description |
In vivo tracking of retrovirus-tagged blood stem and progenitor cells is used to study hematopoiesis. Two techniques have been used most frequently: sequencing the locus of retrovirus insertion, termed integration site analysis (ISA), or retrovirus DNA barcode sequencing (DBS). A key question is how these two techniques compare in their ability to detect and quantify clonal contributions. Here we assessed both methods simultaneously in a nonhuman primate model of autologous, myeloablative transplantation. Our data demonstrate that both methods track abundant clones; however, DBS is at least five-fold more efficient than ISA. We show the sampling depth required for ISA to achieve similar coverage to DBS is likely prohibitive, especially in cases of low gene-modified cell engraftment. Importantly, we observe an early and sustained contribution of primitive clones after transplant in this setting by both methods. Our analysis demonstrates DBS as a useful guide to maximize ISA interpretation in gene therapy patients. |