Genomic origin, fragmentomics, and transcriptional properties of long cell-free DNA molecules in human plasma.

Recent studies have revealed an unexplored population of long cell-free DNA (cfDNA) molecules in human plasma using long-read sequencing technologies. However, the biological properties of long cfDNA molecules (>500 bp) remain largely unknown. To this end, we have investigated the origins of long cfDNA molecules from different genomic elements. Analysis of plasma cfDNA using long-read sequencing reveals an uneven distribution of long molecules from across the genome. Long cfDNA molecules show overrepresentation in euchromatic regions of the genome, in sharp contrast to short DNA molecules. We observe a stronger relationship between the abundance of long molecules and mRNA gene expression levels, compared with short molecules (Pearson's r = 0.71 vs. -0.14). Moreover, long and short molecules show distinct fragmentation patterns surrounding CpG sites. Leveraging the cleavage preferences surrounding CpG sites, the combined cleavage ratios of long and short molecules can differentiate patients with hepatocellular carcinoma (HCC) from non-HCC subjects (AUC = 0.87). We also investigated knockout mice in which selected nuclease genes had been inactivated in comparison with wild-type mice. The proportion of long molecules originating from transcription start sites are lower in Dffb-deficient mice but higher in Dnase1l3-deficient mice compared with that of wild-type mice. This work thus provides new insights into the biological properties and potential clinical applications of long cfDNA molecules.

Universal Targeted Haplotyping by Droplet Digital PCR Sequencing and Its Applications in Noninvasive Prenatal Testing and Pharmacogenetics Analysis.

BACKGROUND: The analysis of haplotypes of variants is important for pharmacogenomics analysis and noninvasive prenatal testing for monogenic diseases. However, there is a lack of robust methods for targeted haplotyping. METHODS: We developed digital PCR haplotype sequencing (dHapSeq) for targeted haplotyping of variants, which is a method that compartmentalizes long DNA molecules into droplets. Within one droplet, 2 target regions are PCR amplified from one template molecule, and their amplicons are fused together. The fused products are then sequenced to determine the phase relationship of the single nucleotide polymorphism (SNP) alleles. The entire haplotype of 10s of SNPs can be deduced after the phase relationship of individual SNPs are determined in a pairwise manner. We applied dHapSeq to noninvasive prenatal testing in 4 families at risk for thalassemia and utilized it to detect NUDT15 diplotypes for predicting drug tolerance in pediatric acute lymphoblastic leukemia (72 cases and 506 controls). RESULTS: For SNPs within 40 kb, phase relation can be determined with 100% accuracy. In 7 trio families, the haplotyping results for 97 SNPs spanning 185 kb determined by dHapSeq were concordant with the results deduced from the genotypes of both parents and the fetus. In 4 thalassemia families, a 19.3-kb Southeast Asian deletion was successfully phased with 97 downstream SNPs, enabling noninvasive determination of fetal inheritance using relative haplotype dosage analysis. In the NUDT15 analysis, the variant status and phase of the variants were successfully determined in all cases and controls. CONCLUSIONS: The dHapSeq represents a robust and scalable haplotyping approach with numerous clinical and research applications.

Dopamine infusion at typical infusion rates does not cause interference on plasma creatinine assays.

a Objectives: Dopamine is known to cause negative interference on enzymatic creatinine measurement. However, its effect on the Jaffe reaction, and its concentration required to interfere with enzymatic reactions, remain uncertain. This study was designed to study the interference of stable dopamine infusion on Jaffe and enzymatic creatinine assays, as well as the effect of dopamine infusion drip arm contamination on both creatinine assays. b Design and Methods: For the first part of the study, dopamine was spiked into pooled plasma samples at different concentrations to mimic the scenario of patients on dopamine infusion at an infusion rate between 2 and 20 µg/kg/min. For the second part, dopamine preparation of 2 g/L (same as the preparation used clinically) was mixed with pooled plasma samples at different proportions to mimic drip arm contamination. Creatinine concentrations were measured using Jaffe and enzymatic reactions. c Results: The first part showed that creatinine measurements were not interfered by dopamine infusion at an infusion rate between 2 and 20 µg/kg/min. The second part showed that dopamine could negatively interfere with enzymatic creatinine assays, even with minute drip arm contamination. The effect on the Jaffe reaction was less significant. d Discussion: Creatinine concentration could be reliably measured by Jaffe or enzymatic reactions if samples are from venous access sites other than the site of dopamine infusion. When dopamine interference on enzymatic creatinine assays is suspected, using the Jaffe reaction to cross-check may provide additional useful information.