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 observation 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 adoption. 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.

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.

Cell survival and DNA damage repair are promoted in the human blood thanatotranscriptome shortly after death.

RNA analysis of post-mortem tissues, or thanatotranscriptomics, has become a topic of interest in forensic science due to the essential information it can provide in forensic investigations. Several studies have previously investigated the effect of death on gene transcription, but it has never been conducted with samples of the same individual. For the first time, a longitudinal mRNA expression analysis study was performed with post-mortem human blood samples from individuals with a known time of death. The results reveal that, after death, two clearly differentiated groups of up- and down-regulated genes can be detected. Pathway analysis suggests active processes that promote cell survival and DNA damage repair, rather than passive degradation, are the source of early post-mortem changes of gene expression in blood. In addition, a generalized linear model with an elastic net restriction predicted post-mortem interval with a root mean square error of 4.75 h. In conclusion, we demonstrate that post-mortem gene expression data can be used as biomarkers to estimate the post-mortem interval though further validation using independent sample sets is required before use in forensic casework.

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.