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

{"sci_version": "1.2", "schema": "https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json", "id": "pmid:35178872", "kind": "science-record", "revision": 4, "content_hash": "sha256:86b684e37125c9ab36e36315cef851890c17a8d117dacf219b63acdd283e811d", "created_at": "2026-11-01T17:36:56Z", "updated_at": "2026-11-01T21:27:16Z", "generator": {"name": "corpus-assay-engine", "run_id": "reading-sci-2026-11-01"}, "paper": {"state": "complete", "title": "epiGBS2: Improvements and evaluation of highly multiplexed, epiGBS-based reduced representation bisulfite sequencing.", "doi": "10.1111/1755-0998.13597", "pmid": "35178872", "authors": ["Fleur Gawehns", "Maarten Postuma", "Morgane van Antro", "Adam Nunn", "Bernice Sepers", "Samar Fatma", "Thomas P van Gurp", "Niels C A M Wagemaker", "A Christa Mateman", "Slavica Milanovic-Ivanovic", "Ivo Groβe", "Kees van Oers", "Philippine Vergeer", "Koen J F Verhoeven"], "journal": "Molecular ecology resources", "year": 2022, "abstract": "Several reduced-representation bisulfite sequencing methods have been developed in recent years to determine cytosine methylation de novo in nonmodel species. Here, we present epiGBS2, a laboratory protocol based on epiGBS with a revised and user-friendly bioinformatics pipeline for a wide range of species with or without a reference genome. epiGBS2 is cost- and time-efficient and the computational workflow is designed in a user-friendly and reproducible manner. The library protocol allows a flexible choice of restriction enzymes and a double digest. The bioinformatics pipeline was integrated in the Snakemake workflow management system, which makes the pipeline easy to execute and modular, and parameter settings for important computational steps flexible. We implemented bismark for alignment and methylation analysis and we preprocessed alignment files by double masking to enable single nucleotide polymorphism calling with Freebayes (epiFreebayes). The performance of several critical steps in epiGBS2 was evaluated against baseline data sets from Arabidopsis thaliana and great tit (Parus major), which confirmed its overall good performance. We provide a detailed description of the laboratory protocol and an extensive manual of the bioinformatics pipeline, which is publicly accessible on github (https://github.com/nioo-knaw/epiGBS2) and zenodo (https://doi.org/10.5281/zenodo.4764652).", "claims": [{"id": "claim-method", "statement": "The performance of the epiGBS2 data was evaluated with correlation plots that were created in R (version 4.0.1) (R Core Team, 2021), using ggplot2 version 3.3.3 (Wickham, 2016). The coefficient of determination (R 2) was calculated using the ggpmisc package version 0.3.9 (Aphalo, 2021).", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:35178872", "locator": "sections[20] \"Parus major samples and description of benchmarking data\" paragraph 4", "quote": "The performance of the epiGBS2 data was evaluated with correlation plots that were created in R (version 4.0.1) (R Core Team, 2021), using ggplot2 version 3.3.3 (Wickham, 2016). The coefficient of determination (R 2) was calculated using the ggpmisc package version 0.3.9 (Aphalo, 2021)."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The demultiplexed A. thaliana epiGBS data were deposited on NCBI with BioProject ID PRJNA764918 and raw data files are accessible on Zenodo (https://doi.org/10.5281/zenodo.5519370). Great tit data can be accessed on NCBI with BioProject ID PRJNA208335. The RRBS reads are available under the SRA accessions SRX10989860, SRX10989861, SRX10989862 and SRX10989863, and the epiGBS2 reads are available under the SRA accessions SRX10994457, SRX10994458, SRX10994459 and SRX10994460. The epiGBS2 laboratory protocol can be found in the Supporting Information. The bioinformatics pipeline and documentation can be accessed on github (https://github.com/nioo‐knaw/epiGBS2) and was deposited on Zenodo (https://doi.org/10.5281/zenodo.4764652). An example data set can also be accessed on Zenodo (https://zenodo.org/record/5878925). All scripts used for benchmarking are available at https://github.com/MaartenPostuma/epiGBS‐Benchmarking.