DRAGEN MRD Pipeline

For command line examples and parameter details, see DRAGEN recipes: DNA Somatic Tumor-Normal-MRD

The DRAGEN MRD (minimal residual disease) pipeline utilizes a tumor-informed whole genome sequencing (WGS) approach to detect trace amounts of circulating tumor DNA (ctDNA) in an individual's bloodstream (plasma) by analyzing high-confidence somatic variant sites and alleles previously established as the individual's tumor fingerprint, thereby enabling the monitoring of treatment efficacy and disease progression. The sensitivity of MRD detection is expected to be below 10^-4 variant allele frequency (VAF) or 100 parts-per-million (PPM), which is significantly lower than what is required for other ctDNA variant calling applications. Therefore, a dedicated MRD Detect module is used for the ultra-sensitive detection of these very rare ctDNA molecules in plasma.

Baseline test: Tumor fingerprint

At initial diagnosis, the individual's fingerprint profile is typically established from a biopsy of the primary tumor tissue paired with a normal sample. The DRAGEN small variant caller is used to identify high-confidence somatic variants unique to the individual's cancer from this matched sample pair. This set of unique variants constitutes the individual's tumor fingerprint. It is recommended to sequence DNA from Formalin-Fixed Paraffin-Embedded (FFPE) or Fresh Frozen tumor tissue to achieve a target raw coverage of 100X, and DNA from buffy coat or plasma-depleted whole blood (BC) as the matched normal to achieve a target raw coverage of 50X.

Recurrent test: Plasma follow-up

After treatment (e.g., surgery, chemotherapy, stem cell transplant), follow-up plasma samples are collected at various time points to detect residual cancer DNA. The tumor fingerprint from the initial diagnosis is used to inform the variant sites where residual disease is monitored. MRD Detect also provides sample QC metrics related to the coverage of informative sites in the individual's fingerprint profile and noise estimates for a given plasma sample. It is recommended to sequence plasma samples to achieve a target raw coverage of 50X.

Pipeline

The DRAGEN MRD pipeline does not include a pre-built workflow script, but rather defines the required computational steps. Data management, sample tracking, and workflow scripts are left to the user.

BCL demultiplexing must be completed prior to running the DRAGEN MRD pipeline to ensure that sample-specific FASTQs are available as input to the pipeline.

The diagram below illustrates the two primary workflows: Fingerprint Workflow and MRD Workflow. Detailed steps are outlined in the accompanying table.

Step	Description
Step 0: Fastq generation	Convert BCL files to FASTQ format for all sample types (FFPE, BC, Plasma).
Step 1: Read alignment and targeted germline variant calling	Map and align reads from the FFPE/BC/Plasma trio samples to generate BAM/CRAM files and call germline variants at ~37K single nucleotide polymorphism (SNP) sites with high population allele frequencies (typically close to 50% VAF) to generate germline VCF files. The selected sites for germline variant calling have high variability and are informative for sample matching and cross-contamination QC (i.e., contamination from a different human) algorithms. This step also provides BC sample cross-contamination QC results.
Step 2: Fingerprint generation + QC	(A) Run the DRAGEN Tumor/Normal somatic small variant caller on the matched FFPE-BC sample pair to generate a fingerprint VCF. This step also provides FFPE normal-aware sample cross-contamination QC results. (B) Run FFPE/BC sample matching QC using the DRAGEN CheckFingerprint module, which compares the FFPE and BC germline VCFs generated in Step 1 to determine whether they are from the same individual using a statistical test based on matching alleles. A positive LOD (Logarithm of the Odds) score means the two samples are from the same individual.
Step 3: MRD detection	Run the MRD Detect module on the plasma sample for ultra-sensitive detection of residual cancer DNA using the tumor fingerprint VCF to inform the variant sites where residual disease is monitored.
Step 4: Plasma QC	(A) Run Plasma/BC sample matching QC using the DRAGEN CheckFingerprint module, which compares the Plasma and BC germline VCFs generated in Step 1 to determine whether they are from the same individual using a statistical test based on matching alleles. A positive LOD (Logarithm of the Odds) score means the two samples are from the same individual. (B) Run the MRD Detect module on the plasma sample using ~37K SNP sites with high population allele frequencies (typically close to 50% VAF) after excluding the individual's BC germline variants as a highly sensitive method for detecting very low levels of contamination from a different human.

Step 0: Fastq generation

Convert BCL files to FASTQ format for all samples (FFPE, BC, Plasma). This step is optional if FASTQ data already exists. If starting from BCL files, this step must be completed before running the pipeline to ensure that sample-specific FASTQs are available as input.

For command line details, see the MRD recipe: Step 0

Step 1: Read alignment and targeted variant calling

Map and align reads from the FFPE/BC/Plasma trio samples to generate BAM/CRAM files and call germline variants at ~37K single nucleotide polymorphism (SNP) sites with high population allele frequencies (typically close to 50% VAF) to generate germline VCF files. These germline VCF files will be used by downstream QC steps, including sample matching and plasma contamination detection steps. While the graph/pangenome reference is generally recommended for germline variant calling, all MRD analyses are performed on the linear reference for consistency.

For FFPE samples, use Step 1A with the --Aligner.hard-clips=7 parameter. For BC and Plasma samples, use Step 1B.

For command line details, see the MRD recipe: Step 1A (FFPE) and Step 1B (BC/Plasma)

Step 2: Fingerprint generation + QC

Step 2A: Fingerprint generation and FFPE normal-aware contamination QC

Run the DRAGEN Tumor/Normal somatic small variant caller on the matched FFPE-BC sample pair to generate a fingerprint VCF. The setting --mrd-fingerprint=true activates additional strict filters, including more aggressive read position filtering. This helps to reduce false positive variants in the tumor fingerprint. This step also provides FFPE normal-aware sample cross-contamination QC results.

For command line details, see the MRD recipe: Step 2A

Step 2B: FFPE/BC sample matching QC

Run FFPE/BC sample matching QC using the DRAGEN CheckFingerprint module, which compares the FFPE and BC germline VCFs generated in Step 1 to determine whether they are from the same individual using a statistical test based on matching alleles. A positive LOD (Logarithm of the Odds) score means the two samples are from the same individual.

For command line details, see the MRD recipe: Step 2B

For more details on sample matching (also referred to as DRAGEN CheckFingerprint, but not to be confused with the tumor fingerprint) please refer to: Check Sample Identity with CheckFingerprint.

Step 3: MRD detection

Run the MRD Detect module on the plasma sample for ultra-sensitive detection of residual cancer DNA using the tumor fingerprint VCF to inform the variant sites where residual disease is monitored. In this step, lower quality reads are removed from consideration, such as those with low Phred scores or where data from only one read direction is available. The number of variant signals seen at the individual's fingerprint sites is 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 the fingerprint sites significantly exceeds the value expected from sequencing "noise", a positive ctDNA detection call is made, indicating the presence of cancer DNA in the plasma.

For command line details, see the MRD recipe: Step 3

Step 4: Plasma QC

Step 4A: Plasma/BC sample matching QC

Run Plasma/BC sample matching QC using the DRAGEN CheckFingerprint module, which compares the Plasma and BC germline VCFs generated in Step 1 to determine whether they are from the same individual using a statistical test based on matching alleles. A positive LOD (Logarithm of the Odds) score means the two samples are from the same individual.

For command line details, see the MRD recipe: Step 4A

Step 4B: Plasma contamination QC

Run the MRD Detect module on the plasma sample using ~37K SNP sites with high population allele frequencies (typically close to 50% VAF) after excluding the individual's BC germline variants as a highly sensitive method for detecting very low levels of contamination from a different human. Specifically, the algorithm analyzes pileups at the specified loci with high population allele frequencies after excluding germline variant sites known to be present in the individual, and then doubles the estimated VAF (eVAF) at these loci to approximate the fractional foreign contamination.

Of note, this module does not inherently adjust for VAF distortions that can occur in copy number variation (CNV)-rich somatic samples. Therefore, it is recommended to include a safety margin when considering reported plasma contamination levels.

For command line details, see the MRD recipe: Step 4B