Abstract
Aim Most laboratories providing chimerism testing use electrophoretic analysis of STR loci or qPCR of SNP polymorphisms. STR assays are limited to 1–5% sensitivity and qPCR requires substantial amounts of DNA which may not be feasible in patients with low blood cell counts or for analysis of rare cell subsets. We developed a chimerism assay based on NGS of SNP and InDel polymorphisms and validated it in a clinical transplant immunology lab supporting a large HCT program. Methods Multiple polymorphic loci were evaluated for specificity, sensitivity and accuracy using artificial admixtures as well as direct comparison to qPCR results from patient specimens post HCT. An initial panel comprised 37 SNPs on 18 chromosomes and 12 InDels on 10 chromosomes. Target generation is performed in 2 multiplexed PCRs with 5 ng sample DNA per reaction. All NGS benchwork is accomplished within 6 h, followed by a 7 h MiSeq run, so that results are available for reporting early on day 2. Results Initial NGS testing used artificial admixtures of unrelated individuals, with mixtures from 10% to 0.1%, see Table 1. On average, 30 informative markers were identified between unrelated individuals and NGS achieved 0.5% sensitivity among all 4 admixture sets. Table 2 shows the results for NGS versus qPCR for 7 patient post HCT specimens. Download : Download high-res image (334KB) Download : Download full-size image Download : Download high-res image (247KB) Download : Download full-size image Conclusions These preliminary results show that NGS technology can provide accurate, informative, and sensitive detection of a minority species with minimal DNA quantity requirements, rapid turnaround and minimal hands on time. As laboratories implement NGS technology for high resolution genotyping of HLA and other genetic loci, extension to engraftment monitoring might also offer workplace efficiencies. M.Z. Askar: 3. Speaker’s Bureau; Company/Organization; Immucor. 4. Scientific/Medical Advisor; Company/Organization; Illumina. S.E. Pereira: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. A.G. Smith: 2. Consultant; Company/Organization; Scisco Genetics Inc. C. Pyo: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. W. Nelson: 5. Employee; Company/Organization; Scisco Genetics Inc. D.E. Geraghty: 6. Stock Shareholder; Company/Organization; Scisco Genetics Inc.
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