A cost-effective approach to DNA methylation detection by Methyl Sensitive DArT sequencing.

Several studies suggest the relation of DNA methylation to diseases in humans and important phenotypes in plants drawing attention to this epigenetic mark as an important source of variability. In the last decades, several methodologies were developed to assess the methylation state of a genome. However, there is still a lack of affordable and precise methods for genome wide analysis in large sample size studies. Methyl sensitive double digestion MS-DArT sequencing method emerges as a promising alternative for methylation profiling. We developed a computational pipeline for the identification of DNA methylation using MS-DArT-seq data and carried out a pilot study using the Eucalyptus grandis tree sequenced for the species reference genome. Using a statistic framework as in differential expression analysis, 72,515 genomic sites were investigated and 5,846 methylated sites identified, several tissue specific, distributed along the species 11 chromosomes. We highlight a bias towards identification of DNA methylation in genic regions and the identification of 2,783 genes and 842 transposons containing methylated sites. Comparison with WGBS, DNA sequencing after treatment with bisulfite, data demonstrated a precision rate higher than 95% for our approach. The availability of a reference genome is useful for determining the genomic context of methylated sites but not imperative, making this approach suitable for any species. Our approach provides a cost effective, broad and reliable examination of DNA methylation profile on MspI/HpaII restriction sites, is fully reproducible and the source code is available on GitHub (https://github.com/wendelljpereira/ms-dart-seq).

METHimpute: imputation-guided construction of complete methylomes from WGBS data.

BACKGROUND: Whole-genome bisulfite sequencing (WGBS) has become the standard method for interrogating plant methylomes at base resolution. However, deep WGBS measurements remain cost prohibitive for large, complex genomes and for population-level studies. As a result, most published plant methylomes are sequenced far below saturation, with a large proportion of cytosines having either missing data or insufficient coverage. RESULTS: Here we present METHimpute, a Hidden Markov Model (HMM) based imputation algorithm for the analysis of WGBS data. Unlike existing methods, METHimpute enables the construction of complete methylomes by inferring the methylation status and level of all cytosines in the genome regardless of coverage. Application of METHimpute to maize, rice and Arabidopsis shows that the algorithm infers cytosine-resolution methylomes with high accuracy from data as low as 6X, compared to data with 60X, thus making it a cost-effective solution for large-scale studies. CONCLUSIONS: METHimpute provides methylation status calls and levels for all cytosines in the genome regardless of coverage, thus yielding complete methylomes even with low-coverage WGBS datasets. The method has been extensively tested in plants, but should also be applicable to other species. An implementation is available on Bioconductor.

Techno-economic evaluation of conditioning with sodium sulfite for bioethanol production from softwood.

Conditioning with reducing agents allows alleviation of inhibition of biocatalytic processes by toxic by-products generated during biomass pretreatment, without necessitating the introduction of a separate process step. In this work, conditioning of steam-pretreated spruce with sodium sulfite made it possible to lower the yeast and enzyme dosages in simultaneous saccharification and fermentation (SSF) to 1g/L and 5FPU/g WIS, respectively. Techno-economic evaluation indicates that the cost of sodium sulfite can be offset by benefits resulting from a reduction of either the yeast load by 0.68g/L or the enzyme load by 1FPU/g WIS. As those thresholds were surpassed, inclusion of conditioning can be justified. Another potential benefit results from shortening the SSF time, which would allow reducing the bioreactor volume and result in capital savings. Sodium sulfite conditioning emerges as an opportunity to lower the financial uncertainty and compensate the overall investment risk for commercializing a softwood-to-ethanol process.

Assessment of Changes in Global DNA Methylation Levels by Pyrosequencing® of Repetitive Elements.

