Residual Disease in Leukemia Patients Identified by ddPCR
By LabMedica International staff writers Posted on 08 Feb 2018 |
Image: The MinION is the only portable real-time device for DNA and RNA sequencing (Photo courtesy of Oxford Nanopore Technologies).
Currently, minimal residual disease for chronic myeloid leukemia (CML) patients is typically monitored using real-time quantitative polymerase chain reaction (RT-qPCR), but often leukemic stem cells with the BCR-ABL1 rearrangement are present below the limit of detect of RT-qPCR.
While DNA-based approaches have previously been shown to be more sensitive identifying the breakpoint of the BCR-ABL1 rearrangement can be challenging because it is highly repetitive. In addition, many diagnostic laboratories do not have next-generation sequencing capabilities. Nanopore sequencing in combination with droplet digital PCR can be used to identify minimal residual disease in some leukemia patients.
Scientists at the University of Bari, (Bari, Italy) included 10 patients with CML in their study. The team designed amplicons to span the BCR-ABL1 junction for 10 patients and then barcoded and sequenced them on the MinION, a portable real-time device for DNA and RNA sequencing. Sequencing took around 24 hours and generated more than 21,000 reads. Average sequencing depth was 400× and the error rate around 8%. For all 10 patients, MinIon sequencing was able to identify the breakpoint and was concordant with Sanger sequencing.
Next, the team used ddPCR to quantify the number of cells that contained the rearrangement. For each patient, they designed a personalized assay based on the sequence of the BCR-ABL1 region. At diagnosis, a median of 87% of the patients' cells contained the rearrangement. The investigators also evaluated sixteen samples at 6, 12, and 15 months from diagnosis. Results from the ddPCR assay were consistent with RT-qPCR in all but one case, and the authors noted that the ddPCR assay was "was more sensitive" for "detecting residual disease" than RT-qPCR.
Chia-Lin Wei, PhD, director of genome technologies at the Jackson Laboratory (Farmington, CT, USA), said, “The study demonstrated a very suitable application for nanopore sequencing. Although the MinION does not yet have the accuracy to evaluate somatic point mutations in a diagnostic setting, sequencing through translocations is a very ideal type of data for the system. The MinIon's long reads are particularly valuable since they enable the entire region to be sequenced through in one read, which is important because breakpoint location can vary patient to patient.” The study was published on January 5, 2018, in the journal Oncotarget.
Related Links:
University of Bari
Jackson Laboratory
While DNA-based approaches have previously been shown to be more sensitive identifying the breakpoint of the BCR-ABL1 rearrangement can be challenging because it is highly repetitive. In addition, many diagnostic laboratories do not have next-generation sequencing capabilities. Nanopore sequencing in combination with droplet digital PCR can be used to identify minimal residual disease in some leukemia patients.
Scientists at the University of Bari, (Bari, Italy) included 10 patients with CML in their study. The team designed amplicons to span the BCR-ABL1 junction for 10 patients and then barcoded and sequenced them on the MinION, a portable real-time device for DNA and RNA sequencing. Sequencing took around 24 hours and generated more than 21,000 reads. Average sequencing depth was 400× and the error rate around 8%. For all 10 patients, MinIon sequencing was able to identify the breakpoint and was concordant with Sanger sequencing.
Next, the team used ddPCR to quantify the number of cells that contained the rearrangement. For each patient, they designed a personalized assay based on the sequence of the BCR-ABL1 region. At diagnosis, a median of 87% of the patients' cells contained the rearrangement. The investigators also evaluated sixteen samples at 6, 12, and 15 months from diagnosis. Results from the ddPCR assay were consistent with RT-qPCR in all but one case, and the authors noted that the ddPCR assay was "was more sensitive" for "detecting residual disease" than RT-qPCR.
Chia-Lin Wei, PhD, director of genome technologies at the Jackson Laboratory (Farmington, CT, USA), said, “The study demonstrated a very suitable application for nanopore sequencing. Although the MinION does not yet have the accuracy to evaluate somatic point mutations in a diagnostic setting, sequencing through translocations is a very ideal type of data for the system. The MinIon's long reads are particularly valuable since they enable the entire region to be sequenced through in one read, which is important because breakpoint location can vary patient to patient.” The study was published on January 5, 2018, in the journal Oncotarget.
Related Links:
University of Bari
Jackson Laboratory
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