# Analysis launch FAQ for the Illumina 5 Base WGS and Enrichment kits **Note**: for run set up and override cycle information, see [Sequencing FAQ for the Illumina 5-Base WGS and Enrichment kits](https://knowledge.illumina.com/library-preparation/multiomics-library-prep/library-preparation-multiomics-library-prep-faq-list/000009949). **Which app is used for analysis of Illumina 5-Base DNA Prep and Illumina 5-Base DNA Prep with Enrichment libraries? Where is analysis supported (BaseSpace (BSSH), Illumina connected Analytics (ICA), local server?)** Analysis will be done in the BaseSpace apps listed below. With the DRAGEN 4.4.6 release, there is a dropdown menu labeled "Methylation for Illumina 5-Base DNA Prep", and checking the "Enable 5-Base Methylation-Aware Algorithms" will activate the proper settings for analysis of 5-Base libraries. The same named options are used in ICA apps as well. * DRAGEN Germline (gDNA or cfDNA) * DRAGEN Somatic (gDNA or cfDNA) * DRAGEN Enrichment (Enrichment libraries) Data can be analyzed on BSSH, ICA, or local server. On-board secondary analysis is not supported, but FASTQs can be generated on-board through BCL Convert, and then used as input for the pipeline in BSSH, ICA, or local server. **Should DRAGEN Germline, Somatic, and Enrichment be used for non-human samples for 5-Base analysis?** * Methylation calling for non-human samples is supported within DRAGEN Germline, Somatic, and Enrichment for 5-Base analysis. Variant calling for non-human samples is not formally validated in these pipelines but can be done experimentally. Some species may have higher CpH methylation; to support such samples, it's recommended to run: **--enable-cpg-methylated-mapping=false** in the "Additional Arguments" section of the pipeline. This setting is not recommended for analysis of samples where the majority of methylation occurs in a CpG context. **Can analysis be performed with a Basic BaseSpace account, or is a Professional BaseSpace account required? Will the app launch require iCredits?** * The DRAGEN apps are paid apps that require iCredits to run; a Basic account cannot be used. **Is a specific 5-Base license required to run 5-base analysis on an on-prem DRAGEN server? Is there a cost for the license?** * A 5-Base license is required for on-prem analysis; this license is provided with no cost/charge. * If the on-prem server is connected to the internet, the 5-Base license will automatically be “pushed” to the server. For offline servers, please contact Illumina’s Customer Care team to get the license installed. **Are there any plans for local analysis?** * Yes, local analysis with a server is available in DRAGEN v4.4+. Illumina recommends using the most current DRAGEN (currently v4.4.6) for local analysis. * There are no plans for on-instrument analysis at this time, it must be run on a standalone DRAGEN server. * Consult the [analysis information site](https://help.connected.illumina.com/dragen-5-base/run-analysis-setup/local-dragen-server-analysis-setup) for details on how to launch. * The Illumina DRAGEN pipelines are designed for the 5-Base library prep kits. **The suggested command-line in the reference manual does not mention anything about read trimming and/or UMIs? Should users enable any options related to these?** * Using the sample sheet template and DRAGEN pipelines as instructed in the user guide should cover the vast majority of a user’s trimming needs. The sample sheet contains trimming setting applied in BCL Convert to remove adapter bases. The 5-base DRAGEN pipelines have soft clipping enabled by default to remove remaining artifacts. * gDNA samples, to trim an additional, custom amount, this can be done in the sample sheet by altering the OverrideCycle setting (eg, “N5” to ignore/trim 5 bases). * The sample sheet template also contains settings ensure proper UMI handling in BCL Convert. For additional details on how to enable UMI collapsing, see the [appropriate DRAGEN recipes for the use case](https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-recipes). UMI collapsing is only recommended for Enrichment datasets to ensure sufficient data yield. **Can data be analyzed with DRAGEN Methylation? Any caveats to results interpretation?** * The DRAGEN Methylation app is not supported or recommended for use. * While DRAGEN Methylation can be run on 5-Base data, it is primarily intended for bisulfite converted DNA (where un-methylated C are converted to T), and not 5-Base libraries (where methylated C are converted to T). Running DRAGEN Methylation with 5-Base data will produce lower alignment and no variant calls. Use DRAGEN Germline, DRAGEN Somatic, or DRAGEN Enrichment after checking the box to “Enable 5-Base Methylation Mode” for Illumina 5-Base data. **Can "Oncoanalyser" be used for analysis?** * Oncoanalyser is intended for DNA or RNA data only, not DNA methylation FASTQ/BAM. Illumina recommends customers use the Illumina DRAGEN pipeline designed for the 5-Base library prep kit. **Why aren't CNV Exome analyses supported? Are PONs for WGS supported?** * The only formally supported features are the ones listed in the user guide. Therefore Illumina is not supporting either of these features for now. **What is the support for structural variant calling?** * SV Calling should be turned on when CNV calling is being performed to improve CNV results, but the SV calling is not independently verified. ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-12db40d75fcc639f85456c0e720fb01f2b3f9e1e%2Fimage1.png?alt=media) **Is a regular DRAGEN reference genome hash table used for analysis or is a special 5-Base version required? Can this 5-Base reference be made in DRAGEN Reference Builder?** * A new DRAGEN reference genome hash is required that was created with DRAGEN Reference Builder v4.4.4+ and with "Include Methylation Data in Reference" checked. For human, this reference has been added to the DRAGEN cloud apps and download site so no customization building should be needed. * Image of option from DRAGEN Reference Builder v4.4.4: ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-beab694d1a5e1ee38ba1f049ce392ec355cdc7a1%2Fimage2.png?alt=media) **What settings are required to create a 5-Base compatible hash table for Map/Align?** The analysis is compatible with all references that are labeled as “methyl\_cg” (which is everything in the DRAGEN v11 Reference folder on ICA). **Regarding reference hash table selection for 5-Base, would there be any reason to pick the graph reference over linear for the hg38 v11 hash table, ie, hg38-alt\_masked.cnv.graph.hla.methyl\_cg.rna-11-r5.0-1 vs hg38-alt\_masked.cnv.hla.methyl\_cg.methylated\_combined.rna-11-r5.0-1 (from** [**here**](https://support.illumina.com/sequencing/sequencing_software/dragen-bio-it-platform/product_files.html)**\*\*\*\*)? There is an additional subdirectory "methyl\_converted/" along with "methyl\_cg/" in the linear reference, but it might not be relevant to 5-Base. Is that correct? This question applies to both germline (with small variant calling) and somatic (tumor only) pipelines.** * Use graph genome reference for germline analyses. * Use linear genome reference for somatic analysis (somatic VC, shouldn't impact mC). * methyl\_cg subdirectory is used for 5-Base data to account for conversion in CpG-dense regions. methyl\_converted subdirectory is specific to 3-base methods (BiS/EMS) and used by the DRAGEN Methylation pipeline. **For somatic variant calling, does Illumina have a recommended file for systematic noise?** The systematic noise file Illumina recommends using (WGS\_hg38\_v2.0.0\_systematic\_noise.snv.bed.gz) is part of the standard DRAGEN Resource files available for DRAGEN v4.4 on Illumina's *DRAGEN Secondary Analysis* [support site](https://support.illumina.com/sequencing/sequencing_software/dragen-bio-it-platform/product_files.html). * See under *Illumina DRAGEN Somatic small variant calling - WGS, WES >* [SNV Somatic Systematic Noise v2.0.0](https://webdata.illumina.com/downloads/software/dragen/resource-files/misc/systematic-noise-baseline-collection-2.0.0.tar) * This is what is contained in the download .tar: ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-b498113058f5c6da92df1bcda8b2e1a84d8bde3f%2Fimage3.jpg?alt=media) **Is there demo data in BaseSpace at this moment or in near future?** * The following demo data sets are expected to be available in Q2 2026 in BaseSpace and ICA: ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-ae9b838727e63de18c79461bfa93abfceb32064d%2Fimage4.png?alt=media) * In BaseSpace, demo data is available under the "Demo Data" tab, and can be found by searching “5-base” or the full dataset name. * In ICA, demo data is available in the DRAGEN v4.4 bundle under "/Illumina DRAGEN 5-Base Methylation Germline Demo Data". **How long does 5-Base analysis take?**\ DRAGEN local runs, < 1 hour for germline. Somatic WGS 100X tumor-only \~3 hours. Somatic WGS 100X/50X T/N \~4 hours. **Is Sample-specific NTD Error Bias Estimation enabled for somatic calling in 5-Base analysis? Are there any changes in the small VC calling in Somatic mode?**\ Yes, it is enabled by default. It's adapted in 5-Base to also be accurate with C>T and G>A Error estimates. **While estimating a kmer uniqueness map for CNV calling, is the methylation status of a base considered? Are the intervals generated based on the post conversion (5mC => T) kmers?**\ No, methylation status is not considered. \ \ \
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