> For the complete documentation index, see [llms.txt](https://help.connected.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://help.connected.illumina.com/dragen-5-base/additional-information/faqs.md). # FAQs ## General Questions * Does this pipeline support ORA compressed FASTQs? * Yes, all iterations of the pipeline support analysis from .fastq.ora files. * Can I load more than one sample at a time? * Yes. When selecting biosamples, multiple can be selected as long as sex is set to auto-detect.
* Is there demo data available? * Yes, there is demo data in BaseSpace, ICA, and ICM. * To access the demo data on BaseSpace, go to the demo data tab and search for "5-Base".
* On ICA, the demo data is included in the DRAGEN 4.4 bundle. After adding the DRAGEN 4.4 bundle to a project, navigate to the data tab, and find the "Illumina DRAGEN 5-Base Methylation Germline Demo Data" folder. * On ICM, create a study, and click "+ Add Data" and "Select from ICA project". Select "Bulk", "Methylation", and then "Illumina 5-Base Solution" as the format. Click "+ Add Demo Data", and navigate to the "Multiomics-Demo-Data" folder. Click into "Methylation", then "Illumina-5-base-solution", and import all samples in that folder.
* How does DRAGEN tell when a site is a genetic variant, or a methylated site? * A genetic variant impacts two strands of the DNA; a methylated site impacts only one.
* What DRAGEN features are 5-base aware? * Only the features and options specified in this documentation are formally supported. All other features should be considered experimental and used at risk. * For additional information, review the DRAGEN documentation: * How does DRAGEN 5-Base in Germline, Somatic, and Enrichment differ from DRAGEN Methylation? * In general, it's not recommended to use DRAGEN Methylation for 5-Base is because DRAGEN Methylation is meant for bisulfite data (unmethylated C -> T), and not our 5-Base solution (where you convert methylated C -> T). DRAGEN Methylation doesn't allow for variant calling with the 5-Base library. Additionally, the alignment isn’t 5-Base aware, and thus will be worse than when running DRAGEN Germline, Somatic, or Enrichment. However, there is a need to do a direct comparison between bisulfite data and 5-base, it is possible to run 5-Base libraries on DRAGEN Methylation. * Should DRAGEN Germline, Somatic, and Enrichment be used for non-human samples? * Methylation calling for non-human samples is supported within DRAGEN Germline, Somatic, and Enrichment. Variant calling for non-human samples is not formally validated in these apps, but can be done experimentally. Some non-mammalian species have high CpH methylation; to support these samples, it's recommended to run `--enable-cpg-methylated-mapping=false` in the "Additional Arguments" section of the App. This setting is not recommended for analysis of samples where the majority of methylation occurs in a CpG context. * How do you build a 5-base specific DRAGEN hashtable? * To build a 5-base specific DRAGEN hashtable, it's required to run `--ht-methylated-cg=true` . This will create a methyl\_cg sub-directory, which the DRAGEN mapper will use automatically. * For additional information, please review the general DRAGEN documentation on non-human reference building: ## Runtime and Cost Summary * How long does the average analysis take and how much does it cost?
PipelineNum SamplesCoverageAnalysis SettingsTimeCompute Cost (iCredits)
DRAGEN Germline1 sample35xGermline, 5-base methylation calling, SNV, CNV, SV~1h~5.5
DRAGEN Somatic1 sample (tumor-only)100xSomatic, 5-base methylation calling, SNV~3h15m~12
DRAGEN Somatic2 samples (tumor-normal pair)100x/50xSomatic, 5-base methylation calling, SNV~4h45m~35
DRAGEN Somatic2 samples (tumor-normal pair)100x/50xSomatic, 5-base methylation calling, CNV, SV~5h~35
DRAGEN Somatic2 samples (tumor-normal pair)100x/50xSomatic, 5-base methylation calling, SNV, CNV, SV~6h30m~40
DRAGEN Enrichment1 sample2500xSomatic UMI Liquid, SNV~30m<1
## Output Files Summary * What output files does DRAGEN produce for 5-base data? The following table describes the key output files produced per sample: | Output File | Filename Pattern | Description | Produced When | | ---------------- | ---------------------------------------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | ----------------------------------------------------------------------------------- | | BAM | `{prefix}.bam (normal) / {prefix}_tumor.bam (tumor)` | Aligned reads with methylation tags (XM, XR, XG). Contains methylation status for all MAPQ>0 proper-pair reads. Control reads marked as unmapped with `ca:Z` tag. | `--enable-map-align-output true` | | VCF | `{prefix}.hard-filtered.vcf.gz` | Small variant calls with integrated methylation reporting (M5mC fields). Reports methylation at ref and alt alleles. To learn more, review the [VCF documentation](https://help.dragen.illumina.com/dragen-v4.5/product-guides/dragen-v4.5/dragen-dna-pipeline/small-variant-calling#small-variant-caller-output). | `--enable-variant-caller true` | | gVCF | `{prefix}.hard-filtered.gvcf.gz` | Genome-wide variant calls including reference-confidence blocks with CpG methylation reporting. To learn more, review the [gVCF documentation](https://help.dragen.illumina.com/dragen-v4.5/product-guides/dragen-v4.5/dragen-dna-pipeline/small-variant-calling#gvcf-output). | `--enable-variant-caller true` | | methyl\_metrics | `{prefix}.methyl_metrics.csv` | Methylation calling QC: total Cs analyzed, methylation rates per context (CpG, CHG, CHH), strand alignment stats, plus lambda/pUC19 control metrics. | Always produced with `--methylation-conversion illumina` | | mapping\_metrics | `{prefix}.mapping_metrics.csv` | Mapping QC: total reads, total bases, mapped %, Q30 %, duplicate %, MAPQ distribution, mismatch rates, soft-clip counts. | Always produced when mapping is enabled | | DRAGEN\_report | `{prefix}.dragen_report.json` | Consolidated JSON metrics report aggregating all pipeline metrics into a single structured file for programmatic access. | Always produced (DRAGEN v4.5 default) | | CX\_report | `{prefix}.CX_report.txt.gz` | Genome-wide per-cytosine methylation status (position, strand, meth/unmeth counts, context). | `--methylation-generate-cytosine-report true` (auto-enabled when no variant caller) | | M-bias | `{prefix}.M-bias.txt` | Per-read-position methylation bias for CpG/CHG/CHH in R1 and R2. Diagnoses end biases. | `--methylation-generate-mbias-report true` (default=true) | **Note:** When `--enable-variant-caller=true` is set, the CX\_report defaults to off (methylation is reported in the VCF/gVCF instead). Set `--methylation-generate-cytosine-report true` explicitly to produce both. ## Output File Presence by Workflow * Which output files are produced for each pipeline configuration? | File | Germline WGS | Somatic TO WGS | Enrichment Germline | Enrichment Germline UMI | Enrichment Somatic TO | | -------------------- | :----------: | :------------: | :-----------------: | :---------------------: | :-------------------: | | BAM | Yes | Yes | Yes | Yes (collapsed) | Yes | | VCF (hard-filtered) | Yes | Yes | Yes | Yes | Yes | | gVCF | Yes | Yes | Yes | Yes | Yes | | CX\_report.txt.gz | Yes | Yes | Yes | Yes | Yes | | methyl\_metrics.csv | Yes | Yes | Yes | Yes | Yes | | mapping\_metrics.csv | Yes | Yes | Yes | Yes | Yes | | CNV VCF | Yes | Yes | No | No | No | | SV VCF | Yes | Yes | Yes | Yes | Yes | | M-bias.txt | Yes | Yes | Yes | Yes | Yes | | umi\_metrics.csv | No | No | No | Yes | No | | Nirvana annotations | Yes | Yes | Yes | Yes | Yes | ## Approximate Disk Usage Per Sample * How much disk space does a single-sample DRAGEN 5-base run consume? The following table shows total disk usage per run (includes all output files plus the loaded reference hash table, which is \~80-90 GB): | Configuration | Coverage | Output File Disk Used | Output Files Included | | ----------------------------------- | -------- | --------------------- | ------------------------------------------------------------------------------------------------------------------------------------- | | Germline WGS | \~35x | \~136 GB | BAM, VCF, gVCF, CX\_report, CNV VCF, SV VCF, annotated VCFs, methyl\_metrics, mapping\_metrics, m-bias, metrics JSON | | Somatic TO WGS | \~100x | \~296 GB | BAM, VCF, gVCF, CX\_report, CNV VCF, SV VCF, annotated VCFs, methyl\_metrics, mapping\_metrics, m-bias, metrics JSON | | Enrichment Germline Panel (non-UMI) | \~30x | \~83 GB | BAM, VCF, gVCF, CX\_report, SV VCF, annotated VCFs, methyl\_metrics, mapping\_metrics, m-bias, metrics JSON | | Enrichment Germline Panel UMI | \~30x | \~1 GB | BAM (collapsed), VCF, gVCF, CX\_report, SV VCF, annotated VCFs, umi\_metrics, methyl\_metrics, mapping\_metrics, m-bias, metrics JSON | | Enrichment Somatic TO Panel | \~30x | \~11 GB | BAM, VCF, gVCF, CX\_report, SV VCF, annotated VCFs, methyl\_metrics, mapping\_metrics, m-bias, metrics JSON | **Note:** Output file disk space does not includes the reference hash table (\~80-90 GB). --- # Agent Instructions This documentation is published with GitBook. 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