epiGBS2: Improvements and evaluation of highly multiplexed, epiGBS‐based reduced representation bisulfite sequencing

Gawehns, Fleur; Postuma, Maarten; Antro, Morgane Van; Nunn, Adam; Sepers, Bernice; Fatma, Samar; Gurp, Thomas P. van; Wagemaker, Cornelis A. M.; Mateman, A. Christa; Milanovic-Ivanovic, Slavica; Große, Ivo; Oers, Kees van; Vergeer, Philippine; Verhoeven, Koen

doi:10.1111/1755-0998.13597

Cited by 23 publications

(40 citation statements)

References 62 publications

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“…An open-source example of a working pipeline for whole genome data is available at https://github.com/EpiDiverse/SNP , which is itself a branch of the EpiDiverse Toolkit [ 22 ]. The presented software is also implemented by epiGBS2 in the analysis of reduced-representation bisulfite data [ 23 ].…”

Section: Methodsmentioning

confidence: 99%

Manipulating base quality scores enables variant calling from bisulfite sequencing alignments using conventional bayesian approaches

Nunn

Otto²,

Fasold³

et al. 2022

BMC Genomics

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Background Calling germline SNP variants from bisulfite-converted sequencing data poses a challenge for conventional software, which have no inherent capability to dissociate true polymorphisms from artificial mutations induced by the chemical treatment. Nevertheless, SNP data is desirable both for genotyping and to understand the DNA methylome in the context of the genetic background. The confounding effect of bisulfite conversion however can be conceptually resolved by observing differences in allele counts on a per-strand basis, whereby artificial mutations are reflected by non-complementary base pairs. Results Herein, we present a computational pre-processing approach for adapting sequence alignment data, thus indirectly enabling downstream analysis on a per-strand basis using conventional variant calling software such as GATK or Freebayes. In comparison to specialised tools, the method represents a marked improvement in precision-sensitivity based on high-quality, published benchmark datasets for both human and model plant variants. Conclusion The presented “double-masking” procedure represents an open source, easy-to-use method to facilitate accurate variant calling using conventional software, thus negating any dependency on specialised tools and mitigating the need to generate additional, conventional sequencing libraries alongside bisulfite sequencing experiments. The method is available at https://github.com/bio15anu/revelioand an implementation with Freebayes is available at https://github.com/EpiDiverse/SNP

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Section: Methodsmentioning

confidence: 99%

Manipulating base quality scores enables variant calling from bisulfite sequencing alignments using conventional bayesian approaches

Nunn

Otto²,

Fasold³

et al. 2022

BMC Genomics

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“…Sequencing data were analysed using the epiGBS2 pipeline (epiGBS2 commit: 5a70433fa) (Gawehns et al, 2022), using the ‘de novo’ option with default parameters (95% sequence identity in the last clustering step and a clustering depth threshold of zero) to generate experiment-specific local genomic references for the epiGBS reads. In brief, the epiGBS2 pipeline first removes all PCR duplicates based on the identity of the UMI inserted in the adapter sequences.…”

Section: Methodsmentioning

confidence: 99%

“…An adapted version of the epiGBS protocol (Van Gurp et al, 2016) was followed, as described by (Gawehns et al, 2022). In brief: After full randomization of all samples, DNA was digested with DNA methylation insensitive restriction enzymes AseI and NsiI (ensuring that the enzymes did not induce a bias by cutting primarily in (non-)methylated regions of the genome).…”

Section: Epigbs Library Preparationmentioning

confidence: 99%

DNA methylation in clonal Duckweed lineages (Lemna minorL.) reflects current and historical environmental exposures

