P072 Adaptation of NGS technology for engraftment monitoring in hematopoietic cell transplantation

Abstract

Aim Increasingly, laboratories that support hematopoietic cell transplantation programs are implementing NGS technology for high resolution HLA genotyping. Extension of NGS to other laboratory services can provide efficiency in staffing and equipment usage. We have evaluated NGS of SNP and InDel polymorphisms for engraftment monitoring. Beyond workplace efficiencies, NGS of InDel polymorphism is not subject to stutter artifacts that can compromise results from the STR technology currently in wide use for chimerism analysis. Methods A panel of 49 polymorphisms on 20 chromosomes, comprising 37 SNPs on 18 chromosomes and 12 InDels on 10 chromosomes, was assembled. Initial amplification for the panel was reduced to 2 multiplexed PCRs with 5 ng of sample DNA per reaction. Total time for library preparation is 6 h, followed by a 7 h sequencing run on the MiSeq platform, with 1 h for chimerism analysis. Hands-on technologist time consists of just 3 of the 14 h time frame. Preliminary evaluation involved 4 sets of artificial mixtures of DNA from 2 unrelated individuals, with mixtures ranging from 10% to 0.1%, see Table. Results On average, 30 informative markers were identified between unrelated individuals. NGS achieved 0.5% sensitivity in all 4 sets of mixtures. The table shows the experimental values and standard deviation, SD, compared with the expected ratios among informative the polymorphisms. Informative InDel polymorphisms showed no false positive artifacts in unmixed specimens. Conclusions The results show that NGS technology provides informative, accurate and sensitive detection of a minority species in mixed specimens. With the 6 h library preparation and 7 h sequencing run, NGS offers rapid turnaround for result reporting. Additional testing is underway to assess the NGS technology in low cell count specimens and in mixtures of 3 or more unrelated individuals, intended to simulate retransplant and double cord donor transplant. Download : Download high-res image (233KB) Download : Download full-size image