DNA methylation plays a fundamental role in the control of gene expression and genome integrity. Although there are multiple tools that enable its detection from Nanopore sequencing, their accuracy remains largely unknown. Here, we present a systematic benchmarking of tools for the detection of CpG methylation from Nanopore sequencing using individual reads, control mixtures of methylated and unmethylated reads, and bisulfite sequencing. We found that tools have a tradeoff between false positives and false negatives and present a high dispersion with respect to the expected methylation frequency values. We described various strategies to improve the accuracy of these tools, including a consensus approach, METEORE (https://github.com/comprna/METEORE), based on the combination of the predictions from two or more tools that shows improved accuracy over individual tools. Snakemake pipelines are also provided for reproducibility and to enable the systematic application of our analyses to other datasets.

The carboxylation kinetic (stable carbon) isotope effect was measured for purified d-ribulose-1,5-bisphosphate carboxylases/oxygenases (Rubiscos) with aqueous CO(2) as substrate by monitoring Rayleigh fractionation using membrane inlet mass spectrometry. This resulted in discriminations (Delta) of 27.4 +/- 0.9 per thousand for wild-type tobacco Rubisco, 22.2 +/- 2.1 per thousand for Rhodospirillum rubrum Rubisco, and 11.2 +/- 1.6 per thousand for a large subunit mutant of tobacco Rubisco in which Leu(335) is mutated to valine (L335V). These Delta values are consistent with the photosynthetic discrimination determined for wild-type tobacco and transplastomic tobacco lines that exclusively produce R. rubrum or L335V Rubisco. The Delta values are indicative of the potential evolutionary variability of Delta values for a range of Rubiscos from different species: Form I Rubisco from higher plants; prokaryotic Rubiscos, including Form II; and the L335V mutant. We explore the implications of these Delta values for the Rubisco catalytic mechanism and suggest that Rubiscos that are associated with a lower Delta value have a less product-like carboxylation transition state and/or allow a decarboxylation step that evolution has excluded in higher plants.

Forensic DNA profiling utilizes autosomal short tandem repeat (STR) markers to establish identity of missing persons, confirm familial relations, and link persons of interest to crime scenes. It is a widely accepted notion that genetic markers used in forensic applications are not predictive of phenotype. At present, there has been no demonstration of forensic STR variants directly causing or predicting disease. Such a demonstration would have many legal and ethical implications. For example, is there a duty to inform a DNA donor if a medical condition is discovered during routine analysis of their sample? In this review, we evaluate the possibility that forensic STRs could provide information beyond mere identity. An extensive search of the literature returned 107 articles associating a forensic STR with a trait. A total of 57 of these studies met our inclusion criteria: a reported link between a STR-inclusive gene and a phenotype and a statistical analysis reporting a p-value less than 0.05. A total of 50 unique traits were associated with the 24 markers included in the 57 studies. TH01 had the greatest number of associations with 27 traits reportedly linked to 40 different genotypes. Five of the articles associated TH01 with schizophrenia. None of the associations found were independently causative or predictive of disease. Regardless, the likelihood of identifying significant associations is increasing as the function of non-coding STRs in gene expression is steadily revealed. It is recommended that regular reviews take place in order to remain aware of future studies that identify a functional role for any forensic STRs.