Abstract
Accurate monitoring of the chimeric status after stem cell transplantation is essential for early detection of relapse and at the moment mainly performed by STR or qPCR. The major disadvantages of STR are the laborious data analysis and poor sensitivity. The qPCR technique enables a much quicker workflow and better sensitivity. However a disadvantage is the need of a pre-transplant sample for each monitoring event. Performing chimerism monitoring by NGS will eliminate these limitations while allowing for a multiplexed setup, reducing the amount of needed lab work and DNA. The workflow can be combined with routinely applied NGS HLA typing in clinical laboratories. PCR amplification primers have been designed for a set of biallelic markers. These markers were tested on a number of artificial chimeric samples in a range between 0.01–100% of positive DNA mixed with negative DNA. Amplicons were applied in the respective NGSgo library preparation workflows and run on Illumina MiSeq, Ion Torrent S5 and Oxford Nanopore MinION systems. NGS data was analysed with customized analysis tools designed to quantitate the two variants of each marker. Each marker was tested on a small panel of non-chimeric samples (Coriell Institute) and resulted in clear homo- or heterozygous patterns for all markers. Results of the different platforms on the artificial chimeric samples in a range of 0.01%–100% were compared. Sensitivity was determined for each sequencing platform. A read depth threshold was established for each of the platforms to achieve a minimum sensitivity of 0.05%. Results including difference between sequencing platforms will be discussed further. The results demonstrate that NGS-based chimerism monitoring is feasible for a wide range of NGS platforms, with a high sensitivity and wide dynamic range. E.E. Bouwmans: 5. Employee; Company/Organization; GenDx. L.A. van de Pasch: 5. Employee; Company/Organization; GenDx. M.T. Penning: 5. Employee; Company/Organization; GenDx. E. Rozemuller: 5. Employee; Company/Organization; GenDx. 6. Stock Shareholder; Company/Organization; GenDx. 7. Other (Identify); Company/Organization; Founder of GenDx.
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