Transposable elements (TE) comprise half of the human genome. LINE-1 and ALU are the most common TE, and they have been used to assess changes in the DNA methylation of repetitive elements in response to intrinsic and extrinsic cellular events. Pyrosequencing(®) is a real-time sequencing technology that enables quantitative assessment of TE methylation at single-base resolution. In Pyrosequencing, a region of interest is first amplified from bisulfite-converted DNA by polymerase chain reaction (PCR), before PCR amplicons are rendered single stranded and annealed with the Pyrosequencing primer prior to sequencing. In this chapter, we provide an overview of the analysis of repetitive element DNA methylation by bisulfite Pyrosequencing, and we describe a protocol that can be used for such purposes.

Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling.

Sequencing-based approaches have led to new insights about DNA methylation. While many different techniques for genome-scale mapping of DNA methylation have been employed, throughput has been a key limitation for most. To further facilitate the mapping of DNA methylation, we describe a protocol for gel-free multiplexed reduced representation bisulfite sequencing (mRRBS) that reduces the workload dramatically and enables processing of 96 or more samples per week. mRRBS achieves similar CpG coverage to the original RRBS protocol, while the higher throughput and lower cost make it better suited for large-scale DNA methylation mapping studies, including cohorts of cancer samples.

Two-color quantitative multiplex methylation-specific PCR.

In recent years, several methylation-specific PCR-based techniques have been developed to identify and characterize hypermethylation of CpG dinucleotides with the primary goal of elucidating a better understanding of the role of DNA methylation in important biological processes, such as chromosome X inactivation and carcinogenesis. The specificity of methylation-specific PCR (MSP) techniques relies on amplifying sodium bisulfite-treated DNA with primers specific to predicted sequences of unmethylated and methylated DNA within the gene of interest. In the past, unmethylated and methylated reactions were singleplex and performed in separate wells. In this paper we report a modification of the real-time quantitative multiplex MSP (QM-MSP) technique of Fackler and colleagues that can be applied to any real-time MSP experiment. Although co-amplification with multiple fluorophores is common in standard reverse transcription PCR (RT-PCR), MSP presents unique challenges both mechanistically and operationally that must be overcome in order to successfully co-amplify two methylation-specific targets. In this two-color modification, unmethylated and methylated primer/probe sets are successfully co-amplified in the same reaction using FAM- and VIC-labeled probes. Our modification decreases the cost and time of each real-time experiment by allowing increased throughput of clinical samples and by doubling either the number of genes or the number of samples that can be analyzed per real-time plate.

Hypertrophic and hyperplastic changes of mucus-secreting epithelial cells in rat airways: assessment using a novel, rapid, and simple technique.

Determination of hyperplastic and hypertrophic changes of mucus-secreting cells in animal airways has been performed in the past by using histologic, immunologic, and/or molecular biologic approaches. Histologic techniques are tedious and time-consuming. The other approaches require specific antibodies and cDNA probes that have proved difficult to develop. Described here is a method for the rapid estimation of hyperplastic and hypertrophic changes of secretory epithelial cells in rat airways. The assay specifically measures acidic and neutral mucoproteins in a linear fashion from 0.5 microgram to at least 10 micrograms. Male Sprague-Dawley rats were exposed to metabisulfite mist (10% wt/vol) for 5 days/wk for 3 wk. The lungs were removed and homogenized in a phosphate-buffered solution containing reducing agents and protease inhibitors. The particulate matter was removed by centrifugation, and the soluble extract was applied to a column packed with Sepharose CL-6B. The material eluting in the void volume was applied to a PVDF membrane and stained for either acidic or neutral mucosubstances using Alcian blue or periodic acid-Schiff (PAS) staining, and the absorbance was read using a 96-well plate reader. Lungs from sodium metabisulfite-exposed animals showed a 7-fold and 3.5-fold increase in PAS-positive and Alcian blue-positive material, respectively. The increase in both PAS and Alcian blue staining was hyaluronidase and chondroitinase insensitive. The observed changes are consistent with morphometric measurements of mucus-containing cells in histologic sections of the tissues. This assay may be useful in determining which neurohumoral mediators might be involved in mucus cell hypertrophy and hyperplasia in animal models of chronic obstructive pulmonary disease.