Towards Sensitive and Cost-Effective Chimerism Assays Using FABCASE: A Fast Approach for Estimating Assay...

Chimerism monitoring is part of the standard of care for patients following an allogeneic hematopoietic stem cell transplantation. There has recently been a move towards sensitive, high-throughput (next-generation) sequencing analysis of biallelic markers for this purpose. Determining the number and properties of the markers to include in an assay to achieve reliable yet cost-effective chimerism quantification is an underexposed but critical part of chimerism assay development, optimization, and validation.

We develop FABCASE (Fast and Accurate Biallelic Chimerism Assay Size Estimation), an approach to estimate the required number of markers to screen to obtain a given informativity rate. We explore several practical examples that illustrate the diverse applications of FABCASE beyond calculating the required number of markers to screen.

FABCASE offers a more than four orders of magnitude speed improvement compared to a previously introduced Monte Carlo simulation approach, unlocking extensive in silico scenario analyses. We find that minor allele frequency (MAF) and informative rate estimation based on small sample series (tens) are reasonably accurate. MAFs may vary drastically between populations, and the number of required markers to attain a preset informativity rate is inflated (here, +28%) when not optimized. Marker subset selection from a pool of candidate markers is little impacted by small-to-medium MAF differences (0%-20% MAF). Prioritizing markers with uniform amplification efficiency reduces sequencing depth requirements and improves cost-effectiveness. A web graphical user interface facilitating marker set informativity evaluation is available at https://mvynck.shinyapps.io/FABCASE.

FABCASE facilitates the design, refinement, and implementation of sensitive and cost-effective chimerism assays. Due attention should be given to an assay's marker MAFs, sensitivities, and amplification efficiencies.