. In it, the scientists describe sequencing samples from six patients who experienced poor response to cancer treatment; samples were collected at diagnosis and at subsequent follow-up periods and sequenced on the PacBio® system.

The team checked for mutations in the BRC-ABL1 fusion transcript, generating on average10,000 full-length sequences of the gene from a single SMRT cell. Short-read sequencers have been tried for this kind of work, the authors note, but their inability to span the entire transcript as well as concerns about bias introduced by nested PCR has limited their utility.

“Here we present for the first time an assay to directly investigate the entire 1,578 bp BCR-ABL1 major fusion transcript, amplified from a single PCR reaction and sequencing on the Pacific Biosciences (PacBio) RS II system,” Cavelier et al. write. “In addition to enabling a rapid workflow at a relatively low cost, the PacBio system produces reads sufficiently long to span across a full length BCR-ABL1 molecule.” They report that the process, which took two to three days to complete, had a 0% false positive rate, attributed to the random error mode of PacBio sequencing data, “which results in highly accurate base calls for molecules that are sequenced at high coverage.”

For each of the six patients studied, the authors report, SMRT Sequencing confirmed the mutations that had already been found with Sanger sequencing. It also detected five low-frequency mutations that were missed by the Sanger pipeline. In one case, the scientists found that PacBio sequencing successfully detected a mutation four months earlier than it was found by Sanger sequencing, indicating that the technology may ultimately accelerate the identification of genetic markers that are important for diagnosis or drug response monitoring.

In addition, long reads from SMRT Sequencing allowed the team to distinguish multiple transcript isoforms for BCR-ABL1 from individual samples. “These results corroborate previous findings that propose alternative splicing as a common mechanism among CML patients undergoing TKI treatment,” the authors write.

Importantly, PacBio data also made it possible to differentiate compound mutations from independent mutations in other molecules, information that cannot be gleaned from Sanger sequencing. “This feature is of major clinical relevance as compound mutations show different resistance profiles compared to individual mutants,” Cavelier et al. report.

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