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:35178872", "locator": "sections[41] \"DATA AVAILABILITY STATEMENT\" paragraph 2", "quote": "The demultiplexed A. thaliana epiGBS data were deposited on NCBI with BioProject ID PRJNA764918 and raw data files are accessible on Zenodo (https://doi.org/10.5281/zenodo.5519370). Great tit data can be accessed on NCBI with BioProject ID PRJNA208335. The RRBS reads are available under the SRA accessions SRX10989860, SRX10989861, SRX10989862 and SRX10989863, and the epiGBS2 reads are available under the SRA accessions SRX10994457, SRX10994458, SRX10994459 and SRX10994460. The epiGBS2 laboratory protocol can be found in the Supporting Information. The bioinformatics pipeline and documentation can be accessed on github (https://github.com/nioo‐knaw/epiGBS2) and was deposited on Zenodo (https://doi.org/10.5281/zenodo.4764652). An example data set can also be accessed on Zenodo (https://zenodo.org/record/5878925). All scripts used for benchmarking are available at https://github.com/MaartenPostuma/epiGBS‐Benchmarking."}], "state": "complete"}, {"id": "claim-target-result", "statement": "For all individuals, the epiGBS2 methylation calls correlated with the RRBS‐derived methylation calls (R 2 ≥ .76) (Figure 9).", "kind": "quantitative", "target": {"value": 2.0, "unit": "correlation coefficient", "tolerance": null, "source": "For all individuals, the epiGBS2 methylation calls correlated with the RRBS‐derived methylation calls (R 2 ≥ .76) (Figure 9)."}, "evidence": [{"asset": "source:pmid:35178872", "locator": "sections[30] \"Performance epiGBS vs. RRBS\" paragraph 2", "quote": "For all individuals, the epiGBS2 methylation calls correlated with the RRBS‐derived methylation calls (R 2 ≥ .76) (Figure 9)."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "For all individuals, the epiGBS2 methylation calls correlated with the RRBS‐derived methylation calls (R 2 ≥ .76) (Figure 9).", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:35178872"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "blocked", "blocked_on": "data:accession_unresolved", "description": "The GEO series accession resolves and its sample accessions are listed. This lane did not download the dataset or read its licence.", "duration_hours": null, "parameters": {}, "inputs": ["data:geo:PRJNA208335"], "outputs": ["the source data with its repository provenance"], "instruments": [], "reagents": []}, {"id": "run_analysis", "order": 3, "name": "Run the published analysis", "kind": "analysis", "state": "complete", "blocked_on": null, "description": "Apply the paper's statistical method to the resolved series and compare the result with the published target.", "duration_hours": null, "parameters": {}, "inputs": ["data:geo:PRJNA208335"], "outputs": ["the replicated result and its comparison"], "instruments": [], "reagents": []}], "materials": [], "instruments": [], "reagents": [], "conditions": {}, "gaps": ["step:obtain_source_data blocked on data:accession_unresolved"]}, "data": {"state": "partial", "assets": [{"id": "source:pmid:35178872", "role": "manuscript", "format": "xml", "media_type": "application/xml", "uri": "https://doi.org/10.1111/1755-0998.13597", "sha256": "sha256:e9e5b036aef269b95336450a025fb19fd4bf87668ee9e7b675e719131c23cdd1", "bytes": 62984, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "10.1111/1755-0998.13597", "version": null, "note": "Full article fetched from PMC; the checksum covers the rendered section body, not the original XML bytes.", "structure": {"pmcid": "PMC9311447", "source": "europe_pmc_fulltext", "coverage": "whole_body_without_references", "text_hash_scope": "rendered_section_body"}}, {"id": "data:geo:PRJNA208335", "role": "raw", "format": "geo-series", "media_type": "application/vnd.ncbi.geo-series+json", "uri": "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=PRJNA208335", "sha256": null, "bytes": null, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "PRJNA208335", "version": null, "note": "The demultiplexed A. thaliana epiGBS data were deposited on NCBI with BioProject ID PRJNA764918 and raw data files are accessible on Zenodo (https://doi.org/10.5281/zenodo.5519370). Great tit data can be accessed on NCBI with BioProject ID PRJNA208335. The RRBS reads are available under the SRA accessions SRX10989860, SRX10989861, SRX10989862 and SRX10989863, and the epiGBS2 reads are available under the SRA accessions SRX10994457, SRX10994458, SRX10994459 and SRX10994460. The epiGBS2 laboratory protocol can be found in the Supporting Information. The bioinformatics pipeline and documentation can be accessed on github (https://github.com/nioo‐knaw/epiGBS2) and was deposited on Zenodo (https://doi.org/10.5281/zenodo.4764652). An example data set can also be accessed on Zenodo (https://zenodo.org/record/5878925). 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An example data set can also be accessed on Zenodo (https://zenodo.org/record/5878925). 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An example data set can also be accessed on Zenodo (https://zenodo.org/record/5878925). 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Great tit data can be accessed on NCBI with BioProject ID PRJNA208335. The RRBS reads are available under the SRA accessions SRX10989860, SRX10989861, SRX10989862 and SRX10989863, and the epiGBS2 reads are available under the SRA accessions SRX10994457, SRX10994458, SRX10994459 and SRX10994460. The epiGBS2 laboratory protocol can be found in the Supporting Information. The bioinformatics pipeline and documentation can be accessed on github (https://github.com/nioo‐knaw/epiGBS2) and was deposited on Zenodo (https://doi.org/10.5281/zenodo.4764652). An example data set can also be accessed on Zenodo (https://zenodo.org/record/5878925). All scripts used for benchmarking are available at https://github.com/MaartenPostuma/epiGBS‐Benchmarking.", "structure": {"repository": "GEO", "access_route": "GEO accession URL", "access_statement_verbatim": "The demultiplexed A. thaliana epiGBS data were deposited on NCBI with BioProject ID PRJNA764918 and raw data files are accessible on Zenodo (https://doi.org/10.5281/zenodo.5519370). Great tit data can be accessed on NCBI with BioProject ID PRJNA208335. The RRBS reads are available under the SRA accessions SRX10989860, SRX10989861, SRX10989862 and SRX10989863, and the epiGBS2 reads are available under the SRA accessions SRX10994457, SRX10994458, SRX10994459 and SRX10994460. The epiGBS2 laboratory protocol can be found in the Supporting Information. The bioinformatics pipeline and documentation can be accessed on github (https://github.com/nioo‐knaw/epiGBS2) and was deposited on Zenodo (https://doi.org/10.5281/zenodo.4764652). An example data set can also be accessed on Zenodo (https://zenodo.org/record/5878925). 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Great tit data can be accessed on NCBI with BioProject ID PRJNA208335. The RRBS reads are available under the SRA accessions SRX10989860, SRX10989861, SRX10989862 and SRX10989863, and the epiGBS2 reads are available under the SRA accessions SRX10994457, SRX10994458, SRX10994459 and SRX10994460. The epiGBS2 laboratory protocol can be found in the Supporting Information. The bioinformatics pipeline and documentation can be accessed on github (https://github.com/nioo‐knaw/epiGBS2) and was deposited on Zenodo (https://doi.org/10.5281/zenodo.4764652). An example data set can also be accessed on Zenodo (https://zenodo.org/record/5878925). All scripts used for benchmarking are available at https://github.com/MaartenPostuma/epiGBS‐Benchmarking.", "structure": {"repository": "GEO", "access_route": "GEO accession URL", "access_statement_verbatim": "The demultiplexed A. thaliana epiGBS data were deposited on NCBI with BioProject ID PRJNA764918 and raw data files are accessible on Zenodo (https://doi.org/10.5281/zenodo.5519370). Great tit data can be accessed on NCBI with BioProject ID PRJNA208335. The RRBS reads are available under the SRA accessions SRX10989860, SRX10989861, SRX10989862 and SRX10989863, and the epiGBS2 reads are available under the SRA accessions SRX10994457, SRX10994458, SRX10994459 and SRX10994460. The epiGBS2 laboratory protocol can be found in the Supporting Information. The bioinformatics pipeline and documentation can be accessed on github (https://github.com/nioo‐knaw/epiGBS2) and was deposited on Zenodo (https://doi.org/10.5281/zenodo.4764652). An example data set can also be accessed on Zenodo (https://zenodo.org/record/5878925). All scripts used for benchmarking are available at https://github.com/MaartenPostuma/epiGBS‐Benchmarking.", "access_statement_kind": "data_availability_statement", "sample_accessions": [], "sample_count": null, "group_sizes": null, "group_sizes_basis": null, "platform": null, "taxon": null, "title": null, "resolution_checked_at": "2026-11-01T18:06:45Z", "resolution_check": "identifier_resolution_only_no_download_no_licence_read", "downloaded": false, "licence_read": false}}, {"id": "data:geo:SRX10989862", "role": "raw", "format": "geo-series", "media_type": "application/vnd.ncbi.geo-series+json", "uri": "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=SRX10989862", "sha256": null, "bytes": null, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "SRX10989862", "version": null, "note": "The demultiplexed A. thaliana epiGBS data were deposited on NCBI with BioProject ID PRJNA764918 and raw data files are accessible on Zenodo (https://doi.org/10.5281/zenodo.5519370). Great tit data can be accessed on NCBI with BioProject ID PRJNA208335. The RRBS reads are available under the SRA accessions SRX10989860, SRX10989861, SRX10989862 and SRX10989863, and the epiGBS2 reads are available under the SRA accessions SRX10994457, SRX10994458, SRX10994459 and SRX10994460. The epiGBS2 laboratory protocol can be found in the Supporting Information. The bioinformatics pipeline and documentation can be accessed on github (https://github.com/nioo‐knaw/epiGBS2) and was deposited on Zenodo (https://doi.org/10.5281/zenodo.4764652). An example data set can also be accessed on Zenodo (https://zenodo.org/record/5878925). All scripts used for benchmarking are available at https://github.com/MaartenPostuma/epiGBS‐Benchmarking.", "structure": {"repository": "GEO", "access_route": "GEO accession URL", "access_statement_verbatim": "The demultiplexed A. thaliana epiGBS data were deposited on NCBI with BioProject ID PRJNA764918 and raw data files are accessible on Zenodo (https://doi.org/10.5281/zenodo.5519370). Great tit data can be accessed on NCBI with BioProject ID PRJNA208335. The RRBS reads are available under the SRA accessions SRX10989860, SRX10989861, SRX10989862 and SRX10989863, and the epiGBS2 reads are available under the SRA accessions SRX10994457, SRX10994458, SRX10994459 and SRX10994460. The epiGBS2 laboratory protocol can be found in the Supporting Information. The bioinformatics pipeline and documentation can be accessed on github (https://github.com/nioo‐knaw/epiGBS2) and was deposited on Zenodo (https://doi.org/10.5281/zenodo.4764652). An example data set can also be accessed on Zenodo (https://zenodo.org/record/5878925). All scripts used for benchmarking are available at https://github.com/MaartenPostuma/epiGBS‐Benchmarking.", "access_statement_kind": "data_availability_statement", "sample_accessions": [], "sample_count": null, "group_sizes": null, "group_sizes_basis": null, "platform": null, "taxon": null, "title": null, "resolution_checked_at": "2026-11-01T18:06:45Z", "resolution_check": "identifier_resolution_only_no_download_no_licence_read", "downloaded": false, "licence_read": false}}, {"id": "data:geo:SRX10989863", "role": "raw", "format": "geo-series", "media_type": "application/vnd.ncbi.geo-series+json", "uri": "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=SRX10989863", "sha256": null, "bytes": null, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "SRX10989863", "version": null, "note": "The demultiplexed A. thaliana epiGBS data were deposited on NCBI with BioProject ID PRJNA764918 and raw data files are accessible on Zenodo (https://doi.org/10.5281/zenodo.5519370). Great tit data can be accessed on NCBI with BioProject ID PRJNA208335. The RRBS reads are available under the SRA accessions SRX10989860, SRX10989861, SRX10989862 and SRX10989863, and the epiGBS2 reads are available under the SRA accessions SRX10994457, SRX10994458, SRX10994459 and SRX10994460. The epiGBS2 laboratory protocol can be found in the Supporting Information. The bioinformatics pipeline and documentation can be accessed on github (https://github.com/nioo‐knaw/epiGBS2) and was deposited on Zenodo (https://doi.org/10.5281/zenodo.4764652). An example data set can also be accessed on Zenodo (https://zenodo.org/record/5878925). All scripts used for benchmarking are available at https://github.com/MaartenPostuma/epiGBS‐Benchmarking.", "structure": {"repository": "GEO", "access_route": "GEO accession URL", "access_statement_verbatim": "The demultiplexed A. thaliana epiGBS data were deposited on NCBI with BioProject ID PRJNA764918 and raw data files are accessible on Zenodo (https://doi.org/10.5281/zenodo.5519370). Great tit data can be accessed on NCBI with BioProject ID PRJNA208335. The RRBS reads are available under the SRA accessions SRX10989860, SRX10989861, SRX10989862 and SRX10989863, and the epiGBS2 reads are available under the SRA accessions SRX10994457, SRX10994458, SRX10994459 and SRX10994460. The epiGBS2 laboratory protocol can be found in the Supporting Information. The bioinformatics pipeline and documentation can be accessed on github (https://github.com/nioo‐knaw/epiGBS2) and was deposited on Zenodo (https://doi.org/10.5281/zenodo.4764652). An example data set can also be accessed on Zenodo (https://zenodo.org/record/5878925). All scripts used for benchmarking are available at https://github.com/MaartenPostuma/epiGBS‐Benchmarking.", "access_statement_kind": "data_availability_statement", "sample_accessions": [], "sample_count": null, "group_sizes": null, "group_sizes_basis": null, "platform": null, "taxon": null, "title": null, "resolution_checked_at": "2026-11-01T18:06:45Z", "resolution_check": "identifier_resolution_only_no_download_no_licence_read", "downloaded": false, "licence_read": false}}, {"id": "data:geo:SRX10994457", "role": "raw", "format": "geo-series", "media_type": "application/vnd.ncbi.geo-series+json", "uri": "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=SRX10994457", "sha256": null, "bytes": null, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "SRX10994457", "version": null, "note": "The demultiplexed A. thaliana epiGBS data were deposited on NCBI with BioProject ID PRJNA764918 and raw data files are accessible on Zenodo (https://doi.org/10.5281/zenodo.5519370). Great tit data can be accessed on NCBI with BioProject ID PRJNA208335. The RRBS reads are available under the SRA accessions SRX10989860, SRX10989861, SRX10989862 and SRX10989863, and the epiGBS2 reads are available under the SRA accessions SRX10994457, SRX10994458, SRX10994459 and SRX10994460. The epiGBS2 laboratory protocol can be found in the Supporting Information. The bioinformatics pipeline and documentation can be accessed on github (https://github.com/nioo‐knaw/epiGBS2) and was deposited on Zenodo (https://doi.org/10.5281/zenodo.4764652). An example data set can also be accessed on Zenodo (https://zenodo.org/record/5878925). 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