FAQs¶
TwistMethylFlow FAQs¶
Pipeline Steps¶
Generate Reference Genome¶
What is the purpose of Generate Reference Genome?
This step creates reference genome index files for Bismark, which are required for bisulfite read alignment.
Raw Data QC¶
How is raw sequencing data quality assessed?
Raw Data QC uses FastQC to evaluate per-base quality, GC content, adapter contamination, and other metrics.
Adapter Trimming¶
How should adapters and low-quality bases be handled?
Trim Galore removes adapters and low-quality bases from reads, ensuring high-quality data for alignment.
Align Reads¶
How are reads aligned to the reference genome?
Bismark (Bowtie2) aligns bisulfite-treated reads to the reference genome.
Accurate alignment is crucial for downstream methylation analysis.
Deduplicate Removal¶
What is deduplicate removal?
Bismark removes PCR duplicates from aligned reads to prevent bias in methylation calling.
Sort and Indexing¶
How are BAM files prepared after alignment?
Samtools sorts and indexes deduplicated BAM files for efficient access in downstream analyses.
Extract Methylation Calls¶
How are methylation calls extracted?
Bismark extracts cytosine methylation calls from aligned reads, generating the input for differential methylation analysis.
Summary Report¶
How is the summary report generated?
Bismark summarizes alignment statistics and methylation extraction results, including conversion efficiency.
Alignment QC¶
How is alignment quality assessed?
Qualimap generates metrics such as coverage, mapping quality, and duplication rate for aligned reads.
QC Reporting¶
How are QC reports aggregated?
MultiQC compiles QC reports from FastQC, Bismark, and Qualimap into a single comprehensive report.
Differential Methylation¶
How is differential methylation analysis performed?
EdgeR and MethylKit identify differentially methylated positions or regions, considering replicates and experimental design.
Post Processing¶
How are results visualized?
ggplot2 creates summary plots like heatmaps, volcano plots, and coverage plots for differential methylation results.
GO Analysis¶
How is Gene Ontology analysis performed?
Functional enrichment analysis identifies pathways associated with differentially methylated genes, providing biological interpretation.
Nextflow Workflow Concepts¶
General Nextflow¶
What is Nextflow?
Nextflow is a workflow management system for reproducible and scalable scientific pipelines.
It allows users to define tasks and data flow using processes and channels.
Processes¶
What are processes in Nextflow?
Processes encapsulate a task with defined inputs, outputs, and commands.
They run independently and can scale across computing resources.
Channels¶
What are channels in Nextflow?
Channels are asynchronous data streams that connect processes, passing input and output data between them.
Outputs¶
How do I define outputs in Nextflow?
Outputs are declared using the output block in processes or workflows.
This defines what data is saved for downstream steps or exported from the workflow.
Parallel Execution¶
How does Nextflow handle parallel execution?
Nextflow automatically runs independent processes in parallel based on available compute resources, enabling scalable execution.
Reproducibility¶
How does Nextflow ensure reproducibility?
Nextflow supports containerization (Docker/Singularity) and environment specification, ensuring the same pipeline produces identical results across systems.
Workflow Profiles¶
What are Nextflow profiles?
Profiles allow users to define environment-specific configurations, such as compute clusters, Docker images, and resource limits, for flexible pipeline execution.
Learning Resources¶
Where can I learn more about Nextflow?
Performance¶
Runtime, Memory, and Storage¶
What are the typical runtime, memory, and storage requirements?
- Runtime: Varies based on dataset size and computational resources. For 24 paired-end samples, it can take several hours to days.
- Memory: Ranges from 6 GB to 200 GB depending on the step. Alignment and differential methylation analysis are more memory-intensive.
- Storage: Approximately 2 TB for 24 paired-end samples, including intermediate files and results.
| Process | Average Process Time | Average Wall Clock Time | Max Peak Memory | Total I/O (Read + Written) |
|---|---|---|---|---|
| FastQC | 7m 3s | 7m 2s | 628.4 MB | 5.3 GB |
| trim galore | 22m 1s | 22m 0s | 348.2 MB | 165.2 GB |
| Bismark Genome Preparation | 2h 24m 38s | 2h 24m 37s | 12.8 GB | 47.8 GB |
| Bismark Alignment | 14h 27m 57s | 14h 27m 56s | 10.2 GB | 229.8 GB |
| Bismark Deduplication | 29m 3s | 29m 1s | 9.8 GB | 95.8 GB |
| Samtools sort | 8m 32s | 8m 31s | 3.3 GB | 14.8 GB |
| Samtools index | 1m 2s | 1m 1s | 21.1 MB | 4.8 GB |
| Bismark Methylation extractor | 2h 56m 23s | 2h 56m 22s | 2 GB | 258.4 GB |
| Qualimap | 6m 29s | 6m 29s | 12.1 GB | 4.7 GB |
| Bismark report | 2.9s | 2.5s | 161.1 MB | 7.1 MB |
| Multiqc | 14.1s | 13.3s | 80.5 MB | 3.0 GB |
| EdgeR analysis | 46m 37s | 46m 36s | 44.9 GB | 8.9 GB |
| Annotate results | 5m 23s | 5m 23s | 6.8 GB | 1.8 GB |
| GO analysis EdgeR | 4m 15s | 4m 15s | 6.4 GB | 1.8 GB |
| Post processing EdgeR | 18m 40s | 18m 40s | 9.3 GB | 7.3 GB |
| MethylKit analysis | 2h 59m 19s | 2h 59m 19s | 37.6 GB | 9.0 GB |
| GO analysis methylKit | 1m 44s | 1m 44s | 3.9 GB | 1.3 GB |
| Post processing methylKit | 8m 4s | 8m 3s | 4.4 GB | 3.5 GB |