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Whole genome bisulfite sequencing (WGBS), with its ability to interrogate methylation status at single CpG site resolution epigenome-wide, is a powerful technique for use in molecular experiments. Here, we aim to advance strategies for accurate and efficient WGBS for application in future large-scale epidemiological studies. We systematically compared the performance of three WGBS library preparation methods with low DNA input requirement (Swift Biosciences Accel-NGS, Illumina TruSeq and QIAGEN QIAseq) on two state-of-the-art sequencing platforms (Illumina NovaSeq and HiSeq X), and also assessed concordance between data generated by WGBS and methylation arrays. Swift achieved the highest proportion of CpG sites assayed and effective coverage at 26x (P < 0.001). TruSeq suffered from the highest proportion of PCR duplicates, while QIAseq failed to deliver across all quality metrics. There was little difference in performance between NovaSeq and HiSeq X, with the exception of higher read duplication rate on the NovaSeq (P < 0.05), likely attributable to the higher cluster densities on its flow cells. Systematic biases exist between WGBS and methylation arrays, with lower precision observed for WGBS across the range of depths investigated. To achieve a level of precision broadly comparable to the methylation array, a minimum coverage of 100x is recommended.

Cultivars of rice (Oryza sativa L.), especially of the type with large spikelets, often fail to reach the yield potential as expected due to the poor grain-filling on the later flowering inferior spikelets (in contrast to the earlier-flowering superior spikelets). The present study showed that the size and grain weight of superior spikelets (SS) was greater than those of inferior spikelets (IS), and the carbohydrate supply should not be the major problem for the poor grain-filling because there was adequate amount of sucrose in IS at the initial grain-filling stage. High resolution two-dimensional gel electrophoresis (2-DE) in combination with Coomassie-brilliant blue (CBB) and Pro-Q Diamond phosphoprotein fluorescence stain revealed that 123 proteins in abundance and 43 phosphoproteins generated from phosphorylation were significantly different between SS and IS. These proteins and phosphoproteins were involved in different cellular and metabolic processes with a prominently functional skew toward metabolism and protein synthesis/destination. Expression analyses of the proteins and phosphoproteins associated with different functional categories/subcategories indicated that the starch synthesis, central carbon metabolism, N metabolism and cell growth/division were closely related to the poor grain-filling of IS. Functional and expression pattern studies also suggested that 14-3-3 proteins played important roles in IS poor grain-filling by regulating the activity of starch synthesis enzymes. The proteome and phosphoproteome obtained from this study provided a better understanding of the molecular mechanism of the IS poor grain-filling. They were also expected to be highly useful for improving the grain filling of rice.

Aims/hypothesis Type 2 diabetes is a complex metabolic disease with increasing prevalence worldwide. Improving the prediction of incident type 2 diabetes using epigenetic markers could help tailor prevention efforts to those at the highest risk. The aim of this study was to identify predictive methylation markers for incident type 2 diabetes by combining epigenome-wide association study (EWAS) results from five prospective European cohorts. Methods We conducted a meta-analysis of EWASs in blood collected 7–10 years prior to type 2 diabetes diagnosis. DNA methylation was measured with Illumina Infinium Methylation arrays. A total of 1250 cases and 1950 controls from five longitudinal cohorts were included: Doetinchem, ESTHER, KORA1, KORA2 and EPIC-Norfolk. Associations between DNA methylation and incident type 2 diabetes were examined using robust linear regression with adjustment for potential confounders. Inverse-variance fixed-effects meta-analysis of cohort-level individual CpG EWAS estimates was performed using METAL. The methylGSA R package was used for gene set enrichment analysis. Confirmation of genome-wide significant CpG sites was performed in a cohort of Indian Asians (LOLIPOP, UK). Results The meta-analysis identified 76 CpG sites that were differentially methylated in individuals with incident type 2 diabetes compared with control individuals (p values <1.1 × 10−7). Sixty-four out of 76 (84.2%) CpG sites were confirmed by directionally consistent effects and p values <0.05 in an independent cohort of Indian Asians. However, on adjustment for baseline BMI only four CpG sites remained genome-wide significant, and addition of the 76 CpG methylation risk score to a prediction model including established predictors of type 2 diabetes (age, sex, BMI and HbA1c) showed no improvement (AUC 0.757 vs 0.753). Gene set enrichment analysis of the full epigenome-wide results clearly showed enrichment of processes linked to insulin signalling, lipid homeostasis and inflammation. Conclusions/interpretation By combining results from five European cohorts, and thus significantly increasing study sample size, we identified 76 CpG sites associated with incident type 2 diabetes. Replication of 64 CpGs in an independent cohort of Indian Asians suggests that the association between DNA methylation levels and incident type 2 diabetes is robust and independent of ethnicity. Our data also indicate that BMI partly explains the association between DNA methylation and incident type 2 diabetes. Further studies are required to elucidate the underlying biological mechanisms and to determine potential causal roles of the differentially methylated CpG sites in type 2 diabetes development. Graphical abstract

Background Total DNA (intracellular, iDNA and extracellular, eDNA) from ancient permafrost records the mixed genetic repository of the past and present microbial populations through geological time. Given the exceptional preservation of eDNA under perennial frozen conditions, typical metagenomic sequencing of total DNA precludes the discrimination between fossil and living microorganisms in ancient cryogenic environments. DNA repair protocols were combined with high throughput sequencing (HTS) of separate iDNA and eDNA fraction to reconstruct metagenome-assembled genomes (MAGs) from ancient microbial DNA entrapped in Siberian coastal permafrost. Results Despite the severe DNA damage in ancient permafrost, the coupling of DNA repair and HTS resulted in a total of 52 MAGs from sediments across a chronosequence (26–120 kyr). These MAGs were compared with those derived from the same samples but without utilizing DNA repair protocols. The MAGs from the youngest stratum showed minimal DNA damage and thus likely originated from viable, active microbial species. Many MAGs from the older and deeper sediment appear related to past aerobic microbial populations that had died upon freezing. MAGs from anaerobic lineages, including Asgard archaea, however exhibited minimal DNA damage and likely represent extant living microorganisms that have become adapted to the cryogenic and anoxic environments. The integration of aspartic acid racemization modeling and metaproteomics further constrained the metabolic status of the living microbial populations. Collectively, combining DNA repair protocols with HTS unveiled the adaptive strategies of microbes to long-term survivability in ancient permafrost. Conclusions Our results indicated that coupling of DNA repair protocols with simultaneous sequencing of iDNA and eDNA fractions enabled the assembly of MAGs from past and living microorganisms in ancient permafrost. The genomic reconstruction from the past and extant microbial populations expanded our understanding about the microbial successions and biogeochemical alterations from the past paleoenvironment to the present-day frozen state. Furthermore, we provided genomic insights into long-term survival mechanisms of microorganisms under cryogenic conditions through geological time. The combined strategies in this study can be extrapolated to examine other ancient non-permafrost environments and constrain the search for past and extant extraterrestrial life in permafrost and ice deposits on Mars.