Antro

Prelovsek

Ivanovic

et al. 2022

Preprint

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While some DNA methylation variants are transgenerationally stable in plants, DNA methylation modifications that are specifically induced by environmental exposure are typically transient and subject to resetting in germ lines, limiting the potential for transgenerational epigenetics stress memory. Asexual reproduction circumvents germlines, and may be more conducive to long-term memory and inheritance of epigenetic marks. This, however, has been poorly explored. Taking advantage of the rapid clonal reproduction of the common duckweed Lemna minor, we tested the hypothesis that a long-term, transgenerational stress memory from exposure to high temperature can be detected in DNA methylation profiles. Using a reduced representation bisulfite sequencing approach (epiGBS), we show that high temperature stress induces DNA hypermethylation at many cytosines in CG and CHG contexts but not in CHH. In addition, a subset of the temperature responsive CHG cytosines, showed differential DNA methylation between in lineages exposed to 30C and 24C, 3-12 clonal generations after subsequent culturing in a common environment, demonstrating a memory effect of stress that persists over many clonal generations and that is reflected in DNA methylation. Structural annotation revealed that this memory effect in CHG methylation was enriched in TEs. We argue that the observed epigenetic stress memory is likely caused by stable transgenerational persistence of high temperature-induced DNA methylation variants across multiple clonal generations. To the extent that such epigenetic memory has functional consequences for gene expression and phenotypes, this result suggests potential for long-term modulation of stress responses in asexual plants and vegetatively propagated crops.

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“…We used the epiGBS2 pipeline (Gawehns et al, 2022) based on the original epiGBS pipeline (van Gurp et al, 2016) to process the raw sequence data. We demultiplexed, quality trimmed sequencing reads, and removed the barcode sequences with the original scripts as previously described (van Moorsel et al, 2019;Mounger et al, 2021b).…”

Section: Data Pre-processingmentioning

confidence: 99%

Genetic and Epigenetic Differentiation Across Intertidal Gradients in the Foundation Plant Spartina alterniflora

et al. 2022

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Ecological genomics approaches have informed us about the structure of genetic diversity in natural populations that might underlie patterns in trait variation. However, we still know surprisingly little about the mechanisms that permit organisms to adapt to variable environmental conditions. The salt marsh foundation plant Spartina alterniflora exhibits a dramatic range in phenotype that is associated with a pronounced intertidal environmental gradient across a narrow spatial scale. Both genetic and non-genetic molecular mechanisms might underlie this phenotypic variation. To investigate both, we used epigenotyping-by-sequencing (epiGBS) to evaluate the make-up of natural populations across the intertidal environmental gradient. Based on recent findings, we expected that both DNA sequence and DNA methylation diversity would be explained by source population and habitat within populations. However, we predicted that epigenetic variation might be more strongly associated with habitat since similar epigenetic modifications could be rapidly elicited across different genetic backgrounds by similar environmental conditions. Overall, with PERMANOVA we found that population of origin explained a significant amount of the genetic (8.6%) and epigenetic (3.2%) variance. In addition, we found that a small but significant amount of genetic and epigenetic variance (<1%) was explained by habitat within populations. The interaction of population and habitat explained an additional 2.9% of the genetic variance and 1.4% of the epigenetic variance. By examining genetic and epigenetic variation within the same fragments (variation in close-cis), we found that population explained epigenetic variation in 9.2% of 8,960 tested loci, even after accounting for differences in the DNA sequence of the fragment. Habitat alone explained very little (<0.1%) of the variation in these close-cis comparisons, but the interaction of population and habitat explained 2.1% of the epigenetic variation in these loci. Using multiple matrix regression with randomization (MMRR) we found that phenotypic differences in natural populations were correlated with epigenetic and environmental differences even when accounting for genetic differences. Our results support the contention that sequence variation explains most of the variation in DNA methylation, but we have provided evidence that DNA methylation distinctly contributes to plant responses in natural populations.

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epiGBS2: Improvements and evaluation of highly multiplexed, epiGBS‐based reduced representation bisulfite sequencing

Cited by 23 publications

References 62 publications

Manipulating base quality scores enables variant calling from bisulfite sequencing alignments using conventional bayesian approaches

Manipulating base quality scores enables variant calling from bisulfite sequencing alignments using conventional bayesian approaches

DNA methylation in clonal Duckweed lineages (Lemna minorL.) reflects current and historical environmental exposures

Genetic and Epigenetic Differentiation Across Intertidal Gradients in the Foundation Plant Spartina alterniflora

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