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‎docs/credits.md‎

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‎docs/faqs.md‎

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# FAQs
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# TwistMethylFlow FAQs
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## Pipeline Steps
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### Generate Reference Genome
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??? question "What is the purpose of Generate Reference Genome?"
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This step creates reference genome index files for **Bismark**, which are required for bisulfite read alignment.
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### Raw Data QC
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??? question "How is raw sequencing data quality assessed?"
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Raw Data QC uses **FastQC** to evaluate per-base quality, GC content, adapter contamination, and other metrics.
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### Adapter Trimming
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??? question "How should adapters and low-quality bases be handled?"
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**Trim Galore** removes adapters and low-quality bases from reads, ensuring high-quality data for alignment.
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### Align Reads
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??? question "How are reads aligned to the reference genome?"
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**Bismark (Bowtie2)** aligns bisulfite-treated reads to the reference genome.
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Accurate alignment is crucial for downstream methylation analysis.
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### Deduplicate Removal
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??? question "What is deduplicate removal?"
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**Bismark** removes PCR duplicates from aligned reads to prevent bias in methylation calling.
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### Sort and Indexing
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??? question "How are BAM files prepared after alignment?"
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**Samtools** sorts and indexes deduplicated BAM files for efficient access in downstream analyses.
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### Extract Methylation Calls
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??? question "How are methylation calls extracted?"
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**Bismark** extracts cytosine methylation calls from aligned reads, generating the input for differential methylation analysis.
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### Summary Report
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??? question "How is the summary report generated?"
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**Bismark** summarizes alignment statistics and methylation extraction results, including conversion efficiency.
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### Alignment QC
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??? question "How is alignment quality assessed?"
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**Qualimap** generates metrics such as coverage, mapping quality, and duplication rate for aligned reads.
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### QC Reporting
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??? question "How are QC reports aggregated?"
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**MultiQC** compiles QC reports from FastQC, Bismark, and Qualimap into a single comprehensive report.
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### Differential Methylation
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??? question "How is differential methylation analysis performed?"
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**EdgeR** and **MethylKit** identify differentially methylated positions or regions, considering replicates and experimental design.
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### Post Processing
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??? question "How are results visualized?"
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**ggplot2** creates summary plots like heatmaps, volcano plots, and coverage plots for differential methylation results.
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### GO Analysis
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??? question "How is Gene Ontology analysis performed?"
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Functional enrichment analysis identifies pathways associated with differentially methylated genes, providing biological interpretation.
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---
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## Nextflow Workflow Concepts
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### General Nextflow
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??? question "What is Nextflow?"
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Nextflow is a workflow management system for reproducible and scalable scientific pipelines.
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It allows users to define tasks and data flow using **processes** and **channels**.
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### Processes
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??? question "What are processes in Nextflow?"
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Processes encapsulate a task with defined inputs, outputs, and commands.
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They run independently and can scale across computing resources.
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### Channels
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??? question "What are channels in Nextflow?"
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Channels are asynchronous data streams that connect processes, passing input and output data between them.
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### Outputs
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??? question "How do I define outputs in Nextflow?"
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Outputs are declared using the `output` block in processes or workflows.
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This defines what data is saved for downstream steps or exported from the workflow.
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### Parallel Execution
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??? question "How does Nextflow handle parallel execution?"
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Nextflow automatically runs independent processes in parallel based on available compute resources, enabling scalable execution.
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### Reproducibility
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??? question "How does Nextflow ensure reproducibility?"
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Nextflow supports containerization (Docker/Singularity) and environment specification, ensuring the same pipeline produces identical results across systems.
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### Workflow Profiles
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??? question "What are Nextflow profiles?"
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Profiles allow users to define environment-specific configurations, such as compute clusters, Docker images, and resource limits, for flexible pipeline execution.
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### Learning Resources
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??? question "Where can I learn more about Nextflow?"
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- [Nextflow Documentation](https://www.nextflow.io/docs/latest/index.html)
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- [Nextflow Tutorials](https://nf-co.re/docs/usage/tutorials/nextflow)
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- [Hello Nextflow Training](https://training.nextflow.io/2.0/hello_nextflow/)
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‎docs/images/JD_logo.png‎

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‎docs/overrides/main.html‎

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{% extends "base.html" %}
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{# ---------------------- Sidebar block ---------------------- #}
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{% block sidebar %}
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{{ super() }}
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<!-- Sidebar GitHub links per page, pushed to bottom -->
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<div class="md-sidebar-footer md-sidebar-footer--custom">
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{% include "partials/sidebar-github.html" %}
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</div>
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{% endblock %}
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{# ---------------------- Footer block ---------------------- #}
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{% block footer %}
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<div style="
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display: flex;

‎docs/overrides/partials/logo.html‎

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{% set repo_url = page and page.meta and page.meta.repo_url or "https://github.com/JD2112" %}
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<a href="{{ repo_url }}" target="_blank" style="display:flex; align-items:center; text-decoration:none;">
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<img src="{{ base_url }}/images/JD_logo.png" alt="Logo" style="height:24px; margin-right:8px;">
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</a>

‎docs/overrides/partials/nav.html‎

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{#-
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This file was automatically generated - do not edit
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-#}
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{% import "partials/nav-item.html" as item with context %}
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{% set class = "md-nav md-nav--primary" %}
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{% if "navigation.tabs" in features %}
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{% set class = class ~ " md-nav--lifted" %}
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{% endif %}
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{% if "toc.integrate" in features %}
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{% set class = class ~ " md-nav--integrated" %}
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{% endif %}
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<nav class="{{ class }}" aria-label="{{ lang.t('nav') }}" data-md-level="0">
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<label class="md-nav__title" for="__drawer">
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<a href="{{ config.extra.homepage | d(nav.homepage.url, true) | url }}" title="{{ config.site_name | e }}" class="md-nav__button md-logo" aria-label="{{ config.site_name }}" data-md-component="logo">
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{% include "partials/logo.html" %}
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</a>
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{{ config.site_name }}
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</label>
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{% if config.repo_url %}
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<div class="md-nav__source">
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{% include "partials/source.html" %}
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</div>
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{% endif %}
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<ul class="md-nav__list" data-md-scrollfix>
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{% for nav_item in nav %}
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{% set path = "__nav_" ~ loop.index %}
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<li class="md-nav__item">
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{{ item.render(nav_item, path, 1) }}
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{# Only add GitHub icon for actual pages #}
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{% if nav_item.__class__.__name__ == "Page" and nav_item.file.src_path.endswith("tutorials.md") %}
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<a href="{% if 'methylr/tutorials.md' in nav_item.file.src_path %}https://github.com/JD2112/methylr
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{% elif 'olinkwrapper/tutorials.md' in nav_item.file.src_path %}https://github.com/JD2112/ShinyOlink
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{% elif 'shinyWGCNA/tutorials.md' in nav_item.file.src_path %}https://github.com/JD2112/Shiny-WGCNA
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{% endif %}"
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target="_blank" class="md-nav__repo-icon" style="margin-left:5px; vertical-align:middle;">
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<img src="https://github.githubassets.com/images/modules/logos_page/GitHub-Mark.png" width="16" height="16" alt="GitHub">
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</a>
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{% endif %}
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</li>
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{% endfor %}
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</ul>
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</nav>

‎docs/overrides/partials/sidebar-github.html‎

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{% if page and page.meta.repo_url %}
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<div class="sidebar-github" style="margin-top:1rem; text-align:center;">
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<a href="{{ page.meta.repo_url }}" target="_blank" title="GitHub Repo">
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<img src="https://github.githubassets.com/images/modules/logos_page/GitHub-Mark.png"
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alt="GitHub" width="32" height="32">
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</a>
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</div>
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{% endif %}

‎docs/references.md‎

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>Huber, W., Carey, V., Gentleman, R. et al. Orchestrating high-throughput genomic analysis with Bioconductor. Nat Methods 12, 115–121 (2015). [https://doi.org/10.1038/nmeth.3252](https://doi.org/10.1038/nmeth.3252).
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### Singularity
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>Kurtzer GM, Sochat V, Bauer MW. Singularity: Scientific containers for mobility of compute. PLoS One. 2017 May 11;12(5):e0177459. doi: 10.1371/journal.pone.0177459. eCollection 2017. PubMed PMID: 28494014; PubMed Central PMCID: PMC5426675.
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>Kurtzer GM, Sochat V, Bauer MW. Singularity: Scientific containers for mobility of compute. PLoS One. 2017 May 11;12(5):e0177459. doi: 10.1371/journal.pone.0177459. eCollection 2017. PubMed PMID: 28494014; PubMed Central PMCID: PMC5426675.
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---
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## 👤 Authors and Maintainers
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| Role | Name & Link | Description |
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|--------------|------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------|
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| **Main Author** | [Jyotirmoy Das](https://github.com/JD2112) | Lead designer, developer, and overall coordinator for the **TwistMethylFlow** pipeline. |
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| **Maintainer** | [Jyotirmoy Das](https://github.com/JD2112) | Actively maintaining the pipeline, fixing bugs, and implementing new features. |
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| **Contributors**| [All Contributors](https://github.com/JD2112/TwistMethylFlow/pulls) | Thanks to everyone who helped in development, testing, and enhancement. Contributions welcome! |
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---
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## 📄 Citation
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If you use **TwistMethylFlow** in your work, please cite:
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> Das, J. (2024). *TwistMethylFlow (v1.0.0)*. Zenodo. [https://doi.org/10.5281/zenodo.14204261](https://doi.org/10.5281/zenodo.14204261)
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---
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## ❓ Help, FAQ, Troubleshooting
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- Refer to the [User Manual](index.md) for detailed usage instructions and guidance.
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- For bug reports, questions, or feature requests, please open an [issue on GitHub](https://github.com/JD2112/TwistMethylFlow/issues).
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---
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## 📜 License
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This project is licensed under the terms of the **GNU General Public License v3.0**.
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---
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## 🙏 Acknowledgements
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### Research Institutions
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- **Core Facility, Faculty of Medicine and Health Sciences, Linköping University**
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[Linköping University Core Facility](https://liu.se/en/organisation/liu/medfak/coref)
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- **Clinical Genomics Linköping, Science for Life Laboratory, Sweden**
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[Clinical Genomics Linköping](https://www.scilifelab.se/units/clinical-genomics-linkoping/)
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- **National Academic Infrastructure for Supercomputing in Sweden (NAISS)**
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> The computations/data storage were enabled by resources provided by the National Academic Infrastructure for Supercomputing in Sweden (NAISS), partially funded by the Swedish Research Council through grant agreement no. 2022-06725.
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These institutions provided invaluable resources and support for the development and testing of the TwistMethylFlow pipeline.
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### Additional Thanks
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- **Open Source Community**: Special thanks to the developers and contributors of [Nextflow](https://www.nextflow.io/), [Bismark](https://www.bioinformatics.babraham.ac.uk/projects/bismark/), [methylKit](https://github.com/observer101/methylKit), [EdgeR](https://bioconductor.org/packages/release/bioc/html/edgeR.html) and other bioinformatics tools integrated into this pipeline.
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- **Users and Testers**: A heartfelt thank you to all the early adopters, testers, and collaborators who helped refine and improve the tool through their feedback and bug reports.
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---

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