> For the complete documentation index, see [llms.txt](https://jd2112.gitbook.io/twistnext/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://jd2112.gitbook.io/twistnext/faqs.md). # faqs ## FAQs ## TwistMethylFlow FAQs ### Pipeline Steps #### Generate Reference Genome ??? question "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 ??? question "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 ??? question "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 ??? question "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 ??? question "What is deduplicate removal?" **Bismark** removes PCR duplicates from aligned reads to prevent bias in methylation calling. #### Sort and Indexing ??? question "How are BAM files prepared after alignment?" **Samtools** sorts and indexes deduplicated BAM files for efficient access in downstream analyses. #### Extract Methylation Calls ??? question "How are methylation calls extracted?" **Bismark** extracts cytosine methylation calls from aligned reads, generating the input for differential methylation analysis. #### Summary Report ??? question "How is the summary report generated?" **Bismark** summarizes alignment statistics and methylation extraction results, including conversion efficiency. #### Alignment QC ??? question "How is alignment quality assessed?" **Qualimap** generates metrics such as coverage, mapping quality, and duplication rate for aligned reads. #### QC Reporting ??? question "How are QC reports aggregated?" **MultiQC** compiles QC reports from FastQC, Bismark, and Qualimap into a single comprehensive report. #### Differential Methylation ??? question "How is differential methylation analysis performed?" **EdgeR** and **MethylKit** identify differentially methylated positions or regions, considering replicates and experimental design. #### Post Processing ??? question "How are results visualized?" **ggplot2** creates summary plots like heatmaps, volcano plots, and coverage plots for differential methylation results. #### GO Analysis ??? question "How is Gene Ontology analysis performed?" Functional enrichment analysis identifies pathways associated with differentially methylated genes, providing biological interpretation. *** ### Nextflow Workflow Concepts #### General Nextflow ??? question "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 ??? question "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 ??? question "What are channels in Nextflow?" Channels are asynchronous data streams that connect processes, passing input and output data between them. #### Outputs ??? question "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 ??? question "How does Nextflow handle parallel execution?" Nextflow automatically runs independent processes in parallel based on available compute resources, enabling scalable execution. #### Reproducibility ??? question "How does Nextflow ensure reproducibility?" Nextflow supports containerization (Docker/Singularity) and environment specification, ensuring the same pipeline produces identical results across systems. #### Workflow Profiles ??? question "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 ??? question "Where can I learn more about Nextflow?" - [Nextflow Documentation](https://www.nextflow.io/docs/latest/index.html)\ \- [Nextflow Tutorials](https://nf-co.re/docs/usage/tutorials/nextflow)\ \- [Hello Nextflow Training](https://training.nextflow.io/2.0/hello_nextflow/) *** ### Performance #### Runtime, Memory, and Storage ??? question "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 | ```