ABSTRACT: Reduced representation libraries from DNA pools analysed with next generation semiconductor based-sequencing to identify SNPs in extreme and divergent pigs for back fat thickness.
Project description: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 as 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.
Project description: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 as 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. Libraries of 96 human samples
Project description:DNA methylation at cytosine-phospho-guanine (CpG) residues is a vital biological process that regulates cell identity and function. Although widely used, bisulfite-based cytosine conversion procedures for DNA methylation sequencing require high temperature and extreme pH, which leads to DNA degradation, especially among unmethylated cytosines. This disproportionate damage to unmethylated cytosines contributes to inaccuracies in GC content representation. EM-seq, an enzyme-based cytosine conversion method, has been proposed as a less biased alternative to methylation profiling. Compared to bisulfite-based methods, EM-seq boasts greater genome coverage with less GC bias and has the potential to cover more CpGs with the same number of reads (i.e., higher signal-to-noise ratio). Reduced representation approaches enrich samples for CpG-rich genomic regions, thereby enhancing throughput and cost effectiveness. We hypothesized that enzyme-based technology could be adapted for reduced representation methylation sequencing to enable high-resolution DNA methylation profiling on low inputs samples. We leveraged the well-established differences in methylation profile between mouse CD4+ T cell populations to compare reduced representation EM-seq (RREM-seq) performance against our previously published modified reduced representation bisulfite sequencing (mRRBS). While the mRRBS method failed to generate reliable DNA libraries when using <2-ng inputs (equivalent to DNA from around 350 cells), the RREM-seq method successfully generated DNA libraries from 1–25 ng of mouse and human genomic DNA. These libraries fell within the expected size range, and primer contamination was not observed. Low-input (<2-ng) RREM-seq libraries’ final concentration, regulatory genomic element coverage, and methylation status within the lineage-defining Treg cell-specific super-enhancers were comparable to mRRBS libraries with more than 10-fold higher DNA input. RREM-seq libraries also successfully detected the methylation differences between alveolar Tconv and Treg cells in mechanically ventilated patients with severe SARS-CoV-2 pneumonia. Our results suggest that the RREM-seq method can generate reliable libraries for single-nucleotide resolution methylation profiling using low input clinical samples.
Project description:In this study, to obtain a clear picture of drought mechanism involved in two distinctive chickpea genotype, the aim was to identify the DNA methylation patterns which potentially regulate drought tolerance/sensitivity of these selected genotypes. The leaf tissues from the shoot apical meristem from drought sensitive and drought tolerant genotypes were used for RRBS (Reduced representation bisulphite sequencing) under drought stress. The sequencing data was analysed using Bismark and methylkit to recall the methylation levels in control and samples for both genotypes and identify differentially methylated regions.
Project description:Reduced Representation Bisulfite Sequencing was used to assess that changes in intestinal DNA methylation associated with NEC outbreak.
