Evaluation of a Microhaplotype Marker Panel for Post-Transplant Chimerism Analysis

Microhaplotypes (MHs) are highly informative genetic markers suitable for forensic and clinical applications, including post-transplant monitoring in liquid biopsy. The aim of this study was to establish and evaluate a highly informative MH marker panel suitable for both chimerism analysis after allogeneic stem cell transplantation and for monitoring organ transplants using cell-free DNA (cfDNA). Two sequencing platforms (Illumina MiSeq and Oxford Nanopore Technologies [ONT]) were compared with regard to their suitability for reliable MH genotyping.

Based on 98 candidates selected from the literature, a panel of 74 MH markers was developed and sequenced in 960 anonymized samples of healthy blood donors. Amplification was performed from genomic DNA in two multiplex PCRs generating short amplicons (95-150 bp). Library preparation and sequencing were carried out on the Illumina MiSeq platform (2 × 150 bp) and the ONT MinION (R10.4.1 flow cells). Bioinformatic analysis included quality filtering, read mapping (Bowtie2 and Minimap2), haplotype determination, and calculation of population genetic parameters such as polymorphism information content (PIC), power of discrimination (PD), expected heterozygosity (He), effective number of alleles (A_e), and Hardy-Weinberg equilibrium.

Evaluation of the sequencing results from 960 samples led to the exclusion of 20 MH markers due to low coverage or nonspecific amplifications. The remaining 54 MH loci were located on 22 chromosomes and comprised a total of 202 single nucleotide variations (SNVs). A balanced allele coverage ratio (>0.6) was observed for 43 loci. In total, 358 different alleles were identified, with most markers exhibiting between four and seven alleles. The PD exceeded 0.6 in 52 loci (mean 0.87), and most markers demonstrated high PIC and He values, indicating substantial genetic variability. Platform comparison revealed that ONT sequencing generated higher absolute read numbers, whereas Illumina sequencing achieved higher relative mapping efficiency and base accuracy. The proportion of uniquely mapped reads was comparable between the platforms. Additionally, previously undescribed SNVs were identified in 23 loci, potentially, further increasing haplotype diversity.

The marker panel with 54 MHs demonstrated high genetic diversity and discriminatory power in the studied population. With the small amplicon size the MH marker panel could also be suitable for cfDNA. Further investigation with regard to the analysis of mixed DNA samples will support the usefulness of MHs as biomarkers for chimerism analysis in post-transplant monitoring.