> For the complete documentation index, see [llms.txt](https://help.bioinsight.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.bioinsight.illumina.com/dragen/dragen-v4.4/product-guide/dragen-v4.4/mrd.md). # DRAGEN MRD Pipeline The DRAGEN MRD (Minimal Residual Disease) pipeline is a tumor-informed whole genome sequencing (WGS) research workflow designed to detect tumor-derived circulating tumor DNA (ctDNA) in plasma samples. To detect trace ctDNA in plasma samples, the analysis targets sites and alleles previously identified as somatic variants in a matched tumor sample. These variants are collectively referred to as the tumor fingerprint. Specialized algorithms identify rare tumor-derived molecules and support detection of ctDNA at very low tumor fractions. The MRD detect component analyzes plasma sequencing data using the tumor fingerprint and generates quality control (QC) metrics to help assess sequencing run and data quality. **Baseline Test: Tumor Fingerprint** The tumor fingerprint profile is established from a primary tumor tissue sample paired with a matched normal sample. The DRAGEN small variant caller identifies high-confidence somatic variants unique to the tumor sample from the matched sample pair. This set of unique markers constitutes the "tumor fingerprint." It is recommended to prepare libraries with greater than 80X average tumor coverage and greater than 30X average normal coverage. Tumor samples can be FFPE (Formalin-Fixed Paraffin-Embedded) or fresh frozen. Buffy Coat (BC) matched normal samples are recommended. **Follow-up Plasma Samples** Follow-up plasma samples are collected over time for analysis of tumor-derived DNA. The tumor fingerprint is used to identify the variant sites analyzed by the MRD workflow. Follow-up samples are also evaluated against QC thresholds to ensure sufficient quality. An inter-sample contamination detection step is included to identify potential sample contamination. It is recommended to sequence plasma samples at approximately 50X average WGS coverage. ## Pipeline The DRAGEN MRD pipeline does not include a pre-built workflow script, but defines the required computational steps. Data management, sample tracking, and workflow scripts are left to the user. BCL demultiplexing must be completed before running the pipeline to ensure that sample-specific FASTQ files (or BAM/CRAM files) are available as input. The following diagram lists the main DRAGEN steps. ![](https://3555446211-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F-MflHs6hPlQAu-m5s2h_%2Fuploads%2Fgit-blob-86505ec2b48eb799a0341040b9518a96bab11599%2Fmrd_simple_flow.png?alt=media) | Step | Description | | ------------------------ | ---------------------------------------------------------------------------------------------------------------------------------------- | | Fingerprint generation | Run the somatic small variant caller on the matched tumor-normal sample pair to generate a once-off fingerprint VCF. | | Germline variant calling | Run the germline small variant caller on the normal sample to generate a germline VCF file that will be used during subsequent QC steps. | | Sample matching | Compare the normal sample germline VCF to the somatic BAM from the fingerprint step. Identify mismatched T/N sample pairs. | | MRD detect | Run the MRD Detect module on the plasma sample to analyze tumor fingerprint sites and generate MRD results. | | Contamination detection | Run the MRD module on the plasma sample to identify cross-contamination from another human sample. | ## Fingerprint Generation The DRAGEN somatic Tumor/Normal small variant caller pipeline is used to generate a fingerprint VCF. The setting `--mrd-fingerprint=true` enables additional strict filters, including more aggressive read position filtering. This helps to improve the specificity of SNV calls in the fingerprint. DRAGEN Fingerprint generation command line: ``` /opt/dragen/$VERSION/bin/dragen # DRAGEN install path --ref-dir $REF_DIR # path to DRAGEN linear hashtable --output-directory $OUTPUT --intermediate-results-dir $PATH --output-file-prefix $PREFIX # Inputs (e.g. FQ lists) --tumor-fastq-list $PATH --tumor-fastq-list-sample-id $STRING --fastq-list $PATH --fastq-list-sample-id $STRING # Mapper --enable-map-align true # optional for BAM/CRAM input --enable-map-align-output true # optionally save the output BAM --enable-duplicate-marking true # default=true --Aligner.hard-clips=7 # removes soft clips to further improve variant calling specificity. # Small variant caller --enable-variant-caller true --mrd-fingerprint=true --vc-target-bed $HIGH_CONFIDENCE_REGIONS --vc-systematic-noise $PATH # e.g. FFPE_WGS_hg38_v2.0.0_systematic_noise.snv.bed.gz # Annotation --enable-variant-annotation=true --variant-annotation-data=PATH --vc-enable-germline-tagging=true # QC --qc-detect-contamination=true ``` For DRAGEN MRD, Illumina recommends using the linear hashtable. DRAGEN hashtables can be downloaded here: [Product Files](https://support.illumina.com/sequencing/sequencing_software/dragen-bio-it-platform/product_files.html) Use the settings `--vc-target-bed` or `--vc-excluded-regions-bed $BED` to limit fingerprint calls to high-confidence regions. The BED file should include easily mapped regions and exclude ALU or highly repetitive regions where recurring noise is more common. Use a systematic noise file to further improve specificity. Prebuilt systematic noise BED files (WES and WGS) can be downloaded here: [Product Files](https://support.illumina.com/sequencing/sequencing_software/dragen-bio-it-platform/product_files.html). | Prebuilt WES/WGS noise files | Description | | -------------------------------------------------- | ------------------------ | | `WGS_hg38_v2.0.0_systematic_noise.snv.bed.gz` | For WGS FF | | `FFPE_WGS_hg38_v2.0.0_systematic_noise.snv.bed.gz` | For WGS FFPE (only hg38) | For more information on systematic noise files, refer to: [SNV systematic noise](/dragen/dragen-v4.4/product-guide/dragen-v4.4/dragen-dna-pipeline/small-variant-calling/somatic-mode.md#systematic-noise-filtering) To download germline annotation files, refer to: [Nirvana](/dragen/dragen-v4.4/product-guide/dragen-v4.4/nirvana.md) ## Germline Small Variant Calling Run the DRAGEN germline small variant calling pipeline on the normal sample to generate a germline small variant VCF. This VCF will be used in downstream QC steps, including sample matching and the plasma cross-sample contamination detection steps. Illumina recommends the graph/pangenome reference for germline calling, but MRD analyses are performed on the linear reference for consistency. DRAGEN germline small variant calling command line: ``` /opt/dragen/$VERSION/bin/dragen # DRAGEN install path --ref-dir $REF_DIR # path to DRAGEN linear hashtable --validate-pangenome-reference=false # required --output-directory $OUTPUT --intermediate-results-dir $PATH --output-file-prefix $PREFIX # Inputs (e.g. FQ list) --fastq-list $PATH --fastq-list-sample-id $STRING # Mapper --enable-map-align true # optional for BAM/CRAM input --enable-map-align-output false # optionally save the output BAM (not required) --enable-duplicate-marking true # default=true # Small variant caller --enable-variant-caller true ``` ## Sample Matching The tumor BAM from the fingerprint generation step can be compared to the normal sample germline VCF to ensure the tumor-normal samples are matched from the same individual. For more details on sample matching (also referred to as DRAGEN checkfingerprint, but not to be confused with the tumor fingerprint), refer to: [Sample Matching](/dragen/dragen-v4.4/product-guide/dragen-v4.4/dragen-dna-pipeline/checkfingerprint.md). Example sample matching command line: ``` /opt/dragen/$VERSION/bin/dragen --ref-dir $REF_DIR # path to DRAGEN linear hashtable --validate-pangenome-reference=false # required -b $TUMOR_SAMPLE_BAM --output-directory $PATH --output-file-prefix $STRING --enable-checkfingerprint true --checkfingerprint-expected-vcf $VCF # normal sample germline VCF ``` ## MRD Detect Run the MRD Detect module on the plasma sample using the tumor fingerprint VCF to analyze tumor fingerprint sites and generate MRD results. In this step, lower-quality reads are excluded from the analysis, such as reads with low Phred scores or reads supported by only one read direction. Variant signals observed at the individual's fingerprint sites are counted and compared to a statistical noise model based on dynamically generated noise sites to produce a statistical log-likelihood score. When the signal at fingerprint sites exceeds the level expected from sequencing noise, the workflow generates a detection result. Higher scores indicate stronger evidence of tumor-derived DNA in the analyzed sample. Example MRD detect command line: ``` /opt/dragen/$VERSION/bin/dragen # DRAGEN install path --ref-dir $REF_DIR # path to DRAGEN linear hashtable --validate-pangenome-reference=false # required --output-directory $OUTPUT --intermediate-results-dir $PATH --output-file-prefix $PREFIX # Inputs (e.g. FQ list) --fastq-list $PATH --fastq-list-sample-id $STRING # Mapper --enable-map-align true # optional for BAM/CRAM input --enable-map-align-output true # optionally save the output BAM (not required) --enable-duplicate-marking true # default=true # MRD settings --enable-mrd=true --mrd-probes-file $FINGERPRINT_VCF --mrd-stats-mode production --mrd-score-threshold $INT # expected to be in the range 4 - 7 ``` The command-line parameters that control MRD detect are: | Parameter Name | Description | | --------------------- | --------------------------------------------------------------------------------------------------------------- | | --enable-mrd | Enables MRD detect. Default = "false". | | --mrd-probes-file | Path to the tumor fingerprint VCF file | | --mrd-score-threshold | Threshold used when generating MRD results. | | --mrd-stats-mode | Set to "production" to include "eVAF" (estimated VAF) and "score" values in the .mrd\_summary.json output file. | The MRD detect module generates an output summary file using the standard DRAGEN output directory and prefix: .mrd\_summary.json. The file is a valid JSON file that contains an array of JSON objects. DRAGEN supports running one sample at a time, so the array will be of length one. When `--mrd-stats-mode=production` is used, the JSON objects contain at least the following key-value pairs: * Run\[i].TumorEstimate.illumina.eVAF * Run\[i].TumorEstimate.illumina.score The eVAF represents the estimated fraction of tumor-derived DNA in the analyzed sample. The score provides a statistical measure associated with the MRD analysis. Higher scores indicate stronger evidence of tumor-derived DNA in the analyzed sample. The score threshold can vary depending on sample quality and sequencing coverage and can be adjusted for a specific workflow. The threshold will typically be between 4 and 7. ## Plasma Contamination Detection The MRD module can be used to detect contamination from another human sample, including very low levels of contamination. Unlike the standard DRAGEN contamination module, this module does not inherently adjust for VAF distortions that can occur in CNV-rich somatic samples. Therefore, it is recommended to include a safety margin when considering reported contamination levels. The module requires a Germline VCF and the plasma FQ files as input. The Germline VCF was generated in an earlier step using the matched normal sample (Buffy Coat). The detection module then analyzes pileups at loci with high population allele frequencies (approximately 50%), after excluding any germline variant sites known to be present in the primary sample. By doubling the estimated variant allele frequency (VAF) at these loci, the module approximates the fractional foreign contamination. Example plasma contamination detection command line: ``` /opt/dragen/$VERSION/bin/dragen # DRAGEN install path --ref-dir $REF_DIR # path to DRAGEN linear hashtable --validate-pangenome-reference=false # required --output-directory $OUTPUT --intermediate-results-dir $PATH --output-file-prefix $PREFIX # Inputs (e.g. FQ list) --fastq-list $PATH --fastq-list-sample-id $STRING # Mapper --enable-map-align true # optional for BAM/CRAM input --enable-map-align-output true # optionally save the output BAM (not required) --enable-duplicate-marking true # default=true # MRD settings --enable-mrd=true --mrd-probes-file=$COMMON_GERMLINE_VCF # VCF with common germline sites (population allele freq. ~50%) --mrd-blocklist=$NORMAL_SAMPLE_VCF # normal sample germline VCF --mrd-stats-mode=production ``` Similar to the MRD detect module, the output JSON will include two fields: * Run\[i].TumorEstimate.illumina.eVAF * Run\[i].TumorEstimate.illumina.score The eVAF (estimated Variant Allele Frequency) can be used as a proxy for DNA contamination from another individual. Since the mrd-probes-file evaluates the signal at common germline sites with population allele frequencies close to 50%, it is expected that only about half of the sites will overlap with germline sites from contaminating individuals. As a result, the eVAF can be multiplied by 2 to estimate the amount of contaminating DNA. --- # Agent Instructions This documentation is published with GitBook. GitBook is the documentation platform designed so that both humans and AI agents can read, navigate, and reason over technical content effectively. Learn more at gitbook.com. ## Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation by asking a question. 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