11 research outputs found

    Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.

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    BACKGROUND: Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing. RESULTS: We perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies. CONCLUSIONS: We show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets.This work was supported by the Biotechnology and Biological Sciences Research Council (CASE studentship to N.O., BB/K010867/1 to W.R.), Wellcome Trust (095645/Z/11/Z to W.R.), EU EpiGeneSys (257082 to W.R.) and EU BLUEPRINT (282510 to W.R.); Babraham Institute/Cambridge European Trust scholarship to N.O.; M.R.B. is a Sir Henry Dale Fellow (101225/Z/ 13/Z), jointly funded by the Wellcome Trust and the Royal Society

    Measuring and interpreting transposable element expression

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    International audienceTransposable elements (TEs) are insertional mutagens that contribute greatly to the plasticity of eukaryotic genomes, influencing the evolution and adaptation of species as well as physiology or disease in individuals. Measuring TE expression helps to understand not only when and where TE mobilization can occur, but also how this process alters gene expression, chromatin accessibility or cellular signalling pathways. Although genome-wide gene expression assays such as RNA-sequencing include transposon-derived transcripts, the majority of computational analytical tools discard or misinterpret TE-derived reads. Emerging approaches are improving the identification of expressed TE loci and helping to discriminate TE transcripts that permit TE mobilization from gene-TE chimeric transcripts or pervasive transcription. Here, we review the main challenges associated with the detection of TE expression, including mappability, insertional and internal sequence polymorphisms, and the diversity of the TE transcriptional landscape, as well as the different experimental and computational strategies to solve them

    Ten things you should know about transposable elements

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    Measuring and interpreting transposable element expression

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    L1 retrotransposition in the soma: a field jumping ahead

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