GMQN: A Reference-Based Method for Correcting Batch Effects and Probe Bias in HumanMethylation BeadChip

The Illumina HumanMethylation BeadChip is one of the most cost-effective methods to quantify DNA methylation levels at single-base resolution across the human genome, which makes it a routine platform for epigenome-wide association studies. It has accumulated tens of thousands of DNA methylation array samples in public databases, providing great support for data integration and further analysis. However, the majority of public DNA methylation data are deposited as processed data without background probes which are widely used in data normalization. Here, we present Gaussian mixture quantile normalization (GMQN), a reference based method for correcting batch effects as well as probe bias in the HumanMethylation BeadChip. Availability and implementation: https://github.com/MengweiLi-project/gmqn.

A bifunctional chemical signature enabling RNA 4-thiouridine enrichment sequencing with single-base resolution

Both sequence enrichment and base resolution is essential for accurate sequencing analysis of low-abundance RNA. Yet it is hindered by the lack of molecular tools. Here we report a bifunctional...

Heterochromatin is a quantitative trait associated with spontaneous epiallele formation

Epialleles are meiotically heritable variations in expression states that are independent from changes in DNA sequence. Although they are common in plant genomes, their molecular origins are unknown. Here we show, using mutant and experimental populations, that epialleles in Arabidopsis thaliana that result from ectopic hypermethylation are due to feedback regulation of pathways that primarily function to maintain DNA methylation at heterochromatin. Perturbations to maintenance of heterochromatin methylation leads to feedback regulation of DNA methylation in genes. Using single base resolution methylomes from epigenetic recombinant inbred lines (epiRIL), we show that epiallelic variation is abundant in euchromatin, yet, associates with QTL primarily in heterochromatin regions. Mapping three-dimensional chromatin contacts shows that genes that are hotspots for ectopic hypermethylation have increases in contact frequencies with regions possessing H3K9me2. Altogether, these data show that feedback regulation of pathways that have evolved to maintain heterochromatin silencing leads to the origins of spontaneous hypermethylated epialleles.

Selective Chemical Labeling and Sequencing of 5-Hydroxymethylcytosine in DNA at Single-Base Resolution

5-Hydroxymethylcytosine (5hmC), the oxidative product of 5-methylcytosine (5mC) catalyzed by ten-eleven translocation enzymes, plays an important role in many biological processes as an epigenetic mediator. Prior studies have shown that 5hmC can be selectively labeled with chemically modified glucose moieties and enriched using click chemistry with biotin affinity approaches. Besides, DNA deaminases of the AID/APOBEC family can discriminate modified 5hmC bases from cytosine (C) or 5mC. Herein, we developed a method based on embryonic stem cell (ESC) whole-genome analysis, which could enrich 5hmC-containing DNA by selective chemical labeling and locate 5hmC sites at single-base resolution with enzyme-based deamination. The combination experimental design is an extension of previous methods, and we hope that this cost-effective single-base resolution 5hmC sequencing method could be used to promote the mechanism and diagnosis research of 5hmC.

RNA-Seq of potato plants reveals a complex of new latent bacterial plant pathogens

The throughput and single-base resolution of RNA-Sequencing (RNA-Seq) have contributed to a dramatic change in diagnostics of viruses and other plant pathogens. A transcriptome represents all RNA molecules, including the coding mRNAs as well as the noncoding rRNA, tRNA, etc. A distinct advantage of RNA-Seq is that cDNA fragments are directly sequenced and the reads can be compared to available reference genome sequences. This approach allows the simultaneous and hypothesis-free identification of all pathogens in the plant. We conducted surveys for potato (Solanum tuberosum L.) -associated phytopathogenic bacteria in 56 original and GenBank RNA-seq data sets for potato breeding material. Bacteria of genera Pseudomonas, Burkholderia, Ralstonia, Xanthomonas, Agrobacterium, and species of family Enterobacteriaceae were most frequently detected in RNA sets from the studied plants. RNA-seq reads identified as Xanthomonas spp. were within X. vesicatoria, and some other species. Xanthomonas spp. covered up to 9,1% of all reads and included the major clades of these bacteria known as pathogens of solanaceous crops, but potato. Bacteria of genus Xanthomonas infect different plant species under artificial inoculation, suggesting that they are shared among wild plants and crops. Our studies indicated that a larger number of solanaceous plants can be occupied by specific Xanthomonas pathovars as endophytes or latent pathogens. Revealing bacteria distribution in the plant breeding material using RNA-seq data improves our knowledge on the ecology of plant pathogens.

Global quantification exposes abundant low-level off-target activity by base editors

Base editors are dedicated engineered deaminases that enable directed conversion of specific bases in the genome or transcriptome in a precise and efficient manner, and hold promise for correcting pathogenic mutations. A major concern limiting application of this powerful approach is the issue of off-target edits. Several recent studies have shown substantial off-target RNA activity induced by base editors and demonstrated that off-target mutations may be suppressed by improved deaminases versions or optimized guide RNAs. Here we describe a new class of off-target events that are invisible to the established methods for detection of genomic variations, and were thus far overlooked. We show that much of the off-target activity of the deaminases is nonspecific, seemingly stochastic, affecting a large number of sites throughout the genome or the transcriptome and accounting for the majority of off-target activity. We develop and employ a different, complementary, approach that is sensitive to the stochastic off-targets activity, and use it to quantify the abundant off-target RNA mutations due to current optimized deaminase editors. Engineered base editors enable directed manipulation of the genome or transcriptome at single-base resolution. We believe that implementation of this computational approach would facilitate design of more specific base editors. We provide a computational tool to quantify global off-target activity, which can be used to optimize future base editors.

Competitive precision genome editing (CGE) assay for functional analysis at single base resolution

The two major limitations of applying CRISPR/Cas9-technology for analysis of the effect of genotype on phenotype are the difficulty of cutting DNA exactly at the intended site, and the decreased cell proliferation and other phenotypic effects caused by the DNA cuts themselves. Here we report a novel competitive genome editing assay that allows analysis of the functional consequence of precise mutations. The method is based on precision genome editing, where a target sequence close to a feature of interest is cut, and the DNA is then repaired using a template that either reconstitutes the original feature, or introduces an altered sequence. Introducing sequence labels to both types of repair templates generates a large number of replicate cultures, increasing statistical power. In addition, the labels identify edited cells, allowing direct comparison between cells that carry wild-type and mutant features. Here, we apply the assay for multiplexed analysis of the role of E-box sequences on MYC binding and cellular fitness.