Chimerism monitoring using biallelic single nucleotide or insertion/deletion polymorphisms : how many markers to screen?

Background/aims: Chimerism monitoring by means of high-throughput sequencing or quantitative PCR of biallelic single nucleotide and insertion/deletion polymorphisms has shown potential for improved patient care when compared to the gold standard capillary electrophoresis assays. When designing chimerism assays the number of markers to screen needs consideration: it determines the informativity rate and accuracy of the assay, but screening too many markers increases the assay's cost and complexity. The minimal number of biallelic markers to screen is currently unstudied.Materials/methods: A simulation framework accounting for marker minor allele frequencies, the number of markers screened, marker allelic constellations and donor-recipient relatedness was constructed. The framework was validated through analysis of 324 clinical samples.Results: Empirical clinical data confirm the validity of the simulation framework. With guidelines suggesting to monitor at least three informative markers, we demonstrate that, for optimized assays, at least 40 biallelic markers need to be screened to achieve enough informative markers in over 99% of cases. We propose and discuss several assay optimization strategies.Conclusion: Currently used chimerism assays often screen too little or too many markers, leaving room for optimization. Through support of the simulation framework here introduced and validated, more informative, cost-effective chimerism assays can be designed

Bias reduction improves accuracy and informativity of high-throughput sequencing chimerism assays

Background and aims: Chimerism monitoring by means of high-throughput sequencing of biallelic polymorphisms has shown promising advantages for patient follow-up after hematopoietic stem cell transplantation. Yet, the presence of method bias precludes achievement of an assay's theoretically attainable informativity rate, as method bias necessitates the exclusion of some markers. This method bias arises because of preferential obser-vation of one allele over the other, and for some allelic constellations because of stochasticity.Results: This paper suggests how preferential allelic observation may lead to method bias, and when and why such bias necessitates the exclusion of markers. It is shown that also markers that remain informative suffer a reduction in trueness and precision due to method bias. A bias reduction approach in the data analysis phase is introduced and shown to improve trueness and precision under all circumstances, meriting its universal adop-tion. This bias reduction furthermore allows to achieve an assay's theoretically achievable informativity rate, though at the cost of reduced sensitivity. Several strategies to consider in the assay design phase that may lower biases are proposed.Conclusion: Improved design and data analysis of chimerism assays increase the accuracy, applicability, and cost-effectiveness of high-throughput sequencing chimerism assays

The development of a forensic clock to determine time of death

The time and date of a person’s death are of great value in forensic investigations. The current time of death estimation techniques however provide large error ranges, especially when post-mortem interval exceeds 24 h. Circadian biomarkers have previously proven their potential use as forensic molecular clock markers to estimate blood-deposition time. More intriguingly, circadian biomarkers identified from the brain transcriptome were also able to estimate time of death. In order to develop a forensic molecular clock for post-mortem blood samples, we designed a study to identify circadian markers in three different classes of circadian molecules using post-mortem blood samples obtained from deceased individuals with a known time of death: mRNA transcripts using RNA-Seq, metabolites with LC–MS/MS and hormone concentrations using ELISA. The current manuscript discusses the melatonin and cortisol results.status: publishe

Performance assessment of the Devyser high-throughput sequencing-based assay for chimerism monitoring in patients after allogeneic hematopoietic stem cell transplantation

Chimerism analysis is widely used to aid in the clinical management of patients after allogeneic hematopoietic stem cell transplantation. Many laboratories currently use assays based on PCR followed by capillary electrophoresis, with a limit of quantification of 1% to 5%. Assays with a lower limit of quantification could allow for earlier relapse detection, resulting in improved patient care. This study investigated the analytical, clinical, technical, and practical performance of the Devyser next-generation sequencing chimerism assay, a commercial high-throughput sequencing-based assay for chimerism analysis. Performance of this assay was compared with that of the Promega PowerPlex 16 HS assay, a commercial capillary electrophoresis-based assay. A limit of quantification of 0.1% was achievable with the Devyser assay. The repeatability, reproducibility, trueness, and linearity of the Devyser assay were acceptable. The Devyser assay showed potential for earlier relapse detection compared with the Promega assay. Conclusive analysis was not possible for 3% of donor-recipient pairs with the Devyser assay due to an insufficient number of informative markers; this factor was not an issue for the Promega assay. Further improvements in assay design or data analysis may allow the assay's applicability to be extended to all donor-recipient pairs studied. Technical performance criteria for chimerism analysis by high-throughput sequencing were suggested and evaluated. Both assays were found to be practical for use in a clinical diagnostics laboratory. (J Mol Diagn 2021, 23: 1116-1126; https://doi.org/10.1016