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 (⌬) of 27.4 ؎ 0.9‰ for wild-type tobacco Rubisco, 22.2 ؎ 2.1‰ for Rhodospirillum rubrum Rubisco, and 11.2 ؎ 1.6‰ for a large subunit mutant of tobacco Rubisco in which Leu 335 is mutated to valine (L335V). These ⌬ 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 ⌬ values are indicative of the potential evolutionary variability of ⌬ 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 ⌬ values for the Rubisco catalytic mechanism and suggest that Rubiscos that are associated with a lower ⌬ value have a less product-like carboxylation transition state and/or allow a decarboxylation step that evolution has excluded in higher plants.

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.

Current forensic ancestry-informative panels are limited in their ability to differentiate populations in the Asia-Pacific region. MAPlex (Multiplex for the Asia-Pacific), a massively parallel sequencing (MPS) assay, was developed to improve differentiation of East Asian, South Asian and Near Oceanian populations found in the extensive cross-continental Asian region that shows complex patterns of admixture at its margins. This study reports the development of MAPlex; the selection of SNPs in combination with microhaplotype markers; assay design considerations for reducing the lengths of microhaplotypes while preserving their ancestry-informativeness; adoption of new population-informative multiple-allele SNPs; compilation of South Asian-informative SNPs suitable for forensic AIMs panels; and the compilation of extensive reference and test population genotypes from online whole-genome-sequence data for MAPlex markers. STRUCTURE genetic clustering software was used to gauge the ability of MAPlex to differentiate a broad set of populations from South and East Asia, the West Pacific regions of Near Oceania, as well as the other globally distributed population groups. Preliminary assessment of MAPlex indicates enhanced South Asian differentiation with increased divergence between West Eurasian, South Asian and East Asian populations, compared to previous forensic SNP panels of comparable scale. In addition, MAPlex shows efficient differentiation of Middle Eastern individuals from Europeans. MAPlex is the first forensic AIM assay to combine binary and multiple-allele SNPs with microhaplotypes, adding the potential to detect and analyze mixed source forensic DNA.