Identification of Marker Immunoglobulin Rearrangements for Use by HAT-PCR in Myeloma

Libraries for next-generation sequencing were prepared using primers directed to framework 2, framework 3 and D and the 6 J regions of the rearranged immunoglobulin gene from 50 diagnosis myeloma bone marrow samples. After sequencing and bioinformatic analysis, a marker sequence suitable for minimal residual disease analysis by HAT-PCR was obtained for 90% of the 50 samples studied. In 88% of samples a complete VDJ sequence was obtained. Failure to detect a marker sequence was principally due to a low percentage of plasma cells in the marrow sample. Evaluation of 24 pairs of primers showed that they were highly specific and, when used in HAT-PCR, they provided accurate quantification. The high frequency of detection of a suitable marker sequence indicates that minimal residual disease can be monitored by HAT-PCR in the great majority of